KR100415826B1 - Pharmaceutical preparations containing CIBOTII RHIZOMA and Harpagophytum procumbens DC. as main ingredients - Google Patents

Abstract

The present invention is a powder of oral water, lower alcohol, ethyl acetate, aromatic hydrocarbon, chlorination of CIBOTII RHIZOMA (dried roots of Cibotium barometz (L.) and Harpagophytum procumbens DC.) It contains at least one component selected from X extracted with a solvent selected from hydrocarbons, and contains malt (HORDEI FRUCTUS GERMINIATUS), Ussel (ACHYRANTHIS BIDENTATAE RADIX), Angelica GIGANTIS RADIX, REHMANNIAE RADIX PREPARAT, (EUCOMMIAE CORTEX), broiler (CINNAMOMI CORTEX), safflower (CARTHAMI FRUCTUS), old plate (or growing up: TESTUDINS PLASTRUM OR TRIONYCIS CARAPAX), SCOLOPENDRA, OGAPI (ACANTHOPANACIS CORTEX), windproof (AEHH RADIC) LYCII FRUCTUS, DIPSACI RADIX, CERVI CORNU, GREEN GREEN, SALVIAE MILTIORRHIZAE RADIX, CRATAEGII FRUCTUS, HYUNDAI GREEN, SCROPHULARIAE RADIX, LONURED HERMASS ICATA FERMENTATA), SOJAE SEMEN, PHASEOLI SEMEN is a composition consisting of X extracted with a solvent selected from water, lower alcohol, ethyl acetate or at least one supplementary herbal medicine, and the composition is osteoporosis, rheumatoid arthritis, It has an excellent effect on the prevention and treatment of disc symptoms.

Description

Pharmaceutical preparations containing CIBOTII RHIZOMA and Harpagophytum procumbens DC. as main ingredients}

The present invention relates to a pharmaceutical formulation containing CIBOTII RHIZOMA (dried root-shaped stem of Cibotium barometz (L.)) and / or Cinnamon Root (Harpagophytum procumbens DC.) As a main component.

At present, the cases of complaints with various bone diseases in Korea are known to increase every year. However, chemotherapy and surgery, which are used for the treatment of bone diseases, have not yet achieved perfect results. These bone diseases are progressing from chronic to degenerative due to aging and the medical burdens as well as the pain accompanying them are required to develop new materials that can be utilized in the clinical field as a whole.

Rheumatoid arthritis and degenerative arthritis, known as adult disease and senile disease, are known as refractory diseases (Fig. 2a), and these diseases are known to have pathogenesis due to the activation of synovial cells of the joints and the factors caused by aging and autoimmunity. (FIG. 2 b). Rheumatoid and degenerative arthritis can be cured by developing drugs that kill or inhibit arthritis cells or inhibit or inhibit the expression of cyclooxygenase II (COX-II) (Fig. 2c), an enzyme produced by these cells. It is expected.

The present inventors have conducted a long study on herbal medicines that can be used for rheumatoid arthritis, degenerative arthritis, and disc diseases of the waist and neck. As a result (CIBOTII RHIZOMA: dried roots of Cibotium barometz (L.)) and / or

It contains powder or X as the main ingredient of Harpagophytum procumbens DC. (EUCOMMIAE CORTEX), broiler (CINNAMOMI CORTEX), safflower (CARTHAMI FRUCTUS), old plate (or growing up: TESTUDINS PLASTRUM OR TRIONYCIS CARAPAX), SCOLOPENDRA, OGAPI (ACANTHOPANACIS CORTEX), windproof (AEHH RADIC) LYCII FRUCTUS, DIPSACI RADIX, CERVI CORNU, GREEN GREEN, SALVIAE MILTIORRHIZAE RADIX, CRATAEGII FRUCTUS, HYUNDAI (SCROPHULARIAE RADIX) ), X-rays extracted with one or more auxiliary herbs selected from SOJAE SEMEN and PHASEOLI SEMEN or solvents selected from water, lower alcohol, ethyl acetate, aromatic hydrocarbons or chlorinated hydrocarbons. In the experiments and animal experiments to discover the amazing facts to suppress arthritis and inflammation, the present invention was completed.

As a result of active research on bone disease worldwide, as a result of the rapid increase in the bone marrow immunity, the pathogenesis of bone disease and the pathogenesis of arthritis have been revealed. Recently, several osteoporosis treatments have been developed and allenradates. , Tamoxifen, Vitamin D ₃ (Vitamin D ₃ ), Parathyroidhormone (PTH), and COX-II inhibitors have already been commercialized to treat rheumatoid arthritis. Sulfasalazine, an anti-inflammatory drug (Becker K, Gromer S) , Schirmer RH, Muller S. Thioredoxin reductase as a pathophysiological factor and drug target.Eur.J. Biochem., 2000 Oct 15; 267 (20): 6118-6125), thioredoxin reductase (a pathophysiological factor and drug target.Eur. J. Biochem. 2000 Oct 15; 267 (20): 6118-6125 and the like are known and are in clinical or research and development, and as the treatment for osteoporosis, alendronate and raloxifene Calcitonin (Moraghan TJ, Perez EA. Mayo Clin Proc. 2000 Aug; 75 (8): 821-9.), Estradiol (Andersson TL, Stehle B, Davidsson B, Hoglund P. Maturitas. 2000 Jun 30; 35 (3): 245-52), genistein (Mazurek AP. Polkowski K. Acta Pol Pharm. 2000 Mar-Apr; 57 (2): 135-55., 1,25-dihydroxyvitamin D (3) ( Am. J. Physiol.Endocrinol. Metab. 2000 Jul; 279 (1): E213-20), parathyroid hormone (Hunziker J, Wronski TJ, Miller SC. J. Dent. Res. 2000 Jun; 79 (6): 1431-8), alendronate (Kashyap AS, Kashyap S. Postgrad Med J. 2000 Jul; 76 (897): 417-8), estrogen receptor modulators, calcitonin, and bisphosphonates (Wimal-awansa SJ. J. Clin. Densitom. 2000 Summer; 3 (2): 187-201).

Gucheok is a citrusium barometz J. Smith that lives in the tropics. It uses root stems for medicinal purposes. 1988], belonging to the Dicksoniaceae. According to the Chinese literature, it is recorded that the vertebrae are effective in strengthening the musculoskeletons in the private sector, and they are orally transmitted. The components of the umbilical cord are onitin and onitine 4-O-beta-di-alolopranoside. (onitin 4-O-β-D-allopyranoside), onitin 4-O-beta-di-glucopyranoside, pterosin R; 4- deoxy, 4-chloro-onitin) is known and onitin has been shown to have smooth muscle relaxation [Murakami, Takao; Satake, Toshiko; Ninomiya, Katsumi; Iida, Hideki; Yamauchi, Kazuhiko; Tanaka, Nobotoshi; Saiki, Yasuhisa; Chen, Chiu-Ming, Pterosin-derivate aus der Famile Pteridaceae. Phytochemistry , 19, 1743 (1980). Yang, Meei-Shieu, Studies on the Twian fork medicine Ⅵ. Studies on onitin. Planta Medica , p 25 (1986)].

Chun Soo- Keun , nicknamed the Harpagophytum procumbens DC, inhabiting the Kalahari desert in Africa, uses rhizome for arthritis in Africa and Europe [Schmidt, S.; Rothenfelde, B., The great significance of Harpagophytum root. Zeitschrift fur Naturheilkunde. 22, 48 (1978). Seeger, PC, Harpagophytum, an effective plant remedy. Erfahrungsheilunde , vol. 8, 1978. Soulimani, R .; Younos, C .; Mortier, F .; Derrieu, C., The role of stomachal activity of plant extracts, using as an example extracts of Harpagophytum procumbens. Canad. J. Physiol. Pharm. 72 (12), 1532] Pelaliaceae family. As a component of Chun Kun-geun (harpagide), harpagoside (harpagoside), procumbide (procumbide) [Kampf, R., Schweiz. Apoth.-Ztg., 114, 377 (1976). Sticher, O., Dtsch. Apoth.-Ztg., 117. 1279 (1977). Caprasse, M., J. Pharm. Belg., 35, 143 (1980).], 8-O- (Para-Cumanoyl) -Harphazide [8-O- (p-coumaroyl) -harpagide], 6'-O- (Para-Cumanoyl) ) -Procumbide [6'-O- (p-coumaroyl) -procumbide], procumboside is known [Kikuch, Tohru; Matsuda, Satoko; Kubo, Yoko; Namba, Tsuneo, New iridoid glucosides from Harpagophytum procumbens DC. Chem. Pharm. Bull., 31 (7), 2296 (1983)] The pharmacological action of these components is unknown.

The present inventors chromatographed an effective fraction of butanol fraction (CBB) in Sephadex LH-20 and divided the three small fractions CBB-10, CBB-20 and CBB-30 into animal experiments. All small fractions of the species were valid. CBB-20 did not separate the active ingredient into the fraction containing sugar, and CBB-30 did not separate the component because it contained a polyphenolic compound. However, three single substances CBB-11, CBB-12 and CBB-13 could be separated and purified from the CBB-10 subfraction. As a result of the chemical structure, CBB-11 was known as onitin and CBB-12 was known as daucosterol, but since CBB-13 is a novel compound, it was named shinbarometzin. The structure of this compound is as follows and its chemical structure is 2-O- (9z, 12z-octadecadienyl) -3-O- [α-galactopyranosyl- (1 "-6 ')-O-β-D-galactopyranosyl] It was glycerol.

