KR20130059622A - Pharmaceutical composition comprising extract of poria cocos showing a bone formation - Google Patents

Abstract

PURPOSE: A pharmaceutical composition containing a Poria cocos extract which promotes osteoporosis is provided to enhance osteogenesis, and to prevent and treat osteoporosis. CONSTITUTION: A pharmaceutical composition for preventing and treating osteoporosis or promoting growth contains a crude extract, a polar solvent-soluble extract, or a non-polar solvent-soluble extract of Poria cocos as an active ingredient. The crude extract is prepared using water including purified water, C1-C4 alcohols, or a mixture thereof. The polar solvent-soluble extract is prepared using a solvent selected from water, methanol, ethanol, butanol, or a mixture thereof. The non-polar solvent-soluble extract is prepared using a solvent selected among hexane, chloroform, and ethyl acetate. The composition contains 0.1-50wt% of Poria cocos extract.

Description

Pharmaceutical composition comprising extract of Poria cocos showing a bone formation}

The present invention relates to a pharmaceutical composition for promoting growth growth or preventing and treating osteoporosis, containing the extract of Bokryeong as an active ingredient.

[Reference 1] Synthetic Theology (below), Doo-kyung Do, Sungbosa, pp1239, 1241, 1243, 1248, 2003.

[Ref. 2] The Korean Society of Pathology: Pathology PATHOLOGY, Advisor, pp1001 ~ 1008, 2000.

Glucocorticoid-induced osteoporosis., Trends Endocrinol . Metab., Manelli, F. and Giustina, A., 11 , pp 79-85, 2000.

Canalis E., Mechanisms of glucocorticoid Action in Bone: Implications to glucocorticoid-induced Osteoporosis. J. Clin . Endocri . Met., 81 , pp 3441-3447, 1996.

Perspectives on glucocorticoid-induced osteoporosis., Bone, Canalis, E. et al., 34 , pp 593-598, 2004.

Document 6 Park YH, Son IH, Kim B, Lyu YS, Moon HI, Kang HW. Poria cocos water extract (PCW) protects PC12 neuronal cells from beta-amyloid-induced cell death through antioxidant and antiapoptotic functions. Pharmazie. 2009; 64 (11): 760-4

7 Tohda C, et al. Inhibitory effects of methanol extracts of herbal medicines on substance P-induced itch-scratch response. Biol Pharm Bull., 23 (5) , pp 599-601, 2000.

Document 8 Li TH, Hou CC, Chang CL, Yang WC Anti-Hyperglycemic Properties of Crude Extract and Triterpenes from Poria cocos . Evid Based Complement Alternat Med. 2011; pii: 128402

9, Harborne JB, A guide to modern techniques of plant analysis. 3 , pp 6-7, 1998.

Document 10 Kapur S, Mohan S, Baylink DJ, Lau KH. Fluid shear stress synergizes with insulin-like growth factor-I (IGF-I) on osteoblast proliferation through integrin-dependent activation of IGF-I mitogenic signaling pathway. J Biol Chem. 2005; 280 (20): 20163-70.

[11] Canalis E., Mechanisms of Glucocorticoid Action in Bone: Implications to glucocorticoid-induced Osteoporosis. J. Clin.Endocri. Met., 81 , pp3441-3447, 1996).

12. Akihisa T, Uchiyama E, Kikuchi T, Tokuda H, Suzuki T, Kimura Y. Anti-tumor-promoting effects of 25-methoxyporicoic acid A and other triterpene acids from Poria cocos. J Nat Prod. 2009; 72 (10): 1786-92

[Reference 13] 上海 中 醫學院.中草藥 學, 香港, 商務印書館, 1990: 40-43

Document 14 鄭普燮, 鄭普燮 民 敎.鄕 藥 大事 典, Seoul, 氷 林 社, 1990: 40-43

Osteoporosis can be divided into type I osteoporosis due to estrogen deficiency, type II osteoporosis in the elderly, and secondary osteoporosis due to drugs and other diseases. In women with postmenopausal type I osteoporosis, estrogen deficiency increases the action of parathyroid hormone (PTH) on osteoclasts, leading to increased destruction of bone matrix and calcium absorption. As a result, elevated blood calcium is inhibited by PTH release through feedback inhibition, resulting in a decrease in Vit D-activated 1,25 (OH) D and decreased intestinal calcium absorption. On the other hand, the causes of senile osteoporosis type II osteoporosis are known to be caused by decreased Iα-hydroxylase activity in the kidneys, decreased activation of Vit D, resulting in decreased intestinal calcium absorption, and increased PTH (Korean Pathology: Pathology). PATHOLOGY, Advisor, pp 1001-1008, 2000).

