US20210000900A1 - Compositon for preventing or treating osteoporosis - Google Patents

Compositon for preventing or treating osteoporosis Download PDF

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Publication number
US20210000900A1
US20210000900A1 US16/976,367 US201916976367A US2021000900A1 US 20210000900 A1 US20210000900 A1 US 20210000900A1 US 201916976367 A US201916976367 A US 201916976367A US 2021000900 A1 US2021000900 A1 US 2021000900A1
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extract
composition
gentiana scabra
sambucus williamsii
gene
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Hong Sik Kim
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Hong Sik Kim
Pens Co Ltd
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Hong Sik Kim
Pens Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/35—Caprifoliaceae (Honeysuckle family)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/51—Gentianaceae (Gentian family)
    • A61K36/515—Gentiana
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
    • A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation, decoction
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/37—Extraction at elevated pressure or temperature, e.g. pressurized solvent extraction [PSE], supercritical carbon dioxide extraction or subcritical water extraction
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00

Definitions

  • the present invention relates to a composition for preventing or treating osteoporosis, comprising a Gentiana scabra extract and a Sambucus williamsii extract.
  • Bone metabolism disease is emerging as a major problem as we enter the aging society, and osteoporosis accounts for the largest proportion of bone metabolic diseases.
  • osteoporosis is a disease in which the composition of the skeleton is changed due to the imbalance of calcium metabolism, thereby reducing bone mass and easily causing fractures of the radius, femoral, and vertebrae, and in particular has the highest incidence in post-menopausal women, and estrogen secretion has been reported as the main cause.
  • Bone remodeling continues to occur even after bone growth is over, homeostasis is maintained between bone formation by osteoblasts and bone resorption by osteoclasts through this process, and bone tissue under normal conditions has a balance of bone formation and bone resorption.
  • Osteoblast progenitor cells are differentiated by various factors (for example, BMP, ALP, and TGF- ⁇ ) to transform into osteoblasts, and fully differentiated osteoblasts are present in the form of osteocytes in the bone matrix by non-protein and protein components.
  • osteoblasts produce a receptor activator of NK-kB ligand (RANKL) and its induction receptor (decoy receptor), OPG (osteoprotegerin).
  • RNK-kB ligand RNK-kB ligand
  • OPG osteoprotegerin
  • osteoclast progenitor cells A very important process for bone resorption is differentiation into osteoclasts, and the main ligand that mediates differentiation is RANKL, and when RANKL binds to RANK, which is a receptor on the surface of osteoclast progenitor cells, osteoclast progenitor cells mature into osteoclasts, resulting in bone resorption.
  • genes that play an essential role in osteoclast differentiation are substances such as NF-kB and c-FOS.
  • osteoclasts produced from blood cells (hematopoietic stem cells), and are used to maintain body function by forming holes in the bone and releasing a small amount of calcium into the bloodstream.
  • osteoblasts produced from bone cells fill the holes with collagen and cover the deposits (hydroxyapatite) of calcium and phosphorus to rebuild the skeleton by making new hard bones.
  • osteoporosis is a representative disease.
  • Korean Patent Laying-Open No. 10-2017-0054115 discloses a pharmaceutical composition for preventing or treating osteoporosis comprising an extract from germinated gemmule of bean
  • Korea Patent No. 10-178311 discloses a composition for preventing or treating bone metabolic diseases, comprising the combined extracts of Schisandra chinensis, Eucommia ulmoides, and Lycium barbarum as active ingredients.
  • the present invention provides a composition for preventing or treating osteoporosis, comprising a Gentiana scabra extract and a Sambucus williamsii extract, according to an embodiment of the present invention.
  • the mixing weight ratio of the Gentiana scabra extract and the Sambucus williamsii extract may be 1:0.1 to 10.
  • the composition may comprise the Gentiana scabra extract and the Sambucus williamsii extract in an amount of 0.001 to 100% by weight, based on the total weight of the total composition.
  • the Gentiana scabra extract and the Sambucus williamsii extract may be extracted by hot water extraction.