Compounds having a structural formula similar to CBB-13 ( sincbarmethine ) have been isolated and reported from Arisaema amurense Max. [Jung, Jee H .; Lee, Hongkun; Kang, Sam Sik, Diacyglycerylgalactosides from Arisaema amurense. Phytochemistry , 42 (2), 447 (1996)], but these reported compounds have ester linkages of various saturated or unsaturated fatty acids at positions 1 and 2, but CBB-13 (sincbarmethine) is a derivative of glycerol. It was proved to be a novel material with no ester bond of saturated or unsaturated fatty acids at position 1.

On the other hand, ethyl acetate fraction (LNE fraction) and butanol fraction (LNB fraction), which are effective fractions of Chunsu Geun, were combined and chromatographed on silica gel to separate and purify LN-1, LN-2 and LN-3, respectively. As a result of the chemical structure, it was identified that LN-1 is harpagoside, LN-2 is starchyose, and LN-3 is harpagide. Harpagoside and harpazide are compounds that have already been isolated and reported in the shallow water [von H. L.ichti :; A. von Wartburg, Die struktur des Harpagosides. Helvetica Chinica Acta, 49, Fasciculus 5, 1552 (1996)], Stachyose is a type of tetrasaccharide that has a relatively high content in cotton and soybeans (Kim Dong-hoon, Food Chem., P. 185, 1978). The inventors separated for the first time.

The present inventors assayed the effectiveness of arthritis for the compounds isolated and purified from the umbilical cord and the medullary muscle. As a result, CBB-13 (sinbarometin, new) and LN-3 (harpazide) had the highest efficacy, while CBB-11 (onitine) and LN-1 (harpagoside) had moderate efficacy. Proved. It has also been known that the heating of the hapagosides in alkaline yields the hapazide [Kikuchi, Tohru; Matsuda, Satoko; Kubo Yoko; Namba, Tsuneo, New iridoid glucosides from Harpagophytum procumbens DC. Chem. Pharm. Bull., 31 (7), 2296 (1983)], the present inventors have obtained that the hapazides are produced from harpagoside even at room temperature without heating in alkaline.

It is an object of the present invention that cinnabarmethine (new) and onitine, which are components of the anti-arthritis effect of the nasolabial fold, are active ingredients, and that the ingredients of harpazide and harpagoside, which are components of the myelium muscle, are effective ingredients having an anti-arthritis effect. Therefore, it is possible to obtain harpazide having a very high anti-arthritis effect from harpagoside having a relatively low anti-arthritis effect.

In the present invention, 10 to 200 parts by weight, and / or 10 to 300 parts by weight of the vertebrae (CIBOTII RHIZOMA: dried roots of Cibotium barometz (L.)), and / or Harpagophytum procumbens DC. It contains as a main ingredient, if necessary, 10 to 200 parts by weight of malt (HORDEI FRUCTUS GERMINIATUS), 50 to 500 parts by weight of cowl (ACHYRANTHIS BIDENTATAE RADIX), 50 to 500 parts by weight of Angelica GIGANTIS RADIX, REHMANNIAE RADIX PREPARAT 50 to 500 parts by weight, 50 to 500 parts by weight of EUCOMMIAE CORTEX, 50 to 500 parts by weight of CINNAMOMI CORTEX, 50 to 500 parts by weight of CARTHAMI FRUCTUS, old plate (or growing up: TESTUDINS PLASTRUM OR TRIONYCIS CARAPAX) 50 to 500 parts by weight, 10 to 200 parts by weight of SCOLOPENDRA, 10 to 200 parts by weight of ACANTHOPANACIS CORTEX, 10 to 200 parts by weight of windproof (LEDEBOURIELLAE RADIX), 10 to 200 parts by weight of liquorice (GLYCYRRHIZAE RADIX) (LYCII FRUCTUS) within 50 500 parts by weight, DIPSACI RADIX 100 to 500 parts by weight, CERVI CORNU 100 to 500 parts by weight, 20 to 200 parts by weight of SALVIAE MILTIORRHIZAE RADIX, 10 to 200 parts by weight of Sansa (CRATAEGII FRUCTUS), 10 to 200 parts by weight of scorching radish, 100 to 500 parts by weight of motherwort (LEONURI HERBA), 50 to 500 parts by weight of MASSA MEDICATA FERMENTATA, 50 to 500 parts by weight of soybean (SOJAE SEMEN), PHASEOLI SEMEN 10 to 200 parts by weight of one or more supplements selected from supplementary medicines or solvents selected from water, lower alcohols, ethyl acetate, aromatic hydrocarbons or chlorinated hydrocarbons are added to the composition to inhibit arthritis and inflammation in molecular biological experiments and animal experiments The present invention was completed by discovering surprising facts.

Accordingly, an object of the present invention is 10 to 200 parts by weight of the spine (CIBOTII RHIZOMA: dried roots of the Cibotium barometz (L.)), and / or 10 to 300 parts by weight or / or Harpagophytum procumbens DC. It provides X extracted with a solvent selected from water, lower alcohol, ethyl acetate, aromatic hydrocarbon or chlorinated hydrocarbon.

Another object of the present invention is 10 to 200 parts by weight, and / or 10 to 300 parts by weight of vertebrae (CIBOTII RHIZOMA: dried roots of Cibotium barometz (L.)), and / or Harpagophytum procumbens DC. Or it contains X as a main ingredient, here 10 to 200 parts by weight of malt (HORDEI FRUCTUS GERMINIATUS), 50 to 500 parts by weight of ACHYRANTHIS BIDENTATAE RADIX, 50 to 500 parts by weight of Angelicae GIGANTIS RADIX, Suhji sulfur (REHMANNIAE) RADIX PREPARAT) 50 to 500 parts by weight, 50 to 500 parts by weight of EUCOMMIAE CORTEX, 50 to 500 parts by weight of CINNAMOMI CORTEX, 50 to 500 parts by weight of CARTHAMI FRUCTUS, old plate (or growing up: TESTUDINS PLASTRUM OR TRIONYCIS CARAPAX) 50 to 500 parts by weight, 10 to 200 parts by weight of SCOLOPENDRA, 10 to 200 parts by weight of ACANTHOPANACIS CORTEX, 10 to 200 parts by weight of windproof (LEDEBOURIELLAE RADIX), 10 to 200 parts by weight of licorice (GLYCYRRHIZAE RADIX) Department, Goji (LYCII FRUCTU) S) 50 to 500 parts by weight, DIPSACI RADIX 100 to 500 parts by weight, CERVI CORNU 100 to 500 parts by weight, Salvia Militare (SALVIAE MILTIORRHIZAE RADIX) 20 to 200 parts by weight, Sansa (CRATAEGII FRUCTUS) 10 to 200 Parts by weight, 10-200 parts by weight of scorphoulea radix, 100-500 parts by weight of LEONURI HERBA, 50-500 parts by weight of MASSA MEDICATA FERMENTATA, 50-500 parts by weight of soybean (SOJAE SEMEN), red beans ( PHASEOLI SEMEN) to provide a composition to which the extract extracted with a solvent selected from one or more auxiliary herbs selected from 10 to 200 parts by weight or water, lower alcohol, ethyl acetate, aromatic hydrocarbon or chlorinated hydrocarbon.

Another object of the present invention is 10 to 200 parts by weight, and / or 10 to 300 parts by weight of the vertebrae ( CIBOTII RHIZOMA: dried roots of Cibotium barometz (L.)), and / or Harpagophytum procumbens DC. Or a component selected from excipients, adjuvants, diluents, preservatives, isotonic agents, suspending agents, etc., which are pharmaceutically acceptable in a composition containing X extracted with a solvent selected from water, lower alcohol, ethyl acetate, aromatic hydrocarbons or chlorinated hydrocarbons thereof. It is to provide a pharmaceutical formulation formulated into a conventional pharmaceutical formulation by mixing and preparing a conventional pharmaceutical formulation.

Another object of the present invention is 10 to 200 parts by weight, and / or 10 to 300 parts by weight of the vertebrae ( CIBOTII RHIZOMA: dried roots of Cibotium barometz (L.)), and / or Harpagophytum procumbens DC. Part or its extract as a main component, and here 10 to 200 parts by weight of malt (HORDEI FRUCTUS GERMINIATUS), 50 to 500 parts by weight of ACHYRANTHIS BIDENTATAE RADIX, 50 to 500 parts by weight of Angelica GIGANTIS RADIX REHMANNIAE RADIX PREPARAT) 50 to 500 parts by weight, 50 to 500 parts by weight of EUCOMMIAE CORTEX, 50 to 500 parts by weight of CINNAMOMI CORTEX, 50 to 500 parts by weight of CARTHAMI FRUCTUS, old plate (or growing) TESTUDINS PLASTRUM OR TRIONYCIS CARAPAX) 50-500 parts by weight, 10-200 parts by weight of SCOLOPENDRA, 10-200 parts by weight of ACANTHOPANACIS CORTEX, 10-200 parts by weight of windproof (LEDEBOURIELLAE RADIX), GLYCYRRHIZAE RADIX 10-200 Parts by weight, wolfberry (LYCII FRU) CTUS) 50 to 500 parts by weight, 100 to 500 parts by weight of DIPSACI RADIX, 100 to 500 parts by weight of CERVI CORNU, 20 to 200 parts by weight of SALVIAEMILTIORRHIZAE RADIX, 10 to 200 parts by weight of Sansa (CRATAEGII FRUCTUS) Tofu, 10 to 200 parts by weight of scalloped radish, 100 to 500 parts by weight of motherwort (LEONURI HERBA), 50 to 500 parts by weight of MASSA MEDICATA FERMENTATA, 50 to 500 parts by weight of soya beans (SOJAE SEMEN), red beans (PHASEOLI) SEMEN) containing from 10 to 200 parts by weight of one or more auxiliary herbs or extracts thereof selected from solvents selected from water, lower alcohols and ethyl acetate, and excipients, adjuvants, diluents, preservatives, isotonics which are pharmaceutically acceptable herein. It is to provide a pharmaceutical formulation formulated into a conventional pharmaceutical preparation by mixing the ingredients selected from a topical agent, a suspending agent and the like and preparing a conventional pharmaceutical preparation.

