KR100380867B1 - Extract of Albizziae Cortex for the prevention and treatment of osteoporosis - Google Patents

Abstract

The present invention relates to an extract of Albizziae Cortex having an effect on the prevention and treatment of osteoporosis. Specifically, the extract of the compound is a large amount of phytoestrogen such as diedzein and formononetin. Since it is included, the haphwanpi extract according to the present invention can be usefully used as a therapeutic or preventive agent for osteoporosis, as well as applied as a functional food.

Description

Extract of Albizziae Cortex for the prevention and treatment of osteoporosis

The present invention relates to an extract of Albizziae Cortex having an effect on the prevention and treatment of osteoporosis. Specifically, the extract of the compound is a large amount of phytoestrogen such as diedzein and formononetin. Since it is included, the haphwanpi extract according to the present invention can be usefully used as a therapeutic or preventive agent for osteoporosis, as well as applied as a functional food.

Osteoporosis is a condition in which the bone tissue is thinned due to reduced lime in bone tissue and thus the bone marrow cavity is widened. Bone mass is influenced by several factors, including genetics, nutrition, hormone changes, differences in exercise and lifestyle, and the causes of osteoporosis include old age, lack of exercise, low weight, smoking, low calcium diet, menopause, Ovarian ablation and the like are known. On the other hand, although there are individual differences, blacks have lower bone resorption levels than whites, which results in higher bone mass. Usually, bone mass is highest at 14-18 years of age and decreases by about 1% per year. Especially in women, bone reduction continues after 30 years of age, and when menopause reaches bone growth rapidly due to hormonal changes. In other words, estrogen concentration rapidly decreases during menopause, when a large amount of B-lymphocytes are produced, as is the case with IL-7 (interleukin-7). cells) accumulate and thereby increase the amount of IL-6, thereby increasing the activity of osteoclasts, which eventually leads to a decrease in bone mass.

As described above, osteoporosis is unavoidable for elderly people, especially postmenopausal women, and as the population ages in developed countries, interest in osteoporosis and its therapeutics is gradually increasing. It is also known that there is a market of about $ 130 billion related to the treatment of bone diseases worldwide and is expected to increase further. Therefore, many research institutes and pharmaceutical companies around the world are investing heavily in the development of bone disease treatments. .

Materials currently used to treat osteoporosis include estrogen, androgen anabolic steroids, calcium preparations, phosphates, fluoride preparations, ipriflavones, and vitamin D ₃ . In 1995, Merck, USA, used aminobisphosphonate, and in 1997, Lilly Co., USA, used raloxifene as a selective estrogen receptor modulator (SERM) for osteoporosis. It was developed as a new drug.

Meanwhile, conventional osteoporosis therapeutic agents are mostly estrogen-based substances, and estrogen-based substances are known to have side effects such as cancer, gallstones, and thrombosis when administered for a long time. Osteoporosis, however, cannot be treated with short-term administration of the drug, and long-term administration of the drug is essential. Therefore, even when long-term administration of the drug is to develop a new substance that does not have the above side effects and excellent efficacy enough to replace estrogen, one of the focus of attention as a substitute for estrogen is soybean isoflavone ( phytoestrogen, such as soybean isoflavone.

Phytoestrogens were first reported in 1946 by Bennett et al., `` Clover disease (red clover, Trifolium subterraneum var. The cause of the disease was identified as isoflavonoids similar to estrogen among the components contained in this plant, and these compounds obtained from plants were named plant estrogens.

Materials known as phytoestrogens include isoflanone compounds such as daidzein, genistein, formononetin, biochanin A, and coumestrol. Counan compounds, such as cumumes compounds, such as enterollactone, and phenol compounds, such as enterodiol. These phytoestrogens are usually present in the form of aglycone, 6'-O-acetylglucoside, 6'-O-malonylglucoside, etc. Dyzezene and Genistein are present in the form of 7-O-glucoside. Among these compounds, sugar compounds are known to be hydrolyzed by β-glucosidase or gastric acid of intestinal bacteria and eventually absorbed in the form of aglycone, which is free isoflavones.

In the isoflavone compound, formononetin is a precursor of dydzein, and when the methoxy group of the formononetin benzene ring is dimethylated, it is converted into diedzein, and when the ketone group of dydzein is removed, it is converted into oecol. do. In addition, biocanin A is a precursor of genistein and is converted to genistein when the methoxy group of the biocanin A benzene ring is dimethylated.

Plant estrogens generally exhibit an action similar to that of animal estrogens, which bind to estrogen receptors to inhibit the growth of breast cancer cells and can be used in place of estrogens in the treatment of cardiovascular disease and other symptoms that appear after menopause. In addition, Asian women who currently consume soy foods are reported to have fewer symptoms of osteoporosis and heart disease due to estrogen deficiency than Americans (Y. Ishimi et al ., Selective Effects of Genistein, a Soybean Isoflavone, on B-Lymphopoiesis). and Bone Loss Caused by Estrogen Deficiency, Endocrinol. , 140 (4): 1893-1900, 1999). Whether this cause is due to the administration of phytoestrogens dyedzein or genistein or genetic factors is not clear, but it is one of the reasons behind the usefulness of phytoestrogens in the prevention and treatment of osteoporosis.

