KR100447607B1 - Extract Method for Resveratrol and Its Derivatives isolated from Paeonia lactiflora Seeds and Its Use for Improved Bone Diseases - Google Patents

Abstract

The present invention relates to a method for preparing resveratrol, which is a stilbene compound, and its derivatives from peony seeds, and the use thereof for the treatment of bone diseases. More specifically, the dried peony seed is evenly ground and then degreased by addition of normal-nucleic acid, methanol is added to the degreasing foil and extracted three times at room temperature for 1 week, followed by filtration and concentration to obtain a methanol extract, Solubilizing the extract with methanol and adsorbing to a DIION HP-20 column previously equilibrated with an aqueous methanol solution, followed by depressurizing and concentrating the aqueous methanol extract obtained by eluting a 20% to 80% aqueous methanol solution in order. And, the concentrated extract was dissolved in methanol and adsorbed onto a Sephadex LH-20 column previously equilibrated with methanol to elute fractions with methanol, and two ultraviolet rays which were sequentially separated through an ultraviolet detector filter. Decompressing and concentrating the absorption fraction (I, II), and dissolving the former fraction with methanol. Then, preparative-liquid chromatography was repeated to separate trans-resveratrol and trans-resveratrol-4′- O -beta-D-glucoside from fraction I, and in the same manner, trans-viniferin and The present invention relates to a method for separating trans-resveratrol and derivatives thereof comprising separating cis-viniferin.

Trans-Resveratrol and its derivative, the viniferin component isolated from the peony seed of the present invention can be provided as a new material of medicines and functional foods for the treatment of bone diseases (fracture and osteoporosis). In addition, the ingredients are prepared in the form of sap, pills, tablets, capsules with the usual excipients can be used for the prevention and treatment of bone diseases, as well as in the form of powder or granule tea and beverage with food additives. It can be used as a functional dietary supplement.

Description

Extract Method for Resveratrol and Its Derivatives isolated from Paeonia lactiflora Seeds and Its Use for Improved Bone Diseases}

The present invention relates to a method for extracting stilbene resveratrol and its derivatives from peony seed, which has bone promoting properties and female hormone estrogen activity, which is useful for use as a therapeutic agent for bone disease in the future.

Peony (芍藥, 0 people: Peony, saengyakmyeong: Paeoniae radix) is Ranunculaceae (Ranuculaceae) peony as medicinal plants belonging to (Paeonia) is divided into herbaceous of peony and neck, I Moran (yunpyeongseop, Korea horticultural plants Encyclopedia, 1989). In terms of plant classification, peony is classified into Paeonia lactiflora Pall., Paeonia obovata Max. And Paeonia japonica Miyabe et Takeda, and varieties of peony are mainly cultivated in Korea (Yunpyeongsup, Korean Horticultural Plant). Illustrated book, 1989).

Peony is the most widely used medicinal crop after ginseng and tangui in Korea. Raw materials of herbal preparations such as Ssanghwatang, Samultang, Dangguijakjaksan, Gyejigapjaktang, Peonygamchotang, Uhwangcheongsimhwan, Socheongyongtang, 1980, it is widely used. Peony's domestic cultivation is 6 major medicinal crops, along with Deodeok, Dangui, bellflower, tofu, and Astragalus, and its output is 2,062 M / T (as of 1998), one of the crops with higher demand than other medicinal crops. Special crop production performance, Ministry of Agriculture and Forestry, 1999).

Peony roots have been widely used for the treatment of analgesic, dysentery, antitussive, gynecological, high blood pressure, abdominal pain, and inflammation in traditional medicine (Han Dae-seok, Herbal Medicine, Dong-myungsa, 1995; Various monoterpene glucosides (Hattori et al., Chem. Pharm. Bull . 33), including peononiflorin, oxypaeoniflorin, and albinoflorin as active substances. 3838-3846, 1985; Kang et al., Kor. J. Pharmacogn . 24: 247-250, 1993; Okubo et al., Biol. Pharm. Bull . 23: 199-203, 2000) and tannins and phenolic acids. This is well known (Sakai et al., Eisei Kagaku 35: 433-443, 1989; Goto et a., Planta Medica 62: 436-439, 1996; Satoh et al., Biochem. Pharmacol . 53: 611-614, 1997; Tanaka et al., Chem. Pharm. Bull . 45: 1891-1897, 1997; Tanaka et al., Chem. Pharm. Bull . 48: 201-207, 2000). As such, many studies have been conducted on the search for various bioactive substances and their physiological and pharmacological effects from the roots of the peony, and the research on the peony seed is inadequate.