Figure 1 relates to a process for recovering the dry weight of the pharmaceutical composition extracted from the constituents with distilled water and organic solvents, concentrated under reduced pressure, freeze-dried (a), shows an organic solvent fraction extraction method according to the extraction process of the active drug ( b, c).

Figure 2 relates to the pathogenesis factors (a), site (b), molecular biological signaling pathways of the pathogenesis in the joint, a representative site of arthritis in bone disease (c).

Figure 3 shows the survival and cytomorphic appearance of synovial cells in the joints, which are representative sites of representative arthritis, chronic and degenerative bone diseases from patients.

Figure 4 shows the treatment of each fraction of the organic solvent fraction of the pharmaceutical composition to oral administration to 75 ㎍ / ml for 2 weeks to show the phenomenon of edema inhibition 70 days after induction of edema which is a part of the symptoms of chronic and degenerative bone disease of arthritis .

5 is treated with an organic solvent as an effective drug at a concentration of 10 ㎍ / ml and cultured in a fertilized egg at 37 ℃ incubator for 2 days and then carefully injected syringe with ⁵ × 10 ³ cell number on the CAM with a syringe for 3 days It shows the number and distribution of CAMs observed.

Figure 6 shows the treatment of water extracts of the pharmaceutical composition in the arthritis induced rats after 70 days, the degree of cartilage tissue destruction H / E tissue staining.

7 is a bar graph showing whether the organic solvent fraction of the mono-drug, which is a pharmaceutical composition, is treated for 24 hours at a concentration of 10 ㎍ / ml for 24 hours to inhibit NO production by stimulating LPS in a Raw264.7 cell line. It is a bar graph showing whether NO inhibits NO production by stimulating LPS for fractions CBB and LNE.

Figure 8 shows the phenomenon of inducing cell death by stimulating LPS to synovial cell lines after treatment with an organic fraction of about 20 ㎍ / ml, and observed whether the same pattern appears on the synovial cells based on the same method.

9 is a flow cytometry analysis of the effects on the cell cycle by stimulating LPS in Raw 264.7 cell line after treatment with an organic fraction of about 20㎍ / ml (a), the same on synovial membrane cells based on the same method (B) to observe the appearance of the pattern.

10 shows whether SPS-PAGE electrophoresis is performed to inhibit the expression of COX-II enzyme protein by stimulating LPS to synovial cell lines through an organic solvent, an effective drug.

Figure 11 shows that stimulating LPS to the synovial cell line through the organic solvent as an effective drug to inhibit the synthesis of iNOS and COX-II enzyme by RT-PCR (a). In the same way it is shown to inhibit the synthesis of iNOS (b) and COX-II enzyme in each fraction (c).

FIG. 12 shows that the compound identified as the active ingredient of the pharmaceutical composition induces the inhibitory effect of COX-II of intramucosal synovial cells. After standing for some time, it was observed under a fluorescence microscope.

FIG. 13 shows the treatment of edema, which is a part of chronic and degenerative bone disease symptoms of arthritis, after 70 days of oral administration of each fraction, which is an organic solvent fraction of the pharmaceutical composition, to 75 µg / ml, for 70 days. The result is a photo taken with x-ray.

14 is a CT scan of the water extract of the pharmaceutical composition of the clinical improvement effect in the disc patients of the hospital patient.

15 is a result of MRI imaging the clinical improvement effect of the pharmaceutical composition in the disc patients of the inpatients.

16 is a diagram illustrating the presence of nogo-A on the mechanism of spinal nerve palsy in disc patients of hospital patients.

FIG. 17 illustrates a pathway in which neurotransmission is blocked because injury is caused in oligodendricyte existing around axon, which is related to neurotransmission paralysis, and nogo-A is secreted.

FIG. 18 shows a pathway in which neurotransmission is blocked in nogo-A-expressing brain cells as if injury is caused in oligodendricyte around axon, a neuronal paralysis associated with neurotransmission, and NGF or CBB-13 / LNE-2 Neutrite regenerates when neurotransmission returns to its original state during the treatment.

The herbal ingredients may be powdered, or each may be extracted alone to obtain an extract, and these extracts may be mixed, or the necessary herbal ingredients may be combined and extracted together.

Next, the present invention will be described in more detail with reference to Examples and Experimental Examples, which do not limit the scope of the present invention.

Example 1

1. The main ingredients of windproof, hyssop scabies, tofu, and dong mulberry as raw materials such as gupan (Growa), gojija, hungjihwang, broiler (cinnamon), soybean paste, green tea, sweet ginseng,, Sansa, Hyunsam, motherwort, walnut, gukcheok, goku, etc. The total dry weight is added to 2 kg and mixed with 2 to 5 times the volume of tertiary distilled water (or purified water). After concentrating it under reduced pressure to 200 ml and freezing it, a freeze-dried weight of 300 g was obtained (FIG. 1 a).

2. A composition that is extracted and separated from the water extract. In the present invention, 2 kg of dry weight in each extraction method was mixed with 5,000 ml of distilled water, and then heated in a 150 ° C water bath for 6 hours, filtered with gauze, and extracted from the filtrate under reflux for 8 hours in a boiling water bath. Extracted once more. It was concentrated under reduced pressure again and lyophilized to obtain a dry weight of about 350 g. It is another object of the present invention to provide a process for separating and purifying water extracts X.

The water extract obtained in the above-described drying process has osteoporosis, rheumatoid arthritis, anti-neurotransmission paralysis and anti-inflammatory activity, as demonstrated by the examples described below. Can be used as an active ingredient in the composition of the present invention (Fig. 1).

Example 2

Extraction and fractionation of herbal medicine

1) Sphere (CR): 250 g of water and 250 ml of butanol were added to 50 g of sphere powder, and refluxed for 8 hours in a boiling water bath. The aqueous layer was filtered and lyophilized (CRW) to give 1.88 g. The butanol layer was dried under reduced pressure (CRB) to give 1.46 g.

2) Pore (SS): To 350 g of pore powder, 2 L of 70% ethanol was added, followed by cold extraction for 7 days at room temperature. Cold extraction was carried out two more times to obtain 98% of 70% ethanol. The extract was suspended in 500 ml of water and extracted sequentially with hexane (500 ml x 3) and butanol (500 ml x 3) to obtain 70 g of hexane fraction (SSH) and 12.87 g of butanol fraction (SSB), respectively. The aqueous layer was lyophilized (SSW) to give 11.48 g.

3) Emulsion (AB), Gucheok (CB), Cheon Soo-Geun (LN): 1,507 g of Emulsion Powder, 1,848 g of Gucheok Powder, and 1,205 g Cheonsu-Geun Powder, respectively, add 8 L of 70% ethanol and extract 4 times in water bath for 6 hours. X 408 g, Guol X 404 g, and Cheon Geun X 598 g were obtained, respectively. Each was suspended in 1 L of water and extracted sequentially with hexane (1 L × 3 times) and butanol (1 L × 3 times). Each extract was concentrated under reduced pressure. Yields of each 70% ethanol extract and solvent fractions are as follows (FIG. 1 b).

Wedge (AB) x: 70% ethanol 408 g, ABE 10.3 g, ABB 44 g, ABW 342 g;

CB extract: 70% ethanol 404 g, CBE 52 g, CBB 93 g, CBW 263 g;

LN X: 70% Ethanol 598 g, LNE 49 g, LNB 85 g, LNW 456 g.

Example 3

Preparation of Small Fractions CBB-10, CBB-20, and CBB-30 from Old CBB Fractions

90 g of the CBB fraction isolated in Example 2 was dissolved in 300 ml of 80% methanol, and the solution was triturated and chromatographed on a Sephadex LH-20 column (6 × 90 cm, equilibrated with 80% methanol) in three times. Was carried out. The first fraction (CBB-10) is a collection of eluted as a white turbidity, and the second fraction (CBB-20) is a collection of mainly eluted sugars. The third fraction (CBB-30) was collected by washing the column with 60% acetone. The yield of each small fraction was CBB-10, 2.1 g; CBB-20, 58 g; 23 g of CBB-30.

Example 4

Isolation of Compounds CBB-11, CBB-12, and CBB-13 from Small Fractional CBB-10

Old fractional CBB-10 (2 g) was purified by silica gel column starting with chloroform / methanol (abbreviated as CM) 20: 1 solvent, followed by CM 10: 1 → 5: 1 → 3: 1 → 2: 1 solvent. Eluted. 25 mg (Amorphous Powder in Methanol) of Compound CBB-11 (onitine) were obtained in a CM 10: 1 eluent, and 95 mg (Colorless needles in methanol) were obtained in CM 5: 1 Eluent. The CM 2: 1 eluate was collected and purified in Sephadex LH 20 (solvent as methanol) to give 420 mg (amorphous in chloroform / methanol solvent) of compound CBB-13 (sincbarmethine, novel).