Genistein also affects bone resorption rate and bone formation rate, which was increased in the 5 μg / g group (Fanti, MC et al. , The Phytoestrogen Genistein Reduces). Bone Loss in Short-Term Ovariectomized Rats, Osteoporos Int. 8: 274-281, 1998). The application of genistein to calvarial cell culture did not directly affect osteoclast formation or bone loss. On the other hand, administration of 100-200 μg / kg of genistein increased the alkaline phosphatase (abbreviated as “ALP”), DNA and calcium concentrations in the femoral metaphyseal and femoraldiaphyseal. Administration of 50 ㎍ / ㎏ increased the concentrations of ALP and DNA, but did not change calcium concentrations. However, administration of 5.5 mg / kg zinc with genistein increased both ALP, DNA and calcium concentrations. In other words, it can be seen that genistein has an anabolic effect on bone, which is increased by zinc and promotes the production of bone protein (Gao, YH et al ., Zinc Enhancement of Genistein's Anabolic Effect). on Bone Components in Elderly Female Rats, Gen. Pharmac. 31 (2): 199-202, 1998).

In (in vitro) test in the genistein (10 ^-5 ~10 ^-8 M) that is effective when added with the zinc (10 ^-5 M) sikyeotgo ALP, increasing the concentration of the DNA and calcium goljung executive tissue (metaphyseal tissue) in vitro Increased further. In addition, raloxifene, a precursor of dydzein, prevented bone loss and exhibited an estrogenic effect without affecting the uterus (Ishimi, Y. et al ., Selective Effects of Genistein, a Soybean Isoflavone, on B-Lymphopoiesis and Bone Loss Caused by Estrogen Deficiency, Endocrinol. , 140 (4): 1893-1900, 1999).

On the other hand, the addition of IL-1β to primary cell cultures of human osteoblastic cells and bone marrow osteoprogenitor stromal (HBMS) cells can result in dose-dependent or The amount of IL-8 mRNA increases in time. At this time, glucocorticoids, dexamethasone, and cortisone inhibited the increase of IL-8 by IL-1β, and PTH (parathyroid hormone) (10 ^-7 , 10 ^-8 M ) Had no effect on IL-8. In addition, genistein (100 μM) also inhibited the increase of IL-8 by IL-1β, suggesting that regulation of IL-8 by IL-1β proceeds through a signal transduction pathway ( Chaudhary, LR et al ., Regulation of Interleukin-8 Gene Expression by Interleukin-1β, Osteotropic Hormones and Protein Kinase Inhibitors in Normal Human Bone Marrow Stromal Cells., J. Bio. Chem ., 271 (28): 16591-16596, 1996).

Platelet-derived growth factor (PDGF) and insulin-like growth factor-II (IGF-II) act as potent mitogens in primary cultures of MG-63 cells and osteoblasts. When administered with Genistein, IP3 (inositol trisphosphate) was increased in the test group containing PDGF, but not in the test group containing IGF-II. That is, genistein inhibited PDGF phosphorylation and mitogen but did not affect IGF-II. Therefore, it was found that genistein acted only on specific tyrosine kinases.

In the experiment of bone resorption by osteoclasts of genistein, the IC ₅₀ was measured to be 3 μmol / L and the osteoclasts were tested in vivo and in vitro at 10 μM. Osteoclastic activity was inhibited. Dydzein, on the other hand, had no effect on bone resorption at 30 μM and promoted bone resorption in cell cultures of 10 ^-8 to 10 ^-10 M (J.P. Williams, et al., Tyrosine Kinase Inhibitor Effects on Avian Osteoclastic acid Transport.Am J. Clin.Nutr., 68 (suppl): 1369S-74S, 1998; H. Tobe, et al ., Daidzein Stimulation of Bone Resorption in Pit Formation Assay.Bioscience Biotech. And Biochem , 61 (2): 370-371, 1997).

On the other hand, isoflavones such as Ipriflavone do not directly affect estrogen receptors, but it is known to suppress the loss of bone mass, especially to promote bone production within a short period of time, to reduce the decrease in bone mass.

The effects of phytoestrogens on bone have some opposite effects, but the overall increase in bone mineral density (BMD) has been reported.

A comparison on the basis of estradiol (estradiol) used for ALP activity by a plant estrogens in human cervical adenocarcinoma cells (human endometrial adenocarcinoma cell) as osteoporosis, Ku scalpel trawl 5 × 10 ^-2 times, genistein and Ecole (equol A) 10 ⁻³ times, Dyzedein 7 × 10 ⁻³ times, Biocanin A 1.5 × 10 ⁻⁴ times, and formononetin 10 ⁻⁵ times. However, estrogen activity on the uterus (uterus) (estrogenic activity) is when, based on the best-diethyl steel rolls (diethylstilbestrol), Equus scalpel trolls 3.5x10 ^-4 times, genistein ^10-5 times, daidzein 7.5x10 ^-6 times, non-Oka non-A 4.6 × 10 ^-6 times, formononetin significantly lower extent 2.6x10 ^-6 times. The amount of estrogen effect was 5 g and 9,412 g of alfalfa for cumestrol and formonenetine, and 48 g and 145 g of tofu for genistein and dydzein, respectively.