  • the composition may improve expression of a gene related to bone formation.
  • the gene related to bone formation may be one or more gene selected from the group consisting of ALP, osteocalcin, BMP, TGF- ⁇ , and osteoprotegerin.
  • the composition may inhibit the expression of a gene related to bone resorption.
  • the gene related to bone resorption may be one or more gene selected from the group consisting of NF-kB, c-FOS, NFATC-1, Cathepsin K, and MMP-9.
  • the present invention provides a method of preparing a composition for preventing or treating osteoporosis, according to another embodiment of the present invention.
  • the method of preparing the composition may comprise the steps:
  • the heating temperature in step 1) above may be 80 to 100° C.
  • Step 2) above may be carried out at 30 to 70° C.
  • composition for preventing or treating osteoporosis according to the present invention may provide an effect of increasing bone formation and inhibiting bone resorption.
  • composition for preventing or treating osteoporosis according to the present invention may increase bioaffinity and have no side effects by comprising a Gentiana scabra extract and a Sambucus williamsii extract, which are natural products, as main ingredients, thereby stably providing an effect of preventing or treating osteoporosis.
  • FIG. 1 shows the measured results of ALP activity according to the concentration of the extracts according to an embodiment of the present invention
  • FIGS. 1 a , 1 b , 1 c , 1 d , and 1 e show the results corresponding to Reference Example 1, Reference Example 2, Example 1, Example 2, and Example 3, respectively.
  • FIG. 2 shows the measured results of tartrate-resistant acid phosphate (TRAP) enzyme activity according to the concentration of the extracts according to an embodiment of the present invention
  • FIGS. 2 a , 2 b , 2 c , 2 d , and 2 e show the results corresponding to Reference Example 1, Reference Example 2, Example 1, Example 2, and Example 3, respectively.
  • TRIP tartrate-resistant acid phosphate
  • FIG. 3 shows the measured results of the expression level of ALP gene according to the concentration of the extracts according to an embodiment of the present invention
  • FIGS. 3 a , 3 b , 3 c , 3 d , and 3 e show the results corresponding to Reference Example 1, Reference Example 2, Example 1, Example 2, and Example 3, respectively.
  • FIG. 4 shows the measured results of the expression level of osteocalcin gene according to the concentration of the extracts according to an embodiment of the present invention
  • FIGS. 4 a , 4 b , 4 c , 4 d , and 4 e show the results corresponding to Reference Example 1, Reference Example 2, Example 1, Example 2, and Example 3, respectively.
  • FIG. 5 shows the measured results of the expression level of NFATC-1 gene according to the concentration of the extracts according to an embodiment of the present invention
  • FIGS. 5 a , 5 b , 5 c , 5 d , and 5 e show the results corresponding to Reference Example 1, Reference Example 2, Example 1, Example 2, and Example 3, respectively.
  • FIG. 6 shows the measured results of the expression level of Cathepsin K gene according to the concentration of the extracts according to an embodiment of the present invention
  • FIGS. 6 a , 6 b , 6 c , 6 d , and 6 e show the results corresponding to Reference Example 1, Reference Example 2, Example 1, Example 2, and Example 3, respectively.
  • FIG. 7 shows the measured results of the expression level of MMP-9 gene according to the concentration of the extracts according to an embodiment of the present invention
  • FIGS. 7 a , 7 b , 7 c , 7 d , and 7 e show the results corresponding to Reference Example 1, Reference Example 2, Example 1, Example 2, and Example 3, respectively.
  • composition for preventing or treating osteoporosis comprises a Gentiana scabra extract and a Sambucus williamsii extract as active ingredients.
  • Gentian scabra used in the present invention which is Gentiana scabra Bunge var. buergeri (Miguel) Maxim
  • Gentiana scabra Bunge var. buergeri (Miguel) Maxim is mainly distributed in Korea, Japan, northeastern China, and eastern Siberia, and it is a herbaceous perennial plant belonging to Gentianaceae, Gentianales, Gamopetalous, Eudicots, and grows on the grass in the mountains.