1) CBB-11 (onitine)

Melting Point: 212 ℃

¹ H-NMR (CDCl ₃ ) δ: 1.19 (6H, s, 10, 11-H ₃ ), 2.34 (3H, s, 12-H ₃ ), 2.59 (3H, s, 15-H ₂ ), 2.83 ( 2H, s, 3-H ₂ ), 3.00 (2H, m, 13-H ₂ ), 3.61 (2H, m, 14-H ₂ )

2) CBB-12 (Dow Costolol)

Melting Point: 295 ℃

IRυ (cm ^-1 ): 3409 (OH), 2934, 1464, 1379, 1165, 1074, 1024, 801

3) CBB-13 (Jace Barrometin, New)

Melting Point: 143 ℃

[α] : +0.642 (c = 1.25.%, Methanol)

IRυ (cm ^-1 ): 3400 (OH), 3010 (C = CH), 1735, 1721 (ester), 1650, 1638 (C = C), 1144, 1074 (CO), 669 (CH = CH, cis)

UV limit absorption (methanol)

¹ H-NMR (CD ₃ OD): Table 1

¹³ C-NMR (CD ₃ OD): Table 1

LC / MS m / z: 677.5 [(M-1) ^- ]

EI / MS m / z (%) : 354 (C ₂₁ H ₃₈ O ₄ ⁺ , 3.5) (glycerol linolate), 299 ([354-C ₄ H ₇ ] ⁺ , 8.3), 262 (C ₁₇ H ₃₀ CO ⁺ , 18.5), 163 (C ₁₂ H ₁₉ ⁺ , 5.6), 149 (C ₁₁ H ₁₇ ⁺ , 7.4), 134 (C ₁₀ H ₁₆ ⁺ , 29.6), 123 (C ₉ H ₁₅ ⁺ , 7.4), 109 ( C ₈ H ₁₃ ⁺ , 18.5), 95 (C ₇ H ₁₁ ⁺ , 37.0), 81 (C ₆ H ₉ ⁺ , 54.6), 67 (C ₅ H ₇ ⁺ , 57.4), 55 (C ₄ H ₇ ⁺ , 74.1)

Example 5

Preparation of Compound CBB-13 Acetate

40 mg of compound CBB-13 powder was dissolved in 0.5 ml of pyridine, 1 ml of anhydrous acetic acid was added, mixed well, and the mixture was left overnight at room temperature to be acetonitrile according to a conventional method. The reaction solution was concentrated under reduced pressure, and then subjected to column chromatography on silica gel with chloroform / acetate acetate 3: 1 solvent to obtain 45 mg (amorphous) of acetate of CBB-13.

Melting Point:

IRυ (cm ^-1 ): 2930, 2857, 1752, 1638, 1373, 1229, 1138, 1067, 910, 602

¹ H-NMR (CDCl ₃ ): Table 1

¹³ C-NMR (CDCl ₃ ): Table 1

Table 1. Nuclear magnetic resonance data of CBB-13 and its acetates

Example 6

Isolation of Compounds LN-1, LN-2, and LN-3 from the LNE and LNB Fractions of Chun Soo-Keun

46 g of LNE and 81 g of LNB were combined and suspended in 500 ml of water, 600 ml of chloroform / methanol (CM) 2: 1 solution was added, mixed, and the chloroform layer was taken. The mixture was extracted two more times with a CM 2: 1 solvent to obtain a chloroform layer. The chloroform layer was collected and concentrated to give 38 g of X (Fr. 1), and the aqueous layer was concentrated to obtain about 80 g of X (Fr. 2). Chloroform X (Fr. 1) was purified on a silica gel column and recrystallized in Sephadex LH-20 (methanol solvent) to obtain a compound (12 g). Aqueous layers (Fr. 2) were chromatographed on a silica gel column with CM 20: 1 → 10: 1 → 8: 1 → 4: 1 → 2: 1 and methanol solvent. Compound LN-1 (5.1 g) was obtained again in a CM 10: 1 eluate. The methanol eluate was collected (6.5 g) and chromatographed with a 50% acetone aqueous solution on a Sephadex LH-20 column three times, and water was chromatographed with a solvent to obtain compound LN-2 (4.5 g).

The above CM 4: 1 eluate was collected and chromatographed with CM 4: 1 on a silica gel column to obtain LN-3 (0.36 g). Compounds LN-1, LN-2 and LN-3 were harpagoside, starchiose and harfazide, respectively.

1) LN-1 (Harpagoside)

Melting Point: 120 ℃ (Amorphous)

MS m / z (%): 148 ([C ₉ H ₈ O ₂ ] ⁺ , 74.8), 147 (79.1), 77 (100)

IRυ (cm ^-1 ): 3430 (OH), 1690 (C = O), 1638 (C = C), 1578, 1561, 1543, 1508,1499 (benzen), 1186, 1119, 1074, 1015 (CO), 990 , 955 (CH = CH, trans), 770, 716, 685 (CH = CH, cis)

¹ H-NMR (CD ₃ OD) δ: 1.53 (3H, s, 10-H ₃ ), 2.02 (1H, dd, J = 4.2, 15 Hz, 7-H), 2.27 (1H, dt, J = 15 , 1.2 Hz, 7-H), 2.94 (1H, br.s, 9-H), 3.22 (1H, dd, J = 7.8, 9.0 Hz glc-2), 3.35 (1H, d-like, J = 9.9 Hz, glc-4), 3.36 (2H, ddd, J = 2.1, 5.1, 10 Hz, glc-5), 3.41 (1H, dd, J = 8.7, 9.3 Hz, glc-3), 3.72 (1H, dd , J = 5.7, 12 Hz, glc-6), 3.76 (1H, dd, J = 1.2, 4.2 Hz, 6-H), 3.93 (1H, dd, J = 2.4, 12 Hz, glc-6), 4.62 (1H, dd, J = 8.1 Hz, glc-1), 4.94 (1H, dd, J = 1.5, 6.3 Hz, 4-H), 6.18 (1H, d, J = 1.2 Hz, 1-H), 6.41 (1H, d, J = 6.3 Hz, 3-H), 6.51 (1H, d, J = 15.9 Hz, C = CH-C = O), 7.38-7.42 (3H, m, benzen-3H), 7.6- 7.61 (2H, m, benzen-2H), 7.67 (1H, d, J = 15.9 Hz, CH = CC = O)

2) LN-2 (Starchios)

Melting Point: 148 ℃

[α] : +138.6

¹ H-NMR (D ₂ O) δ: 3.33 (2H, s, β-Fru (f) -1-H ₂ ), 4.20 (1H, d, J = 9.0 Hz, β-Fru (f) -3H) , 4.97 (2H, d, J = 2.4 Hz, 2 × α-Gal (p) -anomeric proton), 5.41 (1H, d, J = 3.9 Hz, α-Glu (p) -anomeric proton) (abbreviation: β -Fru (f), β-fructofuranosyl; α-Gal (p), α-glactopyranosyl; α-Glu (p), α-glucopyranosyl)

¹³ C-NMR (D ₂ O) δ: Table 2

Table 2. Nuclear Magnetic Resonance Data for LN-2 (Starchios)

location LN-2 Standard location LN-2 Standard sucrose α-Gla (p) α-Glu (p) α-Gal (p) One' 92.86 92.89 One" 99.13 93.7 2' 70.25 71.79 2" 69.20 69.9 3 ' 73.48 73.30 3 " 70.25 70.2 4' 69.56 69.95 4" 70.10 70.2 5 ' 72.04 73.12 5 " 71.75 71.7 6 ' 66.63 62.09 6 " 67.24 62.5 β-Fru (f) α-Gal (p) One 63.22 63.08 One'" 98.79 93.7 2 104.56 104.41 2'" 69.04 69.9 3 82.10 82.09 3 '" 70.13 70.2 4 74.76 74.73 4'" 70.00 70.2 5 77.12 77.16 5 '" 71.44 71.7 6 61.90 60.85 6 '" 62.20 62.5

Example 7

Complete Acid Hydrolysis of LN-2

2.4 ml of 4N HCl / dioxane / benzene (3: 1: 2) mixed solution was added to LN-2 (20 mg), followed by heating at 100 ° C. for one hour. After cooling, the aqueous layer was subjected to TLC on a silica gel plate for chloroform / methanol / water (30: 20: 5). As a result, it could be identified that fructose / glucose / calactose was 1: 1: 1 ratio.

Example 8

Partial Acid Hydrolysis of LN-2

5.0 ml of 2N HCl / dioxane / benzene (3: 1: 2) mixed solution was added to LN-2 (500 mg) and heated at 93 ° C. for 40 minutes. After cooling, the reaction solution was concentrated and then molecularly filtered with Sephadex G-25 (solvent: 5% ethanol) to obtain LN-21 (120 mg) by lyophilization. LLC-21 was subjected to TLC with complete acid hydrolysis as in the above method. As a result, only fructose and galactose were detected without fructose.