On the other hand, animals that use phytoestrogens generally cause problems such as infertility because of high blood levels of phytoestrogens. Since phytoestrogens have an anti-estrogenic effect, estrogens such as the uterus This may have a significant effect on tissues or organs with high density of receptors. Bone mineral density (BMD) decreases when the ovary is removed, and administration of zenithine to ovarian-depleted rats (OVX) results in a reduction in the number of bone marrow cells and increased BMD without increasing uterine weight. Increased. The results were also confirmed by microcomputed tomography and histological analysis of trabecular bone. The mechanism of action of genistein may be due to bone loss activity of osteoclasts as well as tyrosine kinase inhibitors. On the other hand, administration of 1 mg / kg or 5 mg / kg of zenithine suppressed the reduction of BMD and increased bone formation rather than bone resorption. Another report shows that genistein has a dose-dependent change in potency. In other words, 25 μg / g of Genistein in H. pylori treated rats affected the uterus, but 5 μg / g did not affect the uterus and increased the BMD. Did not affect.

In addition, the increase in BMD was also observed when menopausal women received 90 mg daily of Genistein for 6 months. Six weeks of soy flour (including daidzein) resulted in a 40% reduction in hot flushes, consistent with the excretion of ethanol in urine. It contains enterolactone and reduces facial flushing by 25%. However, small doses of genistein (0.5 mg / day) show a similar effect to estrogen (which acts as an agonist on the estrogen receptor), but at higher doses (5 mg / kg), the adverse effect is greater. (Anderson, JJB et al ., Biphasic Effects of Genistein on Bone Tissue in the Ovariectomized, Lactating Rat Model. PSEBM ., 217, 345-350, 1998).

When 50 mg of isoflavones are administered, it is known that the highest plasma concentrations of genistein and dyedzein are 130-640 ng / ml and the half-life is about 7-8 hours, respectively. In addition, the bioavailability of glycosides (glycosides) is higher than aglycone, so urine excretion amounts to 24% genistein, 66% dyedzein and 28% ethanol. Absorption half-life of Genistein, Dyzedein and Ecole is 1.5-3 hours, and half-life excreted in urine has been reported to be 7, 4 and 9 hours, respectively (2nd International Symposium on the Role of Soy in Preventing and Treating Chronic Disease, Brussels, Belgium, 1996).

On the other hand, in order to use isoflavones, which are effective for treating and preventing osteoporosis, as a substitute for estrogen, it is necessary to secure a large amount of compounds, and isoflavones are mainly extracted and used in plants. Therefore, it is important to find a source from which large amounts of isoflavones can be obtained.

It is known that isoflavones are mainly contained in the legumes (Leguminosae family) plants, and several researchers in Korea including the present inventors have determined the content of isoflavones contained in soybean processed foods and fermented foods. It was quantified. Specifically, genistein, dyedzein, and formoninetin were quantified from isoflavones contained in traditional Korean foods such as tofu, bean sprouts, soybean paste, soy sauce, cheonggukjang, chunjang, meju, soybeans, peas, medicinal beans, and Gangnam beans. At this time, the blood concentration of isoflavones and the amount excreted in the urine were analyzed, and HPLC (high pressure liquid chromatography) or gas chromatography using a detector such as an ultraviolet spectrometer, an ECD (electrochemical detector) spectrometer or a fluorescence spectrometer was used as a quantitative analysis method. Or RIA (radio immunoassay) analysis.

According to the analysis of zensteine and dyedzein in phytoestrogens of 18 kinds of soybeans, soybeans contained zenithine 318 to 1107 mg / kg and dyedzein 379 to 1242 mg / kg. The highest amount of isoflavones in a single lobe was 2317 mg / kg for soyflour (JS Choi, TW Kwon, and JS Kim: Isoflavone Contents in some Varieties of Soybean, Foods and Biotechology, 5 ( 2): 167-169, 1996). In addition, the soybean processed foods have high phytoestrogens concentrations, and the domestic liquid soymilk is about 309 mg / kg, which is a died agent (unless stated otherwise), about 367 mg / kg, zenistein, and 584 mg, which is died agent in tofu. / Kg, Genistein 568 mg / kg, soft tofu contained 566 mg / kg dyedzein, Genistein 625 mg / kg and soft tofu contained 817 mg / kg dyedzegen and 702 mg / kg Genistein. On the other hand, in the case of soybean sprouts (dry amount), 194 mg / kg of dydzein and 230 mg / kg of zesteine were contained, and it is known that the concentration of isoflavone increases when the soybean germinates (Choi Yeon-bae, Son Heon-soo: Isoflavone in soybean processed foods). Kor. J. Food Sci.Technol . 30 (4): 745-750, 1998).

On the other hand, Albizziae Cortex is a herbal medicine listed in the Korean Pharmacopoeia Standards Collection, and the bark of Albizziae Cortex Daurazzinii is widely used as a favorite food, drink, and functional food in the private sector. Haphwanpi harmonizes the heart and spleen, so it is known that using Haphwanpi in oriental medicine, mental anxiety, forgetfulness, insomnia, depression, and mood swings, stabilizes fractures and intestines and harmonizes the wick to prevent depression. According to the Chinese herbal medicine, Hwanhwan Blood is used to treat tumors and to treat fractures or muscle damage caused by bruises. , 1998).

However, there have been no reported cases of using Hwanhwanpi in connection with the prevention or treatment of osteoporosis.