  • it has the effect of quenching the exuberant fire of the liver and gall bladder as the known efficacy and effects, is used as an agent for eliminating damp-heat, an agent for quenching the fire of the liver and gall bladder, a bitter stomachic, and a cholagogue, and is effective in digestion, hyperfunction of the liver, and anti-inflammatory.
  • the present inventors confirmed the effect of promoting bone formation and inhibiting bone resorption as a new effect of Gentiana scabra , and confirmed that Gentiana scabra may be effectively used for preventing or treating osteoporosis among bone metabolic diseases.
  • Gentiana scabra is effective for a strong stomach, sores with swelling, scabies, epilepsy, night sweat, convulsions, roundworm, heart disease, eczema, gastritis, chlorhydria, hypochlorhydria, gastric catarrh, and gastrosia, but it was first confirmed by the present inventors that it is effective in preventing or treating osteoporosis in connection with bone metabolic disease.
  • the Gentiana scabra may be a leaf, a sprout, a stem, or a root of the Gentiana scabra , but is not limited thereto. It is preferably a root.
  • Sambucus williamsii used in the present invention is a summergreen shrub belonging to Sambucus and has the efficacy of removing pain by activating blood flow, and thus, it is effective for wind-dampness type arthritis, low back pain, fracture, and traumatic hemorrhage, and is mainly used for the elimination of postpartum hematostasis and bruising.
  • the present inventors have confirmed that since the Sambucus williamsii may promote bone formation and inhibit bone resorption, it may be effectively used for preventing or treating osteoporosis among bone metabolic diseases.
  • the term “preventing” refers to all actions that inhibit or delay the onset of osteoporosis through administration of the composition.
  • treating refers to all actions that improve or beneficially alter the symptoms caused by osteoporosis through administration of the composition.
  • the extract may be obtained by any one or more methods selected from a solvent extraction method, an ultrasonic extraction method, a supercritical extraction method, and an extraction method by a fermentation method and a foaming method, but is not limited thereto.
  • the solvent extraction method is characterized by using water, a lower alcohol of C 1 to C 4 , or a mixed solvent thereof.
  • the solvent extraction method is preferably used, and a solvent extraction method using hot water is more preferably used, but is not limited thereto.
  • the solvent extraction method using hot water may not only safely extract the active ingredients contained in the natural product, but also extracting without using compounds, thereby reducing the side effects that may result from the final product or composition and increasing bioaffinity.
  • the mixing weight ratio of the Gentiana scabra extract and the Sambucus williamsii extract may be 1:0.1 to 10, preferably 1:0.4 to 2.5, but is not limited thereto. Wherein, when the mixing weight ratio is 1:0.1 to 10, the improvement effect on bone formation and the inhibition effect on bone resorption may be balanced with each other.
  • the Gentiana scabra extract and the Sambucus williamsii extract may be included in an amount of 0.001 to 100% by weight, preferably 0.001 to 50% by weight, more preferably 0.001 to 20% by weight, based on the total weight of the total composition, but is not limited thereto. If the content of the extracts is less than 0.001% by weight based on the total composition, the concentration may be too low to sufficiently have an effect of inducing bone formation and inhibiting bone resorption.
  • the Gentiana scabra extract may be included in an amount of 0.001 to 99.999% by weight, preferably 0.001 to 80% by weight, more preferably 30 to 70% by weight, based on the total composition, but is not limited thereto.
  • the Sambucus williamsii extract may be included in an amount of 0.001 to 99.999% by weight, preferably 0.001 to 80% by weight, more preferably 30 to 70% by weight, but is not limited thereto.
  • the composition may improve the expression of a gene related to bone formation, wherein the expression of the gene means that it occurs together in the process that osteoblast progenitor cells are differentiated into osteoblasts or in the process that osteoblasts are changed to the osteocyte form, and wherein the gene may be one or more genes selected from the group consisting of ALP, osteocalcin, BMP, TGF- ⁇ , and osteoprotegerin, preferably ALP and osteocalcin, which are the most important factors in the process that osteoblast progenitor cells are differentiated into osteoblasts, but is not limited thereto.