One of the main causes of osteoporosis is secondary corticosteroids. there are reports (Glucocorticoid-induced osteoporosis., Trends Endocrinol . Metab., Manelli, F. and Giustina, A., 11 , pp 79-85, 2000). Corticosteroids act on osteoblasts to inhibit cell proliferation and reduce osteoblast differentiation. In addition, corticosteroids may include growth factor I (IGF-I), insulin like growth factor II (IGF-II), platelet derived growth factor (PDGF), and IGFBP-5. It has been reported to inhibit (4). As a result, osteocalcin biosynthesis that binds to calcium as a comparative protein and inhibition of collagen synthesis and collagen are reduced. Separately, corticosteroids act on osteoclasts to promote cell differentiation and activate collagenase to increase bone resoprtion. Moreover, corticosteroids may cause osteoporosis by suppressing Vit D action and suppress promoting PTH secretion, Minister calcium absorption inhibition, renal tubular calcium resorption inhibiting calcium deposition in bones (Perspectives on glucocorticoid - induced osteoporosis . , Bone , Canalis, E. et al., 34 , pp 593-598, 2004). As such, bone is a dynamic tissue that is constantly formed and absorbed by osteoblasts (osteoblasts) that are bone forming cells and osteoclasts (osteoclast) and osteocytes (osteocytes), which are osteoporosis. Bone formation is relatively lower than bone resorption, such as increased function and action time, or decreased function and action time of osteoblasts (Synthesis Theology, Du Ho Kyung, Seongbosa, pp1239, 1241, 1243, 1248, 2003) .

Fukryeong is a family of Pachyma Hoelen Rumphius belonging to the polyporaceae, which has a flat heart, a heart, a heart, a heart, and a heart. It is known (中草藥 學, 上海 中 醫學院. 1990). Pharmacological studies show that diuretic and hypoglycemic effects (Anti-Hyperglycemic Properties of Crude Extract and Triterpenes from Poria cocos.Li TH et al.Evid Based Complement Alternat Med. 2011; 2011.pii: 128402), treatment of dementia (Poria cocos water extract (PCW) protects PC12 neuronal cells from beta-amyloid-induced cell death through antioxidant and antiapoptotic functions.Park YH et al. Pharmazie. 2009), Anti-tumor-promoting effects of 25-methoxyporicoic acid A and other triterpene acids from Poria cocos.Akihisa T et al. J Nat Prod. 2009; 72 (10): 1786-92). However, the anti-osteoporosis efficacy of Bokryeong has not been reported.

In order to develop a method for preventing and treating osteoporosis, the present inventors have sought to find a drug that increases bone formation by activating osteoblast function. To this end, the ethanol fractions of Bokryeong extract were administered to osteoblasts isolated from the skull of rats, and the effects on osteochemical dividing and osteochemical markers of osteoblasts were measured. The present invention was completed by confirming the effect of increasing the amount of alkaline phosphatase activity and collagen synthesis.

In order to achieve the above object, the present invention provides a pharmaceutical composition for promoting growth growth or prevention and treatment of osteoporosis, containing the crude extract of Bokryeong, polar solvent soluble extract or non-polar solvent soluble extract as an active ingredient.

Crude extract as defined herein means an extract that is soluble in water, including purified water, lower alcohols having 1 to 4 carbon atoms or a mixed solvent thereof, preferably methanol.

The polar solvent soluble extract as defined herein is an extract soluble in water, methanol, ethanol, butanol, or a mixed solvent thereof, preferably butanol, and the nonpolar solvent soluble extract is hexane, chloroform, methylene chloride, ethyl acetate. , Glycerine, butylene glycol, preferably extracts soluble in chloroform.

Hereinafter, the method for obtaining the crude extract, the polar solvent soluble extract or the non-polar solvent soluble extract of the present invention will be described in detail.