Therefore, the present inventors conducted a search for a traditional herbal medicine while trying to find a substitute for soybean as a source of phytoestrogens that can be used as an agent for preventing or treating osteoporosis. The present invention has been completed by finding out that the present invention can be used as a therapeutic or preventive agent for osteoporosis and as a functional food.

An object of the present invention is to provide a haphwanpi extract containing a large amount of phytoestrogens components useful for the prevention and treatment of osteoporosis, and a method of preparing the same.

Still another object of the present invention is to provide a use of the haphwanpi extract as an agent for treating or preventing osteoporosis and as a functional food.

In order to achieve the above object, the present invention provides an extract of Albizziae Cortex having an effect on the prevention and treatment of osteoporosis.

The Hwanhwanpi extract is characterized in that it contains a large amount of phytoestrogen, such as dyedzein (diazein) and formononetin (formononetin).

Comparing the soybean extract, which has been mainly used as a source of phytoestrogens in the past, and the above-mentioned extract of hwanhwanpi, the extract of hwanhwanpi contains a lot of phytoestrogens such as dydzein and formmononetin. In addition, the yield of Hwanhwanpi extract is high as 11.16 ± 0.20% with respect to dried Hwanhwanpi, and the value of Hwanhwanpi is lower than that of soybean. Therefore, Hwanhwanpi extract can be used as a useful source of phytoestrogens than soybean extract.

The present invention also provides a method for preparing the haphwanpi extract.

According to the preparation method of the present invention, the Hwanhwanpi extract may include a plant estrogen compound in the form of a free compound and a form combined with a sugar, and further, the total plant estrogen compound is hydrolyzed in the form of a free compound. It may also be included as.

Specifically, the present invention provides a method of preparing the above-mentioned extract of hwanhwanpi by putting the dried hwanhwanpi in 10-100% aqueous alcohol solution and immersing at 5-40 ° C. According to this method, haphwanpi extract contains both a plant estrogen compound in the form of a free compound and a plant estrogen compound in a form combined with a sugar.

In another aspect, the present invention provides a method for preparing the haphwanpi extract by hydrolyzing the dried hwanhwanpi with acid or base. In this case, the acid or base is preferably 0.1 to 2 N, the acid is hydrochloric acid or sulfuric acid and the base is sodium hydroxide or potassium hydroxide. According to this method, the phytoestrogens compound bound to the sugar is hydrolyzed, and the cyclic extract contains the phytoestrogens compound in a free form.

The present invention also provides a pharmaceutical composition for treating or preventing osteoporosis, containing the above-mentioned extract of the hwanhwanpi as an active ingredient.

Compared to a single compound of phytoestrogens, the haphwanpi extract according to the present invention has a much higher effect of treating or preventing osteoporosis.

The haphwanpi extract can be administered orally or parenterally during clinical administration and can be used in the form of a general pharmaceutical formulation.

That is, the synthetic ring extract of the present invention may be administered in various oral and parenteral formulations during actual clinical administration, and when formulated, diluents such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants that are commonly used Or using excipients. Solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like, which solid preparations contain at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. Are mixed to prepare. In addition to simple excipients, lubricants such as magnesium styrate talc are also used. Liquid preparations for oral administration include suspensions, solvents, emulsions, and syrups, and various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and the suspension solvent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerol, gelatin and the like can be used. In addition, calcium or vitamin D ₃ may be added to enhance the efficacy of osteoporosis prevention and treatment.

Dosage units may contain, for example, one, two, three or four times, or 1/2, 1/3 or 1/4 times the individual dosage. The individual dosage preferably contains an amount in which the effective drug is administered at one time, which usually corresponds to all, 1/2, 1/3 or 1/4 times the daily dose.

The effective dose of Hwanhwanpi extract is 1-600 mg / kg, preferably 10-100 mg / kg, and can be administered 1-6 times a day.

Toxicity experiments of oral administration and intraperitoneal administration of the Hwanhwanpi extract of the present invention showed that the 50% lethal dose (LD ₅₀ ) by oral toxicity test was at least 15.0 g / kg or more.

In addition, the present invention provides a functional food containing the haphwanpi extract as an active ingredient.

In this case, as a functional food containing the extract of hwanhwanpi, tea, jelly, juice, juice, extract, etc., mainly composed of hwanhwanpi, include folk remedies for the purpose of relieving tension. Also, there are nearly paid old-fashioned law, white lead, mica ego (1), mica ego (2), haphwan, twilight bath, etc. It can be used for many of the same prescriptions.

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

However, the following examples are illustrative of the present invention, and the content of the present invention is not limited by the examples.

<Example 1> Preparation of Hailhwanpi extract 1

50 g of dried Hwanhwanpi (Chinese) powder was placed in 500 ml of 70% aqueous methanol solution, and stirred at room temperature for 2 days. After 2 days, the solution was filtered through a filter paper, concentrated under reduced pressure to remove methanol, and freeze-dried to remove moisture. By this method, 5.58 ± 0.11 g of hwanhwanpi extract (yield for the dried hwanhwanpi dried: 11.16 ± 0.20%), in which the phytoestrogens compound is present in both a free compound form and a sugar-bound form.

Example 2 Preparation of Haphwan Phi Extract 2

0.1 g of dried Hwanhwanpi (Chinese) powder was added to 1 mL of 1 N HCl aqueous solution, and reacted at 100 ° C. for 2 hours. After hydrolysis for 2 hours and freeze-dried to remove moisture to prepare a hwanhwanpi extract.