  • the ALP is a gene that is present in bone tissue and is known to increase its activity when bone growth is actively occurring.
  • the osteocalcin refers to a protein that is synthesized by osteoblasts during bone remodeling and is bound to the extracelluar matrix of bone.
  • the composition may inhibit the expression of a gene related to bone resorption, wherein the expression of the gene means that it occurs together in the process of differentiation into osteoclasts.
  • the gene may be one or more genes selected from the group consisting of NF-kB, c-FOS, NFATC-1, Cathepsin K, and MMP-9, preferably is one or more genes selected from the group consisting of NFATC-1, Cathepsin K, and MMP-9, but is not limited to.
  • NFATC-1 is a gene whose expression increases in the differentiation process of osteoclasts
  • Cathepsin K and MMP-9 are genes whose expression level increases when the differentiation process of osteoclasts continues.
  • composition of the present invention may be used as a pharmaceutical composition, and may further comprises one or more additives selected from the group consisting of pharmaceutically acceptable carriers, excipients, and diluents.
  • additives selected from the group consisting of pharmaceutically acceptable carriers, excipients, and diluents.
  • the kind and amount of additives that are further included in the pharmaceutical composition of the present invention are according to those generally known in the related art.
  • the composition of the present invention may comprise a pharmaceutically acceptable carrier.
  • the composition comprising a pharmaceutically acceptable carrier may be various formulations, oral or parenteral. It is prepared using diluents or excipients, such as a filler, an extender, a binder, a wetting agent, a disintegrating agent, a surfactant, and so on, which are usually used when formulated.
  • Solid formulations for oral administration include a tablet, a pill, a powder, a granule, a capsule, and so on, and such solid formulations are prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin, and so on with one or more compounds.
  • Liquid formulations for oral administration include a suspension, a solution for internal use, an emulsion, a syrup, and so on, and may include various excipients, such as a wetting agent, a sweetener, a flavoring agent, a preservative, and so on, in addition to commonly used simple diluents such as water and liquid paraffin.
  • Formulations for parenteral administration include a sterile aqueous solution, a non-aqueous solvent, a suspension, an emulsion, a lyophilized formulation, and a suppository.
  • non-aqueous solvent and the suspension solvent propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, and so on may be used.
  • suppositories witepsol, macrogol, Tween 61, cacao butter, laurin butter, glycerogelatin, and so on may be used.
  • composition of the present invention may be used by formulating appropriately for the application form in the form of an oral administration agent such as a tablet, a pill, a powder, a granule, a capsule, a suspension, a solution for internal use, an emulsion, a syrup, and so on; and a parenteral administration agent such as an aqueous solution as a sterile injectable solution, a non-aqueous solvent, a suspension, and an emulsion, a lyophilized formulation, or a suppository, and so on.
  • an oral administration agent such as a tablet, a pill, a powder, a granule, a capsule, a suspension, a solution for internal use, an emulsion, a syrup, and so on
  • a parenteral administration agent such as an aqueous solution as a sterile injectable solution, a non-aqueous solvent, a suspension, and an emulsion, a lyophilized formulation, or a
  • the composition may be administered through any general route, as long as it can reach a target tissue.
  • the composition of the present invention may be injected by intraperitoneal administration, intravenous administration, intraarterial administration, intramuscular administration, intraosseous administration, intrauterine administration, intrathecal administration, subcutaneous administration, intradermal administration, oral administration, intranasal administration, intrapulmonary administration, intrarectalal administration, intracerebroventricular administration, as desired, but is not limited thereto.
  • the composition may be administered by any device capable of transporting the active substance to a target cell.
  • composition of the present invention may be administered in a pharmaceutically effective amount.
  • a pharmaceutically effective amount refers to an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level may be determined depending on the factors including the kind and severity of individuals, age, sex, and the type of virus to be infected, the activity of the drug, the sensitivity to the drug, the time of administration, the route of administration and the rate of excretion, the duration of treatment, the drugs used simultaneously, and other factors well known in the medical field.