LN-21 (α-di-galactopyranosyl (1 "→ 6 ')-α-di-galactopyranosyl (1' → 6) glucopyranoside

¹ H-NMR (D ₂ O) δ: 4.66 (d, J = 8.1 Hz, β-Glu (p) -anomeric proton), 4.98 (2H, m, 2 × α-Gal (p) -anomeric proton), 5.23 (d, J = 3.6 Hz, α-Gal (p) -anomeric proton) (4.66 ppm area / 5.23 ppm area ratio = 2.14)

3) LN-3

Melting Point: 120 ℃ (Amorphous)

¹ H-NMR (CD ₃ OD) δ: 1.24 (3H, s, 10-H ₃ ), 1.79 (1h, ddd, J = 0.9, 4.2, 13.8 Hz, 7β-H), 1.90 (1H, dd, J = 4.8, 13.8 Hz, 7α-H), 2.54 (1H, br.s, 9-H), 3.21 (1H, dd, J = 7.8, 9.0 Hz, 2'-H), 3.31 (1H, 4'-) H), 3.34 (1H, m, 5'-H), 3.38 (1H, t, J = 9.0 Hz, 3'-H), 3.66 (1H, dd, J = 5.7, 12.0 Hz, 6'-H) , 3.70 (1H, t-like, J = 4.5 Hz, 6-H), 3.90 (1H, dd, J = 1.5, 12.0 Hz, 6′-H), 4.57 (1H, d, J = 7.8 Hz, 1'-H), 4.94 (1H, dd, J = 1.5, 6.3 Hz, 4-H), 5.73 (1H, d, J = 0.9 Hz, 1-H), 6.31 (1H, d, J = 6.6 Hz , 3-H)

Example 9

Preparation of Harpazide (LN-3) from Harpagoside (LN-1)

560 mg of harpagoside (LN-1) was dissolved in 20 ml of methanol, 1 ml of 1N caustic soda was added well, left to stand at room temperature overnight, and passed through a Sephadex LH-20 (solvent: 80% methanol) column. From the eluate, 400 mg of harpazide and 125 mg of sodium cinnamate were obtained.

Example 10

In a similar manner to Example 6, 2% aqueous sodium carbonate solution was added instead of 1N caustic soda, and the same procedure as in Example 6 was conducted to obtain harpazide and sodium cinnamic acid.

Example 11

Manufacturing of large quantities of harpajid from Tenshu

1 Kg of Chun-Geun Powder was leached 4 times at room temperature with 20 L of methanol, and the leachate was collected and concentrated under reduced pressure. 475 g of methanol was dissolved in 1.5 L of purified water, extracted three times with 1.5 L of hexane, and then the aqueous solution of 5% caustic soda in water was 0.1 L was added (pH 11.5-12.5), mixed well, and it was left to stand overnight at room temperature. The reaction solution was extracted twice with 2 L of isopropyl alcohol. Isopropyl alcohol fractions were collected, washed with 1 L of purified water, and the isopropyl alcohol layer was concentrated under reduced pressure to obtain 15 g of powder. The powder was chromatographed on a Sephadex LH-20 column (size 10 × 100 cm) equilibrated with 95% ethanol. The fractions in which harpazide elutes (silica gel thin layer chromatography, solvent: chloroform / methanol / water 70: 30: 4, Rf value = 0.31) were collected and concentrated to obtain 12 g of harfazide.

Example 12

Chunsu Geun 75g, Angelica 150g, Gugija 150g, Tofu 120g, 240g, Broiler 100g, Safflower 100g, Old plate 140g in the same manner as in Example 1 to obtain an X.

Example 13

75g of Cheonsu-geun, Angelica 150g, Gugija 150g, Tofu 120g, Emulsion 240g, Broiler 100g, Safflower 100g, Old plate 140g are combined and powdered.

Example 14

Chunsu Geun 75g, Angelica 150g, Ogapi 50g, Gugijaja 150g, Tofu 120g, Ulsan 240g, Broiler 100g, Safflower 100g, Old plate 140g in the same manner as in Example 1 to obtain X.

Example 15

Gucheok 50g, Gupan 140g, Goku 50g, Ogapi 50g, Tofu 120g, 240g dew, 50g windproof is extracted with water to get X.

Example 16

Malt 50g, Walnut 240g, Angelica 150g, Sookjihwang 100g, Tofu 120g, Broiler 100g, Safflower 100g, Gupan 140g, Goku 50g, Ogapi 50g, Windproof 50g, Gucheok 50g, Licorice 75g, Gugija 150g, Sokcho 150g, Green 150g , 70g of red ginseng, 25.5g of Sansa, 45g of Hyunsam, 150g of motherwort, 100g of ginseng, 80g of red beans, 50g of red beans, 75g of Chunsu root with 70% ethanol and ethanol is removed to obtain X.

Experimental Example 1

Edema Inhibitory Effect of Arthritis

In order to observe the edema inhibitory effect of the pharmaceutical composition of the present invention in order to induce edema in 6 rats per experimental group in the body of 200g body weight ZYMOSAN-A (20 mg / ml / KG) 0.5ml and Freund's Adjuvant 0.5ml Was mixed and injected into the left foot, and the progress of edema was observed for 70 days. Photographs were taken before and after induction of edema, and water extracts and organic solvent fractions were prepared to be 0.6 mg / ml, respectively. Oral administration for 14 days once a day to investigate whether the edema is suppressed. As a sample, ABE, ABB, ABW, CBE, CBB, CBW, LNE, LNB, LNW, CRB, CRW, SSH, SSB, and SSW, which are organic solvent extracts separated from the pharmaceutical composition, were used, respectively. The observed results showed that the edema was induced from day 4, and it reached about 45 days to reach the highest peak, and the edema inhibitory effect of the extract was observed by setting the period up to 70 days. The measured results were examined for edema size using a precision gauge.

Experimental Example 2

Survival of Synovial Cells in Rheumatoid Arthritis

Arthroscopy of synovial synovial cells isolated from patients with rheumatoid artheritis was performed by dispensing 10 ⁴ cells in a 6 well culture dish in a medium containing penicilin / streptomycin in 5% DMEM medium and incubating at 37 ° C for 24 hours. The cells were observed, and the cell death induced cells were examined at 200 times magnification of the phase contrast microscope to observe the presence or absence and cell proliferation state of the cells.

Experimental Example 3

Induction of Synovial Cell Angiogenesis in Fertilized Egg Uterus

To investigate the effect of the organic solvent fraction obtained in Experimental Example 2 on the cells isolated in Experimental Example 4 on the degree of angiogenesis of synovial cells, synovial cells were injected into fertilized eggs and cultured in an incubator for 3 days. The formation of chorioallantoic membrane (CAM) was observed, and in this state, the composition separated by the above method was dispensed on the CAM to be 10 µg / ml, respectively.

Experimental Example 4

Cartilage destruction of arthritis tissue

In Experimental Example 3, the phase difference was determined by hematoxylin / eosin staining to determine the degree of destruction of cartilage tissue and cartilage cells due to oral administration of the pharmaceutical composition Yang Geun-tang and Cheongpajeon to the secretion of cartilage present in the joint area and secretion of meltalloprotease. After observing with a microscope 200 magnification, the picture was taken.

Experimental Example 5

Osteoporosis Suppression in Ovarian Extracted Rats

In order to observe the osteoporosis inhibitory effect of the pharmaceutical composition of the present invention to remove the ovaries in the female body of 200g body weight of the daily dose of 5 mg / ml oral administration to confirm the course of 4 weeks and then based on the thigh After cutting about 1cm of the upper and lower parts, demineralized and parrafin-embedded blocks were prepared, cut to 5㎛ in the microtome, and observed with a phase contrast microscope at 100 magnification through hematoxylin / eosin staining.

Experimental Example 6

NO Formation in Microporous Cells Stimulated with Lipidpolysaccharide

For the extract obtained according to the method of the present invention RAW264.7 which is a macrophage (macrophage) to confirm the inhibition of the synthesis of nitric oxide (NO), a potent source of inflammation in the blood due to edema as described above Dispense 10 ³ cells per well into 96 microplates, incubate for 12 hours, and dispense lipidid polysaccharides (lipidpolysachrride (LPS)) into each well at 50 ng / well, stimulate for 2 hours, and then add organic solvent and each Fractions were added to a final concentration of 10 μg / ml, 50 μl of Greiss reagent solution was added, the reaction was performed at room temperature, and the absorbance was measured at 540 nm of an enzyme-linked immunosorbent assay (ELISA) reader. Phosphorus 0.1, 1, 10, 20, 50, 100, 150μM sodium nitrite was analyzed by color reaction with sodium nitroprusside dihydrate (SNP).

Experimental Example 7

Induction of Cell Death of Macrophage and Synovial Cell Lines

In order to investigate whether the compound identified as an active ingredient of the pharmaceutical composition isolated according to the present invention induces cell death of raw 264.7 cells, which are macrophages involved in the inflammatory response, and synovial membrane cells, penicilin / streptomycin was added to 5% DMEM medium. 10 ⁴ cells were dispensed in 6 well culture dish on the containing medium and incubated at 37 ° C. for 24 hours. The extract was added to 10 μg / ml to observe the reaction. Cell death observation was examined by photographing cells induced cell death at 200-fold magnification of the polarization microscope.

Experimental Example 8

Effects of Macrophage and Synovial Cell Lines on Cell Cycle

Incubate two cells as in Experiment 7, wash them with phosphate-buffered saline (PBS), dispense LPS into each dish at 50 ng / ml, and stimulate for 2 hours. Extract final concentration is 20 After adding the solution to ㎍ / ml for 2 hours, the supernatant was removed, washed with PBS, treated with trypsin, the cells were collected and placed in a 1.5ml eppendorf tube, centrifuged at 1,000 rpm for 5 minutes to remove the supernatant, followed by 100% EtOH. Add 1 ml and fix. At this time, 5 μg / ml propidium iodide and RNAse were mixed and prepared, and the fixed cells were centrifuged to remove the supernatant and then washed once with PBS. At the same time, the dye was added to the immobilized DNA and warmed in an incubator at 37 ° C. for 30 minutes, and the cells stained with propidium iodide were sealed in foil and stored at 4 ° C. and subjected to flow cytometry analysis. Is FACscan (Becton Dicknson, CA).