<Comparative Example 1> Preparation of soybean extract 1

A soybean extract was prepared in the same manner as in Example 1 except for using soybean (Mungyeong, Korea).

Comparative Example 2 Preparation of Soybean Extract 2

A soybean extract was prepared in the same manner as in Example 2 except for using soybean (Mungyeong, Korea).

Example 3 Analysis of Haphwani Extract

By the following method, phytoestrogens contained in the Hwanhwanpi extract obtained in Examples 1 and 2 and the soybean extract obtained in Comparative Examples 1 and 2 were analyzed.

<3-1> Preparation of analytical sample

40 mg of the Hwanhwanpi extract obtained in Example 1 and 300 mg of the soybean extract obtained in Comparative Example 1 were dissolved in 600 µl of methanol, and centrifuged at 14,000 rpm for 20 minutes at 4 ° C. The supernatant was taken and filtered with a 0.45 μm syringe filter to prepare an assay sample.

60 mg of dried medicinal herb extracts obtained in Example 2 and 300 mg of soybean extract obtained in Comparative Example 2 were added to 600 µl of methanol for 100% HPLC reagent, respectively, and then subjected to sonication for 1 hour, followed by extract 1 ML was taken and centrifuged at 4 DEG C and 14,000 rpm for 20 minutes, and 500 µl of the supernatant was collected and filtered with a 0.45 µm syringe filter to prepare an analytical sample.

<3-2> Analysis of Sample

The sample prepared above was subjected to HPLC (High Performance Liquid Chromatography; Thermo Separation Products, Fremont, CA, USA, model name spectra system P1000) to analyze the type and content of the phytoestrogens contained in the extracts of Hwanhwan. A mixed solution of 5 mM NaH ₂ PO ₄ (pH 4.6): methanol = 4: 6 was used as the mobile phase, and Luna 5 μm C18 (250 × 4.6 mm) was used as a column, and the flow rate was 1 ml / min. The internal standard material is aloe-emodin considering the retention time of the formmononetin and the retention time of tailing and other materials in the mobile phase overlap with the retention time of the internal standard material. ) Is selected. In addition, aloe-emodin, genistein, and dyedzein (all of which were used by Sigma, St. Louis, MO, USA) were used as standard substances. Formononetin was extracted and purified by the present inventors, and confirmed by NMR and MASS. It was used later. Diedzein (0.051 to 213.71 μg / ml), Genistein (0.056 to 194.20 μg / ml), and formonenetine (0.025 to 5.00 μg / ml) were each diluted to different concentrations with methanol and internal standard aloe Emodine (4 μg / ml) was added in the same amount as the standard to prepare a solution. 20 μl of this solution was injected into HPLC to give each chromatogram, which was detected as absorbance at 260 nm. The standard calibration curves were prepared by calculating the peak height ratios of Dydzein, Genistein and Formonenetine at various concentrations as the peak height ratio of the internal standard, respectively. Obtained by Sigmaplot ^@ program (Jandel Scientific, Chicago, IL, USA).

As a result, the amount of phytoestrogens contained in the Hwanhwanpi extract of the present invention is shown in Table 1 below. As a comparative example, the soybean extract obtained in the comparative example was used.

Content of phytoestrogens Haphwanpi Extract Soybean Extract Dyed Jane Free Compound (Example 1) 11.15 ± 1.87 14.52 ± 1.75 Total amount (Example 2) 331.87 ± 103.32 1022.15 ± 201.08 Genistein Free Compound (Example 1) ND 22.49 ± 2.81 Total amount (Example 2) ND 408.58 ± 92.05 Formononetine Free Compound (Example 1) 0.379 ± 0.070 0.0606 ± 0.0341 Total amount (Example 2) 3.894 ± 1.780 0.1613 ± 0.132 Unit: mg / kg * ND: not detected

As can be seen in Table 1 , the hwanhwanpi extract according to the present invention contained a much larger amount of phytoestrogens compound formonenetine than soybean extract, about 6 times in the form of a free compound compared to soybean extract, total The amount contained about 24 times or more. In addition, the amount of dyedzein included was similar to the amount of soybean extract.

As such, the hwanhwanpi extract according to the present invention contains a large amount of phytoestrogens, such as genistein and formmononetin, the hwanhwanpi extract of the present invention can be useful as a functional food as well as a therapeutic or preventive agent for osteoporosis.

Experimental Example 1 Osteoblast Proliferation Experiment

In order to examine the effect of the haphwanpi extract according to the present invention on the proliferation of osteoblasts (osteoblast) was carried out the following experiment.

<1-1> Osteoblast Screening and Cell Culture

The Saos-2 cell line, which has similar properties to osteoblasts, a component of bone, was distributed from the Bank of Korea Cell Line at the Cancer Research Institute of Seoul National University.

Saos-2 cells were cultured in 5% CO ₂ incubator at 37 ° C. in wet conditions using RPMI 1640 medium (Gibco BRL, USA) containing 10% FBS, 100 units / ml penicillin, 100 μg / ml streptomycin. Incubated at. Medium was changed 2-3 times a week and subcultured once a week. Since the cell line has a characteristic of growing and forming a monolayer in a culture flask, the sublayer was stripped off using a 0.25% trypsin solution during subculture.