  • composition of the present invention may be administered in an amount of 0.01 g to 20 g per day based on the mixed dosage of Gentiana scabra and Sambucus williamsiis .
  • an extract prepared using Gentiana scabra and Sambucus williamsiis in the above content range may be considered as a daily dosage.
  • composition of the present invention may be administered as an individual therapeutic agent or may be administered in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. In addition, it may be administered single or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve the maximum effect in a minimal amount without side effects, and can be easily determined by those skilled in the art.
  • composition of the present invention may be used alone or in combination with methods using surgery, hormonal therapy, drug therapy, and biological response modifiers, in order to prevent or treat osteoporosis.
  • the present invention provides a food composition for preventing or improving osteoporosis, comprising a Gentiana scabra extract and a Sambucus williamsii extract as active ingredients.
  • the Gentiana scabra extract and the Sambucus williamsii extract may be prepared and intaked in the form of food that can be eaten all the time and improve osteoporosis.
  • the content of the Gentiana scabra extract and the Sambucus williamsii extract to be contained in the food is not particularly limited, but may be included as an example, in an amount of 0.001 to 100% by weight based on the total weight of the food composition.
  • the food is a beverage, they may be included as an example, in a ratio of 0.01 to 60 g, and as another example, in a ration of 0.1 to 60 g, based on 100 ml.
  • the composition may include additional ingredients that may be commonly used in food compositions to improve odor, taste, visual sense, and so on.
  • additional ingredients may include vitamin A, D, E, B1, B2, B6, B12, niacin, biotin, folate, pantotenic acid, and so on.
  • minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu), and so on.
  • it may include amino acids such as lysine, tryptophan, cysteine, valine, and so on.
  • food additives such as preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, and so on), bactericides (bleaching powder and higher bleaching powder, sodium hypochlorite, and so on), antioxidants (butylhydroxyanisole (BHA), butylhydroxytoluene (BHT), and so on), colorants (tar pigment, and so on), color couplers (sodium nitrite, and so on), bleaching agents (sodium sulfite), seasoning agents (MSG sodium glutamate, and so on), sweetening agents (dulcin, cyclamate, saccharin, sodium, and so on), flavoring agents (vanillin, lactones, and so on), leavening agents (alum, D-potassium hydrogen tartrate, and so on), fortified dietary supplements, emulsifying agents, thickeners (paste), coating agents, gum base, foam inhibitors, solvents, improving agents, and so on may be added.
  • functional food for improving osteoporosis may be prepared using the Gentiana scabra extract and the Sambucus williamsii extract.
  • a processed food capable of improving osteoporosis may be prepared using the food composition, and, for example, may be prepared as a functional food in the form of confectionery, beverages, alcoholic beverages, fermented foods, canned foods, milk processed foods, meat processed foods, or noodles processed foods.
  • the confectionery includes biscuits, pies, cakes, bread, candy, jelly, chewing gum, cereals (including meal replacement products such as cereal flakes, and so on), and so on.
  • the beverages include drinking water, carbonated beverages, functional sports drinks, juices (for example, apple, pear, grape, aloe, tangerine, peach, carrot, tomato juices, and so ons), Sikhye (sweet rice drink), and so on.
  • the alcoholic beverages include clear rice wine, whiskey, Soju (distilled spirits), beer, liquor, fruit wine, and so on.
  • the fermented foods include soy sauce, soybean paste, red chilli paste, and so on.
  • the canned foods include canned seafoods (for example, canned tuna, canned mackerel, canned saury, canned top shell, and so on), canned meats (for example, canned beef, canned pork, canned chicken, canned turkey, and so on), and canned produce (for example, canned corn, canned peach, canned pineapple, and so on).
  • the milk processed foods include cheese, butter, yogurt, and so on.
  • the meat processed foods include pork cutlet, beef cutlet, chicken cutlet, sausage, sweet and sour pork, nuggets, Neobiani (marinated grilled beef slices), and so on.
  • Noodles processed foods include dried noodles, thin noodles, instant noodles, udong noodles, Naengmyeon (cold noodles), seal packed wet noodles, and so on.