Experimental Example 9

Inhibition of expression at the transcription level of COX-II and iNOS proteins via RT-PCR

In order to identify inhibitory components of COX-II and inducible nitric oxide synthetase (iNOS), which are the causes of rheumatoid arthritis, extracts used as active ingredients in the pharmaceutical composition of the present invention as in the seventh experiment. Dispense to 10 ⁵ cells in a T75 flask, incubate for 7 days, dispense LPS to 50 ng / ml in each flask, add stimulus for 2 hours, and add the final concentration to 50 µg / ml. Collected in ml eppendorf tubes and centrifuged at 15,000 rpm for 5 minutes to remove supernatant, add 200 µl of RNAzol solution, add 50 µl of chloroform and carefully pippet lyse the cells and centrifuge at 15,000 rpm for 15 minutes at 4 ° C. by a number of times, total RNA, and then put into the same volume isopropanol to precipitate at 4 ℃ 15 bungan was washed once with 75% EtOH and dried and then put 20㎕ the RNase free dH ₂ 0 60 For 30 minutes and then dissolved by adding total RNA 10 mM dNTP 5㎕, 25mM MgCl in _{5㎕ 2 6㎕, 10x RNA PCR buffer} 5㎕, RNase inhibitor 1㎕, AMV-Optimized Taq 1㎕, AMV reverse Transcriptase XL 1㎕, 50 1 μl of pM specific primer (sense / antisense) and 26 μl of RNase free dH ₂ 0 were added to perform reverse transcription reaction at 50 ° C. for 20 minutes, and the reaction was stopped at 94 ° C. for 2 minutes to polymerase chain reaction (PCR). The reaction conditions were 35 cycles at 94 ° C 1min, 55 ° C 45sec, 70 ° C 60sec, and finally the elogation reaction at 70 ° C for 5 minutes, followed by elute the PCR product on 1% agarose gel. Based on the size marker, the presence or absence of band around 395 bp and 278 bp was confirmed.

Experimental Example 10

Confirmation of Protein Expression of COX-II by SDS-PAGE

In order to determine whether COX-2 activity is inhibited, the synovial cells (synovial membrane cells) isolated from chronic rheumatoid arthritis patients were dispensed to 10 ⁵ cells in a T75 flask to the above-described extract obtained according to the method of the present invention. Incubate daily and dispense LPS into each flask at 50 ng / ml, add stimulus for 2 hours, add extract final concentration to 50 µg / ml, and then lysis buffer (0.1M SDS, sodium azide, PMSF, pepstatin). , leupstatin, pH8.0), dissolved in 0.5ml, collected in 1.5ml eppendorf tube, heated to 100 ° C, inactivated by water, inactivated, cooled at room temperature for 5 minutes, electrophoresed on 10% polyacrylamide gel (SDS-PAGE), and then dyed. Observe by decoloring in destaining solution.

Experimental Example 11

Confirmation of COX-II Protein Expression by Immunocytochemical Analysis

In order to investigate whether the compound identified as the active ingredient of the pharmaceutical composition isolated according to the present invention induces the inhibitory effect of COX-II of intramucosal synovial membrane cells, the cells were cultured on a medium containing penicilin / streptomycin in 5% DMEM medium. 10 ⁴ cells were dispensed in a 6 well culture dish containing a slim cover glass and incubated at 37 ° C. for 24 hours. The extract was added to 10 ㎍ / ml and the reaction was observed. After washing with PBS, the cells were fixed on the cells with metanol, and then washed with PBS. After washing with PBS, labeled with the primary antibody COX-II, it was left at 4 ° C. for about 1 hour and the secondary antibody phosphor (fluorescein isothiocyanate: FITC) is labeled and shaded with foil and left for about 1 hour and observed under a fluorescence microscope.

Experimental Example 12

Decomposition of Joint Site in Edema-inhibited Rats with X-ray

As in Experiment 1, the water extract and the organic solvent fractions were orally administered for 14 days to be 75 µg / ml, respectively, to investigate whether edema was suppressed. X-ray photographs were taken of anesthetized rats during edema.

Experimental Example 13

Pre / post effect of ruptured disc treatment by CT scan

In order to investigate the clinical pharmacological effect through the separated and purified pharmaceutical composition as in Example 2, the patient was taken for two months through an example in which the disc was eluted in a patient with a ruptured disc such that the daily dose was 500 mg. Before and after CT scans were performed.

Experimental Example 14

MRI scan before / after ruptured disc treatment

As in Example 2, an example patient was taken for two months after the disc was eluted to examine the clinical pharmacological effect through the separated and purified pharmaceutical composition, and then pre and post-treatment MRI was performed.

Experimental Example 15

Therapeutic Effect on Spinal Nerve Palsy Block

In order to investigate whether the compound isolated in Example 2 is a bone disease induced neuronal phenomena by nogo-A and induces an inhibitory effect on the resulting neuronal palsy, glia originated from brains sold by the Bank of Korea Cell Line Glioblastoma cells (U-373 MG) were dispensed in 6 well culture dishes with 10 ⁴ cells on a medium containing penicilin / streptomycin in 10% DMEM medium, incubated at 37 ° C for 24 hours and well expressed in nogo-A. Stable transfection of the vector was performed to determine whether neuronal neurites were reduced or renal suppressed.At the same time, reconstitution of neuronal growth factor (NGF) or whether the same phenomena were induced in the treatment of the fractions were performed. It was observed by photographing at 200 magnification.

Experimental Example 16

As a result of observation in the cultured container as in Experimental Example 2, the cells did not grow in patients with degenerative arthritis cultured on a daily basis. After 7 days, the cells were observed at 200 times magnification of the phase contrast microscope. Therefore, in patients with degenerative arthritis, the entire synovial membrane was not surrounded by the cartilage tissue and was worn out, and the absence of the synovial membrane cells promoted the destruction of the cartilage tissue. Progress pathologically (FIG. 3).

Experimental Example 17

When the fractions isolated from Example 2 were applied to the edema induction experiment, CBB-12, 13, and LNE-1, 3 showed strong edema inhibitory effect, especially CBB-12 and LNE-3 had the strongest significance. It was. The results observed for the edema inhibitory effect on the effect of the compound in the elapsed 70 days as described below are shown in Tables 3, 4 and 4.

Table 3. Degree of inhibition of edema induction by organic solvent extraction (unit: mm)

Sample Before drug treatment After drug treatment Sample Before drug treatment After drug treatment ABE 15.5 ± 2 8.5 ± 2 LNB 15.6 ± 1 5 ± 1 ABB 14.6 ± 3 10.1 ± 1 LNW 14.5 ± 2 8 ± 1 ABW 14.4 ± 1 12.2 ± 2 CRB 15.5 ± 1 9 ± 2 CBE 16.3 ± 2 4.1 ± 1 CRW 15.6 ± 1 9 ± 1 CBB 14.6 ± 1 3.6 ± 1 SSH 14.5 ± 2 12 ± 2 CBW 15.5 ± 2 7.2 ± 2 SSB 13.3 ± 1 13 ± 1 LNE 14.5 ± 3 5.3 ± 1 SSW 14.4 ± 2 13 ± 1

Table 4. Organic Solvent Fractions with the Best Suppression of Edema

Fraction sample Before drug treatment After drug treatment CBBCBB- 13CBB- 20CBB-30 14.6 ± 214.4 ± 214.5 ± 114.3 ± 1 3.6 ± 12.5 ± 13.1 ± 23.6 ± 1 LNELNE-1 LNE-3 15.6 ± 115.1 ± 215.2 ± 1 5.2 ± 15.1 ± 23.8 ± 1

Experimental Example 18

The effect of the extract obtained as in Experimental Example 3 on the degree of angiogenesis of synovial cells was observed as a decrease in CAM. As a result, the formation of CAM was slightly slow in normal fertilized eggs, but strongly in the cells injected with synovial cells. The formation of CAM was induced and compared with the control group in the CBB-13 and LNE-3 fractions. Therefore, when the composition of the composition inhibits the proliferation of synovial cells as well as inhibits CAM induction of angiogenesis (angiogenesis) by the cells of the synovial membrane, it can be seen that the drug has an excellent effect in the treatment of arthritis (Fig. 5).

Experimental Example 19

In order to confirm the degree of destruction of cartilage tissue and cartilage cells due to the wear state of cartilage present in the joint area or secretion of meltalloprotease as in Experimental Example 4, the result was photographed after observation with a phase contrast microscope of 100 magnification through hematoxylin / eosin staining. Results In the normal group of the drug, the distribution of cartilage was uniformly distributed evenly in the distribution of bone tissues, but in the control group, bone destruction progressed and bone tissues were disintegrated. It can be seen that this water extract was found to have cartilage tissue destruction to cartilage regeneration (Fig. 6).

Experimental Example 20

The results of inferring the relative values of the drug by comparative analysis as in Experimental Example 6 showed that the control group was about 98 μM high as shown in the bar graph, while showing significant NO inhibitory effect in the CBB, CBE, LNE, and LNB fractions. It can be seen (Fig. 7a), especially in the CBB and LNE fractional series showed a stronger inhibitory effect in CBB-13 and LNE-3. This is believed to be a result of the present composition by inhibiting the synthesis of iNOS inducing NO formation (Fig. 7b).