<1-2> Cell proliferation experiment according to drug concentration: MTT experiment

The cell line was inoculated in a 96-well plate at 20,000 cell numbers / well, and the Hwanhwanpi extract obtained in Examples 1 and 2 was dried at a concentration of 5 to 5 x 10 ^-7 mg / ml for the dry medicine. Was added to the wells. Since Hwanhwanpi extract is non-polar, it was dissolved in DMSO (dimethylsulfoxide), and the final concentration of DMSO was 1% under culture conditions. On the other hand, the control group was not added to the hwanhwanpi extract, as a comparative group NaF and 17β-estradiol (E2) which is currently mainly used as a therapeutic agent for osteoporosis and formmono identified as a component of the hwanhwanpi extract through the above examples Netine and genistein, and biocanine A and 1,25 (OH) ₂ D ₃ were added to the wells by concentration, respectively.

The prepared above was incubated for 3 days in an incubator at 37 ° C., and MTT (3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium bromide; Triazolyl Blue) was added at a concentration of 0.05 mg / ml. Incubated further 4 hours under conditions. The resulting formazan crystals were dissolved in DMSO and the absorbance at 550 nm was measured with an ELISA reader.

Cell proliferation (%) was calculated as the ratio of the absorbance of the extract addition wells to the absorbance of the control wells without addition of the Hwanhwan bark extract, as shown in Equation 1 below. Values were averaged.

As a result, the osteoblast proliferation rate (%) by the hwanhwanpi extract is shown in Table 2 below.

Proliferation Results of Saos-2 Cells by MTT Assay Saos-2 cell proliferation (%) Test group Comparison Concentration (mg / ml) Haphwanpi extract of Example 1 Haphwanpi extract of Example 1 Concentration (mg / ml) NaF E2 Formononetine Genistein Dyed Jane Biocanine A 1,25 (OH) ₂ D ₃ 5 × 10 ^-7 99.97 97.73 1 × 10 ^-7 113.52 103.17 104.54 121.88 107.44 97.89 93.58 5 × 10 ^-6 95.77 98.06 1 × 10 ^-6 112.62 118.11 100.85 143.58 114.59 103.14 96.01 5 × 10 ^-5 101.25 101.43 1 × 10 ^-5 92.02 93.79 104.04 134.77 88.37 106.11 101.55 5 × 10 ^-4 100.08 99.78 1 × 10 ^-4 102.93 96.48 98.33 121.37 93.99 75.75 103.93 5 × 10 ^-3 97.22 101.94 1 × 10 ^-3 103.56 100.07 82.14 122.95 87.12 105.16 120.88 5 × 10 ^-2 72.96 98.06 - - - - - - - - 5 × 10 ^-1 16.72 98.14 - - - - - - - - 5 0.97 113.23

As can be seen in Table 2 , the hwanhwanpi extract according to the present invention showed an excellent proliferation rate for osteoblast-like cells such as Saos-2 cells. In addition, the osteoblast proliferation rate is superior to that of NaF and E2, which are conventionally used as a therapeutic agent for osteoporosis, and also show a higher cell proliferation rate than a single compound such as forfononetin and dydzein. Therefore, haphwanpi extract according to the present invention can be usefully used in drugs and functional foods associated with the treatment or prevention of osteoporosis.

<1-3> Active Search of ALP

Since osteoblasts exhibit ALP activity, the effect of the Hwanhwanpi extract according to the present invention on ALP activity in osteoblasts was examined by the following method.

Saos-2 cell lines of the same cell number as in the MTT experiment were treated with the same concentrations and harvested after 3 days of incubation under the same conditions. At this time, NaF, E2, formonenetine, Zenithine, dydzein, biocanine A, 1,25 (OH) ₂ D ₃ were used as a comparison group, respectively. Meanwhile, ALP activity was measured using a change in absorbance at 405 nm by decomposing p-nitrophenylphosphate to p-nitrophenol and phosphate. The results are shown in Table 3 below.

Results of ALP Activity in Saos-2 Cells % ALP Activity of Saos-2 Cells Test group Comparison Concentration (mg / ml) Haphwanpi extract of Example 1 Haphwanpi extract of Example 1 Concentration (mg / ml) NaF E2 Formononetine Genistein Dyed Jane Biocanine A 1,25 (OH) ₂ D ₃ 5 × 10 ^-7 91.56 91.43 1 × 10 ^-7 94.81 96.18 95.73 110.43 101.94 104.74 192.15 5 × 10 ^-6 125.08 94.48 1 × 10 ^-6 94.56 100.92 264.12 134.82 99.87 103.52 93.92 5 × 10 ^-5 70.08 119.61 1 × 10 ^-5 93.02 98.71 111.38 123.09 103.00 131.27 99.51 5 × 10 ^-4 68.48 105.11 1 × 10 ^-4 87.17 90.85 105.69 124.71 93.01 101.94 182.73 5 × 10 ^-3 134.43 134.52 1 × 10 ^-3 87.05 91.03 90.59 121.47 98.00 98.35 167.46 5 × 10 ^-2 48.03 111.32 - - - - - - - - 5 × 10 ^-1 58.79 111.18 - - - - - - - - 5 64.34 79.00 - - - - - - - -

As can be seen in Table 3 , the Hwanhwanpi extract according to the present invention showed excellent ALP activity rate against Saos-2 cells. In addition, the ALP activity rate is superior to that of NaF and E2, which are conventionally used as a therapeutic agent for osteoporosis, and also exhibited higher activity rates than single compounds such as phorononetin, dydzein, and genistein. Therefore, Hwanhwanpi extract according to the present invention can increase the activity of osteoblasts to promote the production of bone, and thus can be useful for drugs and functional foods related to the treatment or prevention of osteoporosis.