The crude extract of Bokryeong of the present invention is chopped dried Bokryeong, about 1 to 20 times the weight (kg), preferably about 5 to 15 times the water, C ₁ to C ₄ lower alcohol or their Mixed solvent, preferably methanol, cold extraction, hot water extraction, ultrasonic extraction, for about 0.5 to 20 hours, preferably about 1 to 10 hours at an extraction temperature of 20 ℃ to 100 ℃, preferably 50 ℃ to 100 ℃ By reflux extraction, preferably by reflux extraction, the obtained extract can be obtained by filtration, concentration under reduced pressure, or drying.

In addition, the non-polar solvent soluble extract of the present invention, after suspending the crude extract in distilled water, the suspension is about 1 to 100 times, preferably about 1 to 5 times the volume of hexane, chloroform, methylene chloride, ethyl acetate, preferably Extracts and fractionates the nonpolar solvent soluble layer once to 10 times, preferably 2 to 5 times by adding a nonpolar solvent such as chloroform to obtain a nonpolar solvent soluble extract available in the nonpolar solvent, and a polar solvent soluble in the polar solvent. Soluble extract can be separated. It is also possible to further carry out conventional fractionation processes (Harborne JB, A guide to modern techniques of plant analysis . 3 , pp 6-7, 1998).

The Furk crude extract, polar solvent soluble extract or non-polar solvent soluble extract obtained by the above method exhibits an effect of increasing cleavage capacity, alkaline phosphatase activity and collagen synthesis amount in bone cells isolated and cultured from the skull of rats. In addition, it was confirmed that the effect of promoting growth growth or preventing and treating osteoporosis.

The present invention provides a pharmaceutical composition for promoting growth growth or prevention and treatment of osteoporosis, containing the Fukryeong extract obtained by the production method as an active ingredient.

The composition for promoting growth growth or preventing and treating osteoporosis of the present invention comprises the extract in an amount of 0.1 to 50% by weight based on the total weight of the composition.

However, the composition is not limited thereto, and may vary depending on the condition of the patient, the type of disease, and the progress of the disease.

The composition comprising the Bokryeong extract of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

The composition containing the extract according to the present invention may be formulated in the form of powders, granules, tablets, capsules, oral preparations such as suspensions, emulsions, syrups and aerosols, external preparations, suppositories and sterilized injection solutions, Examples of the carrier, excipient and diluent which can be contained in the composition containing the extract include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin , Calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid preparations may include at least one excipient such as starch, calcium carbonate, sucrose in the extract. ) Or lactose, gelatin and the like are mixed. In addition to simple excipients, lubricants such as magnesium stearate talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

Preferred dosages of the extracts of the present invention vary depending on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art. However, for the desired effect, the extract of the present invention is preferably administered at a dose of 0.01 mg / kg to 10 g / kg per day, preferably 1 mg / kg to 1 g / kg per day. The administration may be carried out once a day or divided into several doses. Thus, the dosage amounts are not intended to limit the scope of the invention in any manner.

The composition of the present invention may be administered to mammals such as rats, mice, livestock, humans, and the like in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

The crude extract, polar solvent or non-polar solvent soluble extract of the present invention is a drug that can be used with confidence even when taken for a long time for prophylactic purposes because there is little toxicity and side effects.

Fukryeong extract of the present invention shows an effect of increasing the division capacity, alkaline phosphatase activity and collagen synthesis amount in bone cells isolated and cultured from the skull of the rat, to promote the growth growth or to prevent and treat osteoporosis composition It can be usefully used.

1 is a diagram showing the measurement of bone cell division ability when the treatment of the extract of the present invention (PC),
Figure 2 is a diagram showing the measurement of alkaline phosphatase activity during the treatment of the Bokryeong extract (PC) of the present invention,
Figure 3 is a diagram showing the measurement of collagen synthesis amount when the treatment of the extract of the present invention (PC),
Figure 4 is a diagram showing the measurement of cell viability of the bone cells during the treatment of the extract of the present invention (PC).

Hereinafter, the present invention will be described in detail with reference to Examples and Experimental Examples.

However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the present invention is not limited to the following Examples and Experimental Examples.

Example  1. Refuge Crude extract  Produce

1-1. Preparation of Bokryeong Methanol Extract

1,000 ml of methanol was added to 100 g of Bokryeong obtained from Kyungwon University Oriental Hospital, and the mixture was heated and refluxed for 6 hours or more. After filtration using filter paper, the filtrate was concentrated under reduced pressure using an evaporator (EYERA, Japan) and dried to obtain 18 g of Bokyeong methanol extract (yield: 18%).