  • the composition may be used for retort foods, soups, and so on.
  • the term “functional food” in the present invention is the same term as food for special health use (FoSHU), and refers to food with high medical and clinical effects, which is processed to efficiently exhibit the biological control function in addition to nutrition supply, wherein the food may be prepared in various forms such as tablets, capsules, powders, granules, liquids, pills, and so on, in order to obtain useful effects in improving osteoporosis.
  • the present invention may provide a method of preparing a composition for preventing or treating osteoporosis.
  • the method of preparing the composition comprises the steps:
  • Step 1) above is a step for extracting the active ingredients of the Gentiana scabra and the Sambucus williamsiis , and is a step of effectively extracting the active ingredients by heating the Gentiana scabra and the Sambucus williamsiis with a solvent extraction method.
  • it may be a step of putting and heating the Gentiana scabra and the Sambucus williamsii in water, respectively.
  • the heating temperature in step 1) above may be 80 to 100° C. If the heating temperature is carried out at 80 to 100° C., it is possible to prevent the active ingredients from volatilizing or vaporizing during the execution while quickly extracting the active ingredients.
  • Step 2) above is a step of concentrating each of the Gentiana scabra extract and the Sambucus williamsii extract obtained from step 1) above under reduced pressure.
  • step 2) above may be carried out at 30 to 70° C., when concentrated in its temperature range, it is possible to reduce the concentration time.
  • the method of preparing a composition for preventing or treating osteoporosis, after step 2) above, may further comprise the steps of:
  • Step 3) above is a step of lyophilizing the concentrated extracts from step 2) above, and the Gentiana scabra extract and the Sambucus williamsii extract may be obtained in powder form through this step.
  • step 4) above is a step of obtaining a mixture of the Gentiana scabra extract and the Sambucus williamsii extract, and may be easily changed by those skilled in the art.
  • MC3T3-L1 cells 1 ⁇ 10 4 MC3T3-L1 cells were seeded into a 96-well plate, and DMEM containing 10% FBS was used as the culture medium. After incubation for 12 hours, each extract (Examples 1 to 5) was treated at 100-1000 ⁇ g/ml and incubated for 24 hours. Then, 20 ⁇ l of WST reagent was added thereto, light was blocked with foil to minimize WST reduction by light, and the reaction was carried out for 3 hours. Optical density was measured at a wavelength of 560 nm using an iMARKTM Microplate Reader (Bio-Rad Laboratories Headquarters, Hercules, Calif., USA).
  • alkaline phosphatase (ALP; basic phosphatase) is present in most tissues, and ALP present in bone tissue is known to increase its activity when bone growth is actively occurring.
  • 5 ⁇ 10 3 MC3T3-L1 cells were seeded into a 6-well plate, and 50 ⁇ g/ml ascorbic acid and 10 mM ⁇ -glycerophosphate were added to induce differentiation into osteoblasts. After they were treated with each of Reference Examples 1 and 2 and Examples 1 to 3 at concentrations of 50 ⁇ g/ml, 100 ⁇ g/ml, and 200 ⁇ g/ml, and incubated for 48 hours at 37° C. in 5% CO2 incubator, and then washed twice with PBS, and 0.2% triton X-100 was added thereto, and subsequently, lysis was carried out in a 37° C. incubator for 30 minutes.
  • the NC in FIG. 1 is an experimental group in which nothing was treated as a control group.
  • FIG. 1 it was confirmed that ALP activity increases as the concentration of each extract increases, and it was confirmed that Examples 1 to 3 ( FIGS. 1 c to 1 e ) exhibits better ALP activity compared to Reference Example 1 ( FIG. 1 a ) or Reference Example 2 ( FIG. 1 b ). That is, it was confirmed that the ALP activity is remarkably higher when treated with the Gentiana scabra extract and the Sambucus williamsii extract than when treated with the Gentiana scabra extract or the Sambucus williamsii extract alone. Accordingly, it was confirmed that the Gentiana scabra and the Sambucus williamsii of the present invention have excellent bone growth inducing effect.