Experimental Example 2 Animal Experiments on Ovariectomized Rats

Animal studies on osteoporosis were performed in females of SD (Sprague-Dawley) rats with type I osteoporosis after menopause.

<2-1> Experimental Animal and Experimental Step

Sprague Dawley rats of 200-300 g body weight, 10 weeks old, were used as experimental materials. Experimental procedures can be largely divided into ovarian extraction in rats, drug administration according to each group, weight change measurement, histological observation and histomorphologic analysis at the sacrifice of rats at regular intervals after extraction.

<2-2> Ovarian Extraction and Drug Administration

Ovarian extraction was performed in all female rats of the control group and the test group except for the sham group (normal group). Female rats were anaesthetized by intramuscular injection of 5 mg / 100 g of ketamine (Yuhan, Korea) and 1 mg / 100 g of xylazine (Bayer, South Korea) into the left and right hind femurs of the rat. . After removing the hair of the lower abdomen, the surgical site was sterilized with potadidine solution (Samil Pharm .: Iodine, Korea) while lying on the animal's body. The skin, abs and peritoneum were incised about 2 cm from the lower abdomen around the midline, and the ovaries were exposed with sterile tweezers. 0.3 ml of antibiotic (Sulpaporte-4, Unichem Co., Ltd.) was injected intraperitoneally to prevent infection, and the peritoneum, abs and skin were sutured with silk and nylon yarns.

The Sham group is a control group for ovarian extraction and no drug administration, except for ovarian extraction, in order to compare the ovarian extraction with the control group. The control group, on the other hand, is for comparing the changes due to drug administration compared to animals of the administration groups that underwent ovarian extraction and drug administration.

One week after ovarian extraction, the Sham group and the control group received 10% Tween 80 solution, the E2 group received 17β-estradiol at 1 μg / kg / day, and the test drug group received 500 mg / ml of the test drug for 9 weeks. Animal experiments were conducted by intraperitoneal injection or subcutaneous injection at a concentration. Lumbar and right tibia (5 and 6) were isolated for bone mineral density and stored in 4% formalin solution (10% dilution).

<2-3> Measurement of Changes in Weight and Tissue Weight by Drug Administration

When the weight and tissue weight of the experimental animal to which the drug was administered after the ovary was extracted according to Experimental Example <2-2>, before and after administration for a predetermined period of time (before and after ovarian extraction, drug administration 1-9 Week)) was compared to the weight and tissue weight of the Sham group and all ovaries, except for ovarian extraction, and control group without drug administration. The measurement results of the weight and tissue weight of the experimental animals of each group are shown in Tables 4 and 5 below.

Weight Change of Experiment Animals by Drug Administration Week Control Sham E2 Haphwanpi Extract 10 233.47 ± 6.38 233.29 ± 5.71 244.97 ± 8.06 233.84 ± 9.95 11 256.74 ± 6.10 242.99 ± 5.35 262.41 ± 6.91 257.56 ± 7.65 12 285.14 ± 6.03 ^** 251.69 ± 5.52 ^# 272.33 ± 7.13 287.93 ± 8.04 ^* 13 310.79 ± 5.94 ^** 260.17 ± 5.64 ^## 281.00 ± 6.30 312.57 ± 11.46 ^** 14 326.45 ± 7.09 ^** 270.05 ± 6.76 ^{** ##} 293.05 ± 7.74 ^** 330.59 ± 12.56 ^** 15 336.18 ± 7.97 ^** 275.66 ± 5.70 ^{** ##} 302.64 ± 8.78 ^** 339.16 ± 12.95 ^** 16 345.38 ± 7.95 ^** 278.34 ± 6.46 ^{** ##} 299.82 ± 7.76 ^** 333.28 ± 8.27 ^** 17 343.13 ± 8.12 ^** 282.68 ± 7.31 ^{** ##} 306.27 ± 8.47 ^** 335.75 ± 7.69 ^** 18 357.26 ± 8.58 ^** 285.88 ± 6.72 ^{** ##} 308.97 ± 8.56 ^{** #} 341.69 ± 8.13 ^** 19 364.77 ± 8.59 ^** 292.07 ± 6.83 ^{** ##} 319.14 ± 9.06 ^** 362.53 ± 16.88 ^** 20 371.21 ± 8.45 ^** 300.32 ± 6.95 ^{** ##} 327.96 ± 8.22 ^** 368.52 ± 16.79 ^**

Comparison with the boneferroni multiple comparison method,

* As compared to 10 weeks of age; p <0.05, **; p <0.01,

# Compared to control; p <0.05, ##; p <0.01

Changes in Tissue Weight of Laboratory Animals by Drug Administration Uterus (mg) Liver (g) Height (g) Tibia (g) Control 83 ± 6 9.78 ± 0.30 1.77 ± 0.05 0.56 ± 0.02 Sham County 499 ± 35 ^** 9.27 ± 0.27 1.90 ± 0.07 0.55 ± 0.02 E2 259 ± 14 ^** 9.78 ± 0.27 2.03 ± 0.06 0.55 ± 0.01 Blood 86 ± 2 9.13 ± 0.53 1.82 ± 0.10 0.56 ± 0.02

Comparison with Bonferroni multiple comparison method,

* In comparison to the control; p <0.05, **; p <0.01,

As shown in Table 4 , the Sham group and the experimental group administered with E2 were significantly lower in weight gain than the control group during the drug administration period, while in the experimental group to which the Hwanhwanpi extract of the present invention was administered, almost identical to the control group. It can be seen that the weight has been increased. These results indicate that in steady state, body weight increases with increasing age, and administration of Hwanhwanpi extract has no effect on weight gain with increasing age.