Example  2. Refuge Nonpolar solvent  And preparation of a polar solvent soluble extract

2-1. Ghost Butanol  Preparation of Soluble Extracts

18 g of the methanol-soluble extract of Example 1-1 was dissolved in 500 ml of water, extracted and fractionated three times with chloroform, and the remaining layer (supernatant) was extracted and fractionated three times with butanol, and the butanol soluble layers were combined. The solvent was evaporated using a rotary evaporator to obtain 5.7 g of butanol soluble extract (yield: 5.7%), and freeze-dried and stored at -20 ° C. until freeze-dried. When the test solution (butanol extract fraction powder) in the experiment was dissolved in the medium and passed through a filter paper of the pore size (pore size 0.45μm) was used as a sample of the experimental example (hereinafter referred to as "PC").

Reference Example  1. Preparation of experimental animals

The experimental animals were purchased from Biolink for pregnant female SD rats and used after adapting to the laboratory. During the experimental period, the solid feed and water were supplied with sufficient intake.

Reference Example  2. Drug treatment

The Bokyeong butanol extract sample (PC) obtained in Example 2-1 was added to the normal group (NC), to which the PC was added 1 μg / ml to the normal group (PC1), and to the normal group was added 10 μg / ml of the PC. Administration was divided into groups (PC10), which was used in the experimental example.

Reference Example 3. The fetal skull cell culture (Fetal calvarial cell culture ( FCS ))

The uterus of the 21-day-old rat was excised to remove the fetus, and then the head was cut and the skull was removed. The connective tissue attached to the skull was removed and washed with HBSS. The skull was reacted at 37 ^° C. in 1.5 ml of collagenase, trypsin, 0.5 mM EDTA solution. The supernatant was taken and centrifuged at 500 rpm to obtain precipitated bone cells. After resuspending in PBS and washed by centrifugation at 1500rpm, it was suspended in DMEM medium and incubated at 37 ^° C, 5% CO ₂ . The supernatant was removed and 1.5 ml of enzyme was added to the remaining skull, and the reaction was repeated several times. The medium of cells was changed every three days. The cultured cells were trypsinized 2 weeks later and used for the experiment after measuring the cell number.

Reference Example  4. Statistical processing

All experimental results were statistically analyzed using one way analysis of variance (ANOVA), and significance was tested at p <0.05 level using the Student-Newman-Keuls Test if significant. Was carried out.

Experimental Example  1. Osteoblasts Cleavage ability  Measure

In order to determine the effect of the bone cell division ability of the PC obtained in Example 2-1, the experiment was performed as follows using the method described in the literature (10)

The fetal skull cells (FCS) obtained in Reference Example 3 were divided into 1.0-3.010 ⁵ / well, and the medium of the cells was removed to count the number of cultured cells, and the cells were washed with HBSS. The cells were then separated from the culture plate by addition of collagenase, trypsin, 0.5 mM EDTA. After diluting the cells 20 times with Isoton-2 solution, the number of cells was measured by a cell counter (sysmax F-820), and the results are shown in FIG. 1 (see FIG. 1 below).

Experimental results, as shown in Figure 1, after culturing the bone cells isolated from the skull of the rat fetus for 10 days to count the bone cell number, compared to the normal cells Bokyeong extract at concentrations of 1μg / ml, 10μg / ml All showed a significant increase compared to normal.

Experimental Example  2. Alkali phosphatase ( Alkaline 포스화제 ) Active measurement

In order to measure the alkaline phosphatase activity of the PC obtained in Example 2-1, experiments were performed as follows using the method described in the literature (10).

After culturing the culture plate with cells, the cells were washed with cold PBS, and the cells were scraped with a scraper and suspended in cooled PBS containing Leupeptin. The suspension was digested with an ultrasonic mill under cooling and centrifuged at 3000 rpm for 10 minutes. After centrifugation, the supernatant was taken and contained 0.56 M 2-amino-methyl 1-propanol, 1 mM magnesium chloride (MgCl _{2 )} and 10 mM p-nitrophenylphosphate. The reaction solution was reacted at 37 ^° C. for 10 minutes. After stopping the reaction by adding 1 ml of 0.2 N sodium hydroxide (NaOH), the absorbance was measured at 405 nm, and the experimental results are shown in FIG. 2 (see FIG. 2 below).