  • the activity of the TRAP enzyme increases when osteoclasts act to resorb bone, and mononuclear osteoclast progenitor cells are formed, and then multinucleated mature osteoclasts in which cells are fused are formed, and adhere to the bone surface.
  • TRAP exhibits activity in the presence of nitrophenyl phosphate and ATP, and is an osteolytic enzyme that is uniquely present in osteoclasts.
  • RAW 264.7 osteoclast precursor cells were seeded into a 6-well plate, and RANKL was put in each well at a concentration of 30 ng/ml, and then osteoblasts treated with each of Reference Examples 1 and 2 and Examples 1 to 3 at concentrations of 50 ⁇ g/ml, 100 ⁇ g/ml, and 200 ⁇ g/ml were treated with a conditioned culture medium and incubated for 3 days.
  • FIG. 2 it was confirmed that TRAP activity decreases compared to the experimental group in which nothing was treated as the concentration of each extract increases, and it was confirmed that the TRAP enzyme activity with Examples 1 to 3 ( FIGS. 2 c to 2 e ) is more remarkably lowered compared to Reference Example 1 ( FIG. 2 a ) or Reference Example 2 ( FIG. 2 b ).
  • the TRAP enzyme activity is more remarkably lowered when treated with the Gentiana scabra extract and the Sambucus williamsii extract than when treated with the Gentiana scabra extract or the Sambucus williamsii extract alone, and thus, it was confirmed that the Gentiana scabra extract and the Sambucus williamsii extract of the present invention can inhibit bone resorption.
  • cDNA was synthesized using the iScript cDNA synthesis kit (Bio-Rad Laboratories Headquarters, Hercules, Calif., USA). To measure the expression of genes, real-time quantitative PCR was carried out using SYBR Green (iQ SYBR Green Supermix, Bio-Rad Bio-Rad Laboratories Inc.), and Real-Time PCR (Applied Biosystems, Foster City, Calif., USA) was used as the device. The base sequences of the genes used for the measurement are shown in Table 3 below.
  • PCR amplification step was as follows, and the amplification cycle was conducted at 40 cycles. Heating was carried out for 8 minutes at 95° C. for hot start, and then, denaturation at 95° C. for 15 seconds, annealing at 55° C. for 30 seconds, and extension at 72° C. for 30 seconds were repeated in the amplification step and the value was recorded after extension of each cycle. After all cycles were completed, melting curve analysis was carried out to confirm the specificity of the primer.
  • the results were analyzed with One step system software v2.1 provided by Applied Biosystems.
  • the measured results of the expression level of ALP gene are shown in FIG. 3
  • the measured results of the expression level of osteocalcin gene are shown in FIG. 4 .
  • the NC of FIG. 3 and FIG. 4 above is an experimental group in which no osteoblast differentiation induction experiment was carried out and nothing was treated as a control group.
  • FIG. 3 it was confirmed that the expression of ALP gene increases as the concentration of each extract increases, and it was confirmed that Examples 1 to 3 ( FIGS. 3 c to 3 e ) exhibits more expression of ALP gene compared to Reference Example 1 ( FIG. 3 a ) or Reference Example 2 ( FIG. 3 b ). That is, it was confirmed that the effect of inducing the expression of ALP gene is superior when treated with the Gentiana scabra extract and the Sambucus williamsii extract than when treated with the Gentiana scabra extract or the Sambucus williamsii extract alone. Accordingly, it was confirmed that the Gentiana scabra and the Sambucus williamsii of the present invention have excellent bone growth inducing effect.
  • FIG. 4 it was confirmed that the expression of osteocalcin (OC) increases as the concentration of each extract increases, and it was confirmed that Examples 1 to 3 ( FIGS. 4 c to 4 e ) exhibits more expression of osteocalcin compared to Reference Example 1 ( FIG. 4 a ) or Reference Example 2 ( FIG. 4 b ). That is, it was confirmed that the effect of inducing the expression of osteocalcin is superior when treated with the Gentiana scabra extract and the Sambucus williamsii extract than when treated with the Gentiana scabra extract or the Sambucus williamsii extract alone.