In addition, as shown in Table 4 , the weight of all tissues except the uterus in the experimental animals of each group did not show any difference, but the weight of the uterus was increased by 6 times and 3 times in the Sham and E2 administration groups, respectively, compared to the control group. However, it was almost similar to the control group in the Hwanhwan peel extract group.

<2-4> Histopathological observation

The collected femoral tissue was fixed in 10% formalin solution and demineralized in formic acid. Observation sites of bone tissue were cut with a surgical knife, followed by a dehydration process ranging from 70 to 100% alcohol and acetone. Paraffin-embedded bone tissue was cut to 5 μm with a microtome and subjected to hematoxyline eosin (HE) staining and observed with an optical microscope.

As a result, no pathological difference was observed between the bone tissues of each group, so it was judged that there was no toxicity in vivo due to administration of the test drug.

<2-5> Morphometric Analysis

Morphometric analysis was performed in the lumbar and tibia of each group by the following method.

The quantizer of the Quantitative image analysis system (Wild Leitz Co.) draws the contours of each shochu and obtains an image on a computer screen, and automatically calculates the area of this image using a special computer system. The area of trabecula was obtained by. The length of the straight portion of the lower portion of the growth plate roadside using the average area of Suzhou bone within the interior of the basis area of ² × 10 ⁶ ㎛ 2 is rectangular with a length that is approximately 2/3 of the length of the growth plate of each computer in the proximal tibia After calculating the number of bone shochu inside the rectangle, the average area was multiplied by the number to obtain the small bone area of each bone sample and statistical processing. The results are shown in Table 6 below.

Morphometric Analysis Right tibia Lumbar vertebra Area of the small bone (× 10 ⁴ μm ² ) Area of Soju Bone for Control Group (%) Area of the small bone (× 10 ⁴ μm ² ) Area of Soju Bone for Control Group (%) Control 35.60 ± 1.30 100.00 ± 3.65 61.64 ± 2.70 100.00 ± 4.38 Sham County 101.29 ± 8.79 ^** 284.52 ± 24.69 ^** 88.11 ± 4.02 ^** 142.94 ± 6.52 ^** E2 52.44 ± 2.92 ^* 147.30 ± 8.20 ^* 76.64 ± 2.69 ^* 124.33 ± 4.36 ^* Haphwanpi Extract 58.79 ± 4.69 ^** 165.14 ± 13.17 ^** 82.25 ± 3.31 ^** 133.44 ± 5.37 ^**

Comparison with Bonferroni multiple comparison method,

* In comparison to the control; p <0.05, **; p <0.01,

As can be seen in Table 6 , the control group of the ovary was reduced by 50% of the area of the sub-catheter bone compared to the normal state (Sham group), indicating that osteoporosis was induced. The area of osseous bone caused by ovarian extraction was increased by the administration of E2 or Hwanhwanpi extract (P <0.01), which was lower than that of the Sham group (P <0.01), but significantly increased (P <0.05). The administration of Hwanhwanpi extract increased the area of the minor bone by about 65% compared to the control group, and in particular, increased the area of the small bone than E2, which is currently used for the prevention and treatment of osteoporosis. Therefore, Hwanhwanpi extract according to the present invention can be effectively used for the prevention and treatment of osteoporosis.

As described above, the Hwanhwanpi extract according to the present invention contains a large amount of phytoestrogens, such as dydzein and formonenetine, compared to soybean extracts used as a source of phytoestrogens, and thus is cheaper than soybeans. It is useful as a source of phytoestrogens and can be used as a raw material or health food for the prevention and treatment of various diseases using dydzein and formmononetin. In particular, Hwanhwanpi extract exhibits a superior osteoporosis treatment or prevention effect than a single compound in phytoestrogens, and thus can be used as a therapeutic or prophylactic agent for osteoporosis, but also as a functional food.

Claims (7)

Albizziae Cortex extract, effective in the prevention and treatment of osteoporosis. The method of claim 1, wherein the hwanhwanpi extract is characterized in that the haphwanpi extract (daidzein) or formononetin (formononetin). Method of preparing a hwanhwanpi extract of claim 1 by immersing the dried hwanhwanpi dried in 10-100% aqueous alcohol solution. A method for producing a compound ring extract of claim 1 by hydrolyzing the compound ring dried product with 0.1 to 2 N acid or base solution. The method of claim 4, wherein the acid is hydrochloric acid or sulfuric acid and the base is sodium hydroxide or potassium hydroxide. A pharmaceutical composition for treating or preventing osteoporosis, comprising the extract of claim 1 as an active ingredient. A functional food comprising the extract of Haphwanpi according to claim 1 as an active ingredient.