Experimental Example 21

As in Experiment 7, it was examined whether the compound induces cell death of raw 264.7 cells and synovial cells, which are macrophages involved in the inflammatory response. As a result, the cell proliferation was actively progressed in the control group, whereas the CBB and LNE fractions were used in the drug treatment group. Phosphorylation of CBB-13, 20, and LNE-1, 3 showed a strong effect on inducing cell death. Therefore, this composition is thought to be involved in inducing cell death in two cells and consequently inhibiting inflammation and edema. (Figures 8a, b).

Experimental Example 22

As a result of observing the effect of the fraction of the active ingredient of the pharmaceutical composition in Experimental Example 8 on the cell cycle, the synovial fibroblast cell line and the macrophage raw 264.7 cell line were analyzed. In the macrophage cell line, the control group was typically G ₀ / G ₁ phase. Is relatively highest while S phase has the lowest peak and G ₂ / M phase shows the middle peak. In particular, in the case of CBB-13 in the drug treatment group, apoptosis (cell death) induces G ₀ / G ₁ arrest in LNE-3, and thus, it is predicted that the drugs are drugs that induce cell death (FIG. 9A). On the other hand, in the synovial cell line, the G ₀ / G ₁ phase of the cell is relatively lower than the G ₂ / M phase and the S phase is generally higher than the cell line in which the proliferation is generally strong. Or CBB-13, which is a metastatic cancer cell with slow growth rate or angiogenesis cell, exhibits G ₀ / G ₁ arrest. Inducing apoptosis in LNE-3, it is expected that these drugs are drugs that induce cell death in synovial cells (FIG. 9B). Therefore, it can be seen that the CBB-13 and LNE-3 fractions of the present invention are significant in the treatment of bone diseases by inhibiting the proliferation of cells that cause bone diseases by inducing cell death.

Experimental Example 23

As in Experiment 12, whether the active ingredient inhibits the expression of COX-2, which is the cause of rheumatoid arthritis in synovial cells, was electrophoresed on a 10% polyacrylamide gel (SDS PAGE) and stained to decolorize in a destaining solution. The results showed that COX-II protein had a molecular weight of about 70KD and a strong band in the control group, but a weak band in the CBB-13 and LNE-3 groups, indicating that the drug has a pharmacological effect of inhibiting protein expression. It can be seen (Fig. 10).

Experimental Example 24

PCR was performed by electrophoresis as in Experiment 9, and in case of iNOS, PCR product was confirmed to have a band around 278 bp. Lanes 1, 2, 3, and 4 were control, ABB, SSB, and CRB, respectively. In contrast, lanes 5, 6, 7, and 8 were found to affect protein expression with CBB, CBB-13, LNE, and LNE-3, whereas COX-II was expressed in lanes 1, 2, and 3 In contrast, lanes 4, 5, 6, 7, and 8 showed inhibitory effects on transcriptional levels (11 a). As in Example 15, the extracts used as active ingredients in the pharmaceutical composition of the present invention were examined for the expression of COX-2 and iNOS, which are the causes of rheumatoid arthritis, and lanes 1, 3, and 4 corresponding to the elute lanes of each fraction of the iNOS protein. The control group, ABB and CRB, respectively, had no effect on expression, whereas lanes 2, 5, 6, 7, and 8 were CBB, LNE, SD, DS, and YM, respectively. 11B), COX-2 was also a control group, CBW, ABB and LNW in lanes 1, 2, 3, and 5, respectively, and had no effect on expression. Lanes 4, 6, 7, and 8 were CBB-13, 20 and It can be seen that LNE-1, 3 and COX-II expression is strongly inhibited (Fig. 11c).

Experimental Example 25

In order to investigate whether the pharmaceutical composition isolated according to the present invention induces the inhibitory effect of intraarticular synovial cell COX-II, the cells were fixed on the cells with metanol and then washed with PBS, as in Experiment 11, and washed with PBS. After labeling -II and leaving it at 4 ℃ for 1 hour and labeling the secondary antibody FITC, it was shaded with foil and left for 1 hour, and observed under a fluorescence microscope. The protein was strongly expressed and the expression level of CBB-13 and LNE-3 tended to decrease significantly. This may be due to the fact that protein expression of COX-II inhibits transcription at the transcription level as in Example 16 (FIG. 12).

Experimental Example 26

As in Example 2, the edema inhibitory effect of the pharmaceutical composition was observed. Using the isolated organic solvent extracts CBB-12, 13, 20, 30 and LNE-1, 3, anesthetized rats were anesthetized with X-RAY. Photographs showed that the cartilage of rheumatoid arthritis was severely damaged and the degree of degradation of bone tissue was severely damaged in the control group, and the damage reached its peak until about 70 days. On the other hand, unlike the control group, it was found that the cartilage at the arthritis site was inhibited and the bone synthesis was promoted in CBB-13 and LNE-3 (FIG. 13).

Experimental Example 27

In order to investigate the clinical pharmacological effect as in Experimental Example 13, the water extract group obtained according to the method of the present invention was taken for 2 months through an example in which the disk was eluted from the disk patient, and then CT scans were performed before and after treatment. In the control group before treatment, the ruptured disc was ruptured, but after treatment, the disc was lost, neuritis was eliminated, and the resulting nerve palsy was removed (FIG. 15).

Experimental Example 28

MRI was performed on the patients with the water extract group obtained in Experimental Example 1, but the ruptured disc was ruptured in the control group before the treatment, but after the treatment, the disc was lost in the water extract group and the resulting nerve paralysis was removed. 16).

Experimental Example 29

As shown in Experiment 17, it was confirmed whether the neuronal neurite was reduced or suppressed, and at the same time, when the neuronal growth factor (NGF) was treated, it was confirmed whether the neurite was regenerated or the same phenomenon was induced when the fraction was treated. The photographs were observed at magnification. Nogo-A-expressing U-373 cell line showed the decreased or almost disappeared original elongated neurite. When NGF, CBB-13 and LNE-3 were treated, I could see the longer side. As a result, it was found that the drug promotes the regeneration of neurite so that the nerve regenerates as compared with NGF (FIGS. 18A, B, and C).

Experimental Example 30

The compound identified as the active ingredient of the pharmaceutical composition isolated according to the present invention was identified by chemical structural determination of each compound having an effect on bone disease as a novel substance of the drug CBB-13 was named shinbarometin , LNE-1, and 3 are harpagoside and harpazide, respectively, and the results of efficacy are known for the first time when the structure and pharmacological effects are known.

As confirmed from the above experimental results, the novel composition of the present invention has excellent pharmacological effect.

Therefore, the composition of the present invention can be usefully used as a pharmaceutical.

The composition of the present invention may vary depending on the sex, age, degree of disease, etc. of the patient, but when using the herbal medicine itself without extracting the composition of the present invention with water or an organic solvent, 1.0g to 50g per day Amounts may be used, and when used in the form of X, 10 mg to 1000 mg per day may be administered once to several times a day.

Compounds of the present invention are already used as an agent for osteoporosis, arthritis and ruptured discs, allenrate, tamoxifen, vitamin B ₃ , parathyroid hormone (PTH), sulfasalazine (sulfasalazine) ), Thioredoxin reductase, alendronate, raloxifene, calcitonin, calcitonin, estradiol, genistein, 1,25-dihydroxyvitamin D ₃ , may be used in combination with drugs such as alendronate, estrogen receptor modulators, biphosphonates, harpagide (the drug for which the inventors have developed their use), and the like.

By using the compound of the present invention and the above drug, it is possible to reduce the normal dose of the existing drug, thereby reducing the problems with the existing drugs.

The compounds of the present invention may be mixed with excipients, adjuvants, analgesics, isotonic agents, preservatives, and other pharmaceutically acceptable adjuvants, which are commonly used pharmaceutically, and formulated into pharmaceutically acceptable formulations for pharmaceutical purposes. Suitable formulations may be prepared. Such pharmaceutical preparations may be formulated into injections, solutions, tablets, capsules, powders, syrups and the like.

Next, the present invention will be described in more detail as formulation examples.

Formulation Example 1

50 mg of Guchu extract CBB of Example 2

Appropriate sterile distilled water for injection

pH adjuster

50 mg of Gucheol X CBB fractions of Example 2 were dissolved in distilled water for injection, adjusted to pH about 7.6 with a pH adjuster, and then the total amount was 2 ml, and then filled into 2 ml ampoules and sterilized to prepare an injection.

Formulation Example 2

Tenthmus root X 50mg of Example 2

Appropriate sterile distilled water for injection

pH adjuster

50 mg of Chun-Geun-X of Example 2 was dissolved in sterile distilled water for injection, adjusted to pH 7.2 with a pH adjuster, and the whole was made into 2 ml, and then filled in an ampoule of 2 ml to prepare an injection.

Formulation Example 3

X 200mg of Example 12

Lactose 100mg

Starch 100mg

Magnesium stearate proper amount

The above components are mixed and tableted according to a conventional method for producing tablets to produce tablets.

Formulation Example 4

Harpazide 10mg of Example 11

Lactose 100mg

Starch 50mg

Magnesium stearate proper amount

The above components are mixed and tableted according to a conventional method for producing tablets to produce tablets.

Formulation Example 5

1000 mg of powder of Example 13

Lactose 100mg

2 mg magnesium stearate

After mixing the above components, the inside is filled with a polyethylene coated coated chloride and sealed to prepare a powder.

Formulation Example 6

X 500mg of Example 14

Lactose 50mg

Starch 50mg

Talc 2mg

Magnesium stearate appropriate amount

The capsules are prepared by mixing the above components and filling gelatin capsules according to a conventional method for preparing capsules.