  • osteocalcin is a protein that is synthesized by osteoblasts during bone remodeling and is bound to the extracelluar matrix of bone, and thus, it was confirmed that the Gentiana scabra extract and the Sambucus williamsii extract of the present invention can induce bone remodeling.
  • Reference Examples 1 and 2 and Examples 1 to 3 were treated with each of Reference Examples 1 and 2 and Examples 1 to 3 at concentrations of 50 ⁇ g/ml, 100 ⁇ g/ml, and 200 ⁇ g/ml, and after 48 hours, the cells were recovered and RNA extraction was carried out according to the manufacturer's protocol with the RNeasy extraction kit (Qiagen, Gaithersburg, Md., USA). cDNA was synthesized using the iScript cDNA synthesis kit (Bio-Rad Laboratories Headquarters, Hercules, Calif., USA).
  • Heating was carried out for 8 minutes at 95° C. for hot start, and then, denaturation at 95° C. for 15 seconds, annealing at 55° C. for 30 seconds, and extension at 72° C. for 30 seconds were repeated in the amplification step and the value was recorded after extension of each cycle. After all cycles were completed, melting curve analysis was carried out to confirm the specificity of the primer. The results were analyzed with One step system software v2.1 provided by Applied Biosystems.
  • Osteoblast differentiation is initiated by the binding of osteoblasts, a receptor activatior of nuclear facotr-kb ligand (RANKL), which is an osteoclast inducing factor secreted from activated T lymphocytes, and its receptor RANK expressed in osteoclast progenitor cells.
  • RTKL nuclear facotr-kb ligand
  • RANKL nuclear facotr-kb ligand
  • the expression of c-fos and NFATC-1 increases, and as a result, the expression of Cathepsin K and MMP9 increases, thereby maintaining the differentiation of osteoclasts.
  • FIG. 7 The NC of FIG. 5 and FIG. 7 above is an experimental group in which no osteoclast differentiation induction experiment was carried out and nothing was treated as a control group, and the C is an experimental group in which osteoclast differentiation induction experiment was carried out but nothing was treated as a comparative group.
  • FIG. 5 it was confirmed that the expression of NFATC-1 gene decreases as the concentration of each extract increases, and it was confirmed that the expression level of NFATC-1 gene with Examples 1 to 3 ( FIGS. 5 c to 5 e ) is remarkably lower compared to Reference Example 1 ( FIG. 5 a ) or Reference Example 2 ( FIG. 5 b ). That is, it was confirmed that the effect of inhibiting the expression of NFATC-1 gene is superior when treated with the Gentiana scabra extract and the Sambucus williamsii extract than when treated with the Gentiana scabra extract or the Sambucus williamsii extract alone.
  • FIG. 6 it was confirmed that the expression of Cathepsin K gene decreases as the concentration of each extract increases, and it was confirmed that the expression level of Cathepsin K gene with Examples 1 to 3 ( FIGS. 6 c to 6 e ) is remarkably lower compared to Reference Example 1 ( FIG. 6 a ) or Reference Example 2 ( FIG. 6 b ). That is, it was confirmed that the effect of inhibiting the expression of Cathepsin K gene is superior when treated with the Gentiana scabra extract and the Sambucus williamsii extract than when treated with the Gentiana scabra extract or the Sambucus williamsii extract alone.
  • FIG. 7 it was confirmed that the expression of MMP-9 gene decreases as the concentration of each extract increases, and it was confirmed that the expression level of MMP-9 gene with Examples 1 to 3 ( FIGS. 7 c to 7 e ) is remarkably lower compared to Reference Example 1 ( FIG. 7 a ) or Reference Example 2 ( FIG. 7 b ). That is, it was confirmed that the effect of inhibiting the expression of MMP-9 gene is superior when treated with the Gentiana scabra extract and the Sambucus williamsii extract than when treated with the Gentiana scabra extract or the Sambucus williamsii extract alone.

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