Experimental results, as shown in Figure 2, after culturing the bone cells isolated from the rat fetal skull for 10 days, compared to the ALP activity in the normal cells, Fukryeong extract is normal at the concentration of 1μg / ml, 10μg / ml It can be seen that the significant increase compared to.

Experimental Example  3. Collagen synthesis amount measurement

In order to measure the amount of collagen synthesis of PC obtained in Example 2-1, experiments were performed as follows using the method described in the literature (10).

After culturing the culture plate with cells in PBS and scraping the cells using a scraper (scraper). It was suspended in 50 mM Tris buffer solution containing 5 mM dithiothreitol and then digested with an ultrasonic mill. Thereafter, the supernatant was removed by centrifugation at 100,000xg for 30 minutes, and 6N HCl was added to the pellet and hydrolyzed at 100 ^° C. for 24 hours. It was carefully neutralized with 6N sodium hydroxide (NaOH) and then diluted to the appropriate concentration with water. Hydroxyproline (Hydroxy-proline) was quantified using an amino acid analyzer (amino acid analyzer).

Experimental results, as shown in Figure 3, after culturing the bone cells isolated from the rat fetal skull for 10 days, the collagen biosynthesis was significant at the concentration of 1μg / ml, 10μg / ml of Bokyeong extract compared to normal cells It can be seen that one increase. (See Figure 3 below).

Experimental Example  4. Cell viability measurement

In order to measure the cell viability of the PC obtained in Example 2-1, an experiment was performed using the MTT assay method.

As a result of the measurement, as shown in Figure 4, after culturing the bone cells isolated from the rat fetal skull for 10 days, the survival rate in normal cells and Bokyeong extract is different between the survival rate at a concentration of 1μg / ml, 10μg / ml It was confirmed that there is no (see Figure 4 below).

Hereinafter, the preparation examples of the composition including the extract of the present invention, but the present invention is not intended to limit it, but is intended to explain in detail only.

Formulation example  One. Sanje  Produce

PC 20 mg of Example 2-1

Lactose 100 mg

Talc 10 mg

The above components are mixed and filled in airtight bags to prepare powders.

Formulation example  2. Preparation of tablets

PC 10 mg of Example 2-1

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

After mixing the above components, tablets are prepared by tableting according to the usual preparation method of tablets.

Formulation example  3. Preparation of capsules

PC 10 mg of Example 2-1

Crystalline cellulose 3 mg

Lactose 14.8 mg

Magnesium stearate 0.2 mg

The above components are mixed according to a conventional capsule preparation method and filled in gelatin capsules to prepare capsules.

Formulation example  4. Preparation of injections

PC 10 mg of Example 2-1

Mannitol 180 mg

Sterile distilled water for injection 2974 mg

Na ₂ HPO _{4 ,} 12H ₂ O 26 mg

(2 ml) per 1 ampoule according to the usual injection preparation method.

Formulation example  5. Liquid  Produce

PC 20 mg of Example 2-1

10 g per isomer

5 g mannitol

Purified water

Each component was added to purified water in accordance with the usual liquid preparation method and dissolved, and the lemon flavor was added in an appropriate amount. Then, the above components were mixed, and purified water was added thereto. The whole was adjusted to 100 ml with purified water, And sterilized to prepare a liquid preparation.

Claims (5)

A pharmaceutical composition for promoting growth growth or preventing and treating osteoporosis, which contains crude extract of Bokryeong, polar solvent soluble extract or nonpolar solvent soluble extract as an active ingredient. The pharmaceutical composition according to claim 1, wherein the crude extract is an extract available in a solvent selected from water including purified water, a lower alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof. The pharmaceutical composition of claim 1, wherein the polar solvent soluble extract is an extract soluble in a solvent selected from water, methanol, ethanol, butanol, or a mixed solvent thereof. The pharmaceutical composition according to claim 1, wherein the nonpolar solvent soluble extract is an extract soluble in a solvent selected from hexane, chloroform and ethyl acetate. According to claim 1, wherein the pharmaceutical composition, characterized in that it comprises 0.1 to 50% by weight of the extract of Bokryeong relative to the total weight of the composition.

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