Formulation Example 6

X 400mg of Example 15

Lactose 100mg

Starch 93mg

Tackle 2mg

Magnesium stearate proper amount

The capsules are prepared by mixing the above components and filling gelatin capsules according to a conventional method for preparing capsules.

Formulation Example 7

X 5g of Example 14

20 g of sugar

20 g of isomerized sugar

Lemon flavor

Add 100 ml of purified water

The above components are mixed according to a conventional method for preparing a liquid, and filled into 100 ml of brown bottle and sterilized to prepare a liquid.

Formulation Example 8

X 5g of Example 16

20 g of sugar

20 g of isomerized sugar

Lemon flavor

Add 100 ml of purified water

The above components are mixed according to a conventional method for preparing a liquid, and filled into 100 ml of brown bottle and sterilized to prepare a liquid.

Formulation Example 9

Tensile root extract 50mg of Example 2

Alenate 10mg

Proper sterile distilled water for injection

pH adjuster

50 mg of Chun Geun-X extract of Example 2 was dissolved in distilled water for injection, adjusted to pH about 7.6 with a pH adjuster, and then the total amount was 2 ml, and then filled into 2 ml ampoules and sterilized to prepare an injection.

Formulation Example 10

X 50mg of Example 12

Tamoxifen 10mg

Appropriate sterile distilled water for injection

pH adjuster

X 50 mg of Example 12 was dissolved in sterile distilled water for injection, adjusted to pH 7.2 with a pH adjuster, and the whole was made to 2 ml, followed by filling into a 2 ml ampoule to prepare an injection.

Formulation Example 11

X 200mg of Example 14

20 mg sulfasalin

Lactose 100mg

Starch 100mg

Magnesium stearate proper amount

The above components are mixed and tableted according to a conventional method for producing tablets to produce tablets.

Formulation Example 12

Example 200 x 200mg

Alendronate 50mg

Lactose 100mg

Starch 50mg

Magnesium stearate proper amount

The above components are mixed and tableted according to a conventional method for producing tablets to produce tablets.

Formulation Example 13

X 500mg of Example 16

Lactose 50mg

Starch 50mg

Talc 2mg

Magnesium stearate appropriate amount

The capsules are prepared by mixing the above components and filling gelatin capsules according to a conventional method for preparing capsules.

Formulation Example 14

Guolgi extract 50mg of Example 2

Raloxifene 10mg

Lactose 50mg

Starch 50mg

Talc 2mg

Magnesium stearate appropriate amount

The capsules are prepared by mixing the above components and filling gelatin capsules according to a conventional method for preparing capsules.

Formulation Example 14

Tensile root extract 50mg of Example 2

Pamideronic disodium 20mg

Lactose 100mg

Starch 93mg

Tackle 2mg

Magnesium stearate proper amount

The capsules are prepared by mixing the above components and filling gelatin capsules according to a conventional method for preparing capsules.

Formulation Example 16

X 2000mg of Example 16

Pamideronide Disodium 1000mg

20 g of sugar

20 g of isomerized sugar

Lemon flavor

Add 100 ml of purified water

The above components are mixed according to a conventional method for preparing a liquid, and filled into 100 ml of brown bottle and sterilized to prepare a liquid.

As described in detail above, the main component of the present invention is CIBOTII RHIZOMA: dried hair root stem of Cibotium barometz (L.) and / or powder or X of the myelium root ( Harpagophytum procumbens DC.). If necessary, Malt (HORDEI FRUCTUS GERMINIATUS), Umbrella (ACHYRANTHIS BIDENTATAE RADIX), Angelica (ANGELICAE GIGANTIS RADIX), SUNGJIANG (REHMANNIAE RADIX PREPARAT), CHOAP (EUCOMMIAE CORTEX), Broiler (CINNAMOAMI CORT) , Gupan (or growing up: TESTUDINS PLASTRUM OR TRIONYCIS CARAPAX), Goku (SCOLOPENDRA), Ogapi (ACANTHOPANACIS CORTEX), Windproof (LEDEBOURIELLAE RADIX), Licorice (GLYCYRRHIZAE RADIX) CERVI CORNU, Salvia (SALVIAE MILTIORRHIZAE RADIX), Sansa (CRATAEGII FRUCTUS), Hyunsam (SCROPHULARIAE RADIX), Motherwort (LEONURI HERBA), Singok (MASSA MEDICATA FERMENTATA), Medicinal Beans (SOJAE SEMEN) 1 SEMIPH Paper boy Composition containing X and extracted with herbal medicines or their water, lower alcohols, ethyl acetate, aromatic hydrocarbon, solvents selected from chlorinated hydrocarbon is an excellent effect in the prevention and treatment of osteoporosis, rheumatoid arthritis, disk symptoms.

Claims (5)

Osteoporosis, rheumatoid arthritis, or the like obtained by extracting from powder of citric acid (CIBOTII RHIZOMA: dried root of Cibotium barometz (L.)) or water thereof, lower alcohol having 1-4 carbon atoms or mixed solvents thereof X is effective for the prevention and treatment of disc symptoms. CIBOTII RHIZOMA (extracted from dried roots of Cibotium barometz (L.)) Powder or water, lower alcohols having 1-4 carbon atoms or mixed solvents thereof. And here, Hargogophytum procumbens DC., Malt (HORDEI FRUCTUS GERMINIATUS), Umbrella (ACHYRANTHIS BIDENTATAE RADIX), Angelica (ANGELICAE GIGANTIS RADIX), REHMANNIAE RADIX PREPARAT, Chickpeas (ORTEXMIMI) Caf (DIPSACI RADIX), CERVI CORNU, Salvia (SALVIAE MILTIORRHIZAE RADIX), Sansa (CRATAEGII FRUCTUS), Hyunsam (SCROPHULARIAE RADIX), Motherwort (LEONURI HERBA), Shingok (MASSA MEDICATA Medicinal Soybeans) Choose from (PHASEOLI SEMEN) At least one auxiliary crude drugs or the water thereof, which composition is effective in the prevention and treatment of lower alcohols or contains at least 1 x species to a mixed solvent thereof osteoporosis, rheumatoid arthritis, the symptoms of the disk 1 to 4 carbon atoms. The method according to claim 1 or 2, wherein 10 to 200 parts by weight of CIBOTII RHIZOMA (dried stems of Cibotium barometz (L.) dried) is contained as a main component, and here, Harpagophytum procumbens DC .) 10 to 300 parts by weight, 10 to 200 parts by weight of malt (HORDEI FRUCTUS GERMINIATUS), 50 to 500 parts by weight of cowl (ACHYRANTHIS BIDENTATAE RADIX), 50 to 500 parts by weight of Angelica GIGANTIS RADIX, REHMANNIAE RADIX PREPARAT 50 to 500 parts by weight, 50 to 500 parts by weight of EUCOMMIAE CORTEX, 50 to 500 parts by weight of CINNAMOMI CORTEX, 50 to 500 parts by weight of CARTHAMI FRUCTUS, old plate (or growing up: TESTUDINS PLASTRUM OR TRIONYCIS CARAPAX) 50 to 500 parts by weight, 10 to 200 parts by weight of SCOLOPENDRA, 10 to 200 parts by weight of ACANTHOPANACIS CORTEX, 10 to 200 parts by weight of windproof (LEDEBOURIELLAE RADIX), 10 to 200 parts by weight of liquorice (GLYCYRRHIZAE RADIX) (LYCII FRUCTUS) 50 to 500 Part, 100-500 parts by weight of DIPSACIRADIX, 100-500 parts by weight of CERVI CORNU, 20-200 parts by weight of SALVIAE MILTIORRHIZAE RADIX, 10-200 parts by weight of CRATAEGII FRUCTUS, Hyunsam (SCROPHULARIAE RADIX) ) 10 to 200 parts by weight, 100 to 500 parts by weight of motherwort (LEONURI HERBA), 50 to 500 parts by weight of MASSA MEDICATA FERMENTATA, 50 to 500 parts by weight of SOJAE SEMEN, 10 to 200 parts by weight of PHASEOLI SEMEN A composition effective in the prevention and treatment of osteoporosis, rheumatoid arthritis, disc symptoms, containing X extracted with a solvent selected from at least one auxiliary medicine selected from the above or water, a lower alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof. Osteoporosis formulated in the form of a pharmaceutically acceptable pharmaceutical formulation of the composition of claim 1 together with pharmaceutically conventionally acceptable excipients, adjuvants, diluents, isotonic agents, preservatives, suspending agents, dissolution aids, Pharmaceutical preparations effective for the prevention and treatment of rheumatoid arthritis and disc symptoms. The method of claim 4, wherein the drug is already used as an allenrate, tamoxifen, vitamin B ₃ , parathyroid hormone (PTH), sulfasalazine, thioredoxin reductase, or thioredoxin reductase. ), Alendronate, raloxifene, calcitonin, calcitonin, estradiol, genistein, 1,25-dihydroxyvitamin D ₃ , alendronate, estrogen receptor modulator (etrogen receptor modulator), biphosphonates, harpagide (harpagide is a drug that the inventors developed the use) further contains one or more pharmaceutically acceptable Osteoporosis, which is formulated in the form of a pharmaceutically acceptable pharmaceutical formulation with excipients, adjuvants, diluents, isotonic agents, preservatives, suspending agents, and dissolution aids. The pharmaceutical formulation with efficacy in the treatment and prevention of arthritis and tooth disc symptoms.