On the other hand, postmenopausal women increase the incidence of several physiological disorders such as osteoporosis, cardiovascular disease and Alzheimer's disease due to deficiency of hormone estrogen (Ibrahim and Hortogbagyi, Surgical Oncology 8: 103-123, 1999). In particular, osteoporosis is the most common disease in post-menopausal women. For prevention and treatment, osteoporosis is currently used for hormone replacement therapy, such as ethylnyl estradiol and 17 beta-estradiol. HRT) is effectively and widely used (Grady et al., Ann Intern Med ., 17: 1016-1037, 1992).

HRT administration inhibits bone resorption, increases bone mineral density, greatly reduces the risk of hip fracture (Michaelsson et al., British Med. J. , 316: 1858-1863, 1998), and sustained long-term HRT administration for more than 5 years. Has an excellent effect on the prevention and treatment of osteoporosis, but the risk of developing side effects such as irregular bleeding and breast and uterine cancer is high (Genant et al., Am. J. Obstet. Gynecol ., 161: 1842-1846, 1989). Persson et al., Cancer Causes Control 10: 253-260, 1999). Therefore, there is an urgent need to develop an estrogen replacement material that selectively acts only on bone or cardiovascular tissue and has little effect on the breast or uterus to minimize the side effects of HRT.

The estrogen substitutes developed so far include a selective molecule estrogen receptor modulator (SERM) (Aloysio et al., Gynecol Endocrinol 11: 289-293, 1997) and plant-derived phytoestrogen (Martini). et al., Nutr. Cancer 34: 133-139 , 1999). SERMs are agents that can have two activities as antagonists or agonists for estrogen receptors (ER) (Ibrahim and Hortogbagyi, Surgical Oncology 8: 103-123, 1999). SERMs act primarily as estrogen agonists in bones, blood vessels and liver, and as estrogen antagonists or extremely weakly active agonists in the breast or genital organs (Kauffman and Bryant, DN & P 8: 531-538, 1995).

Raloxifen, a representative compound of SERM, is currently the only certified substance for treating osteoporosis (Mark et al., Am. J. Physiol. Cell Physiol ., 279: C1550-C1557, 2000). It has been reported that there is little effect on the uterus with estrogen action (Black et al., J. Clin. Invest . 93: 63-69, 1994). Larosifene, however, has a high risk of developing thromboembolism, which is problematic as an estrogen replacement (Delmas et al., New Eng. J. Med ., 338: 1313-1314, 1998). Therefore, there is a need for development of safer and more effective phytoestrogens derived from natural products that can replace synthetic estrogens.

Phytoestrogens are plant-derived diphenols with molecular structures similar to the female hormone estrogen (Knight and Eden, Obstet Gynecol ., 87: 897-904, 1996). Isoflavonoids, lignans of coumestan and flaxseed of cloba and alfalfa are representative compounds thereof (Axelson et al., Nature 12: 298: 659-660, 1982; Setchell and Adlercreutz , Academic Press , London, p315-345, 1988; Aldercreutz et al., J. Steroid Biochem. Mol. Biol ., 44: 147-153, 1993; Hutchins et al., J. Am. Diet Assoc ., 95a: 545-551, 95b: 769-774, 1995; Draper et al., J. Nutr ., 127: 1795-1799, 1997; Amnon and Debi.Eur . J. Obstet Gynecol Reprod Biol ., 85: 47-51, 1999; Chiechi, Int. J. Gynecol & Obstet ., 67: 39-40, 1999). These phytoestrogen compounds are transformed into mammalian isoflavones and lignans by human intestinal microorganisms to compete with the 17β-estradiol of the living body to occupy estrogen binding sites so that estrogens and antiestrogens ( antiestrogen) (Miksikek, Steroid Biochem. Mol. Biol ., 49: 153-160, 1994) to prevent hormone-dependent breast cancer, prostate cancer and colorectal cancer, as well as heart disease and hypertension in women after menopause. And osteoporosis (Tang and Adams, J. Endocrinol ., 85: 291-297, 1980; Aldercreutz et al., J. Steroid Biochem. Mol. Biol ., 44: 147-153, 1993; Dwyer et al., J. Am. Diet.Assoc., 94: 739-743, 1994; Draper et al., J. Nutr . 127: 1795-1799, 1997; Adlercreutz, Baillieres Clin.Endocrinol . Metab. 12: 605-623, 1998).

In particular, isoflavonoids in soybeans have been shown to maintain bone density by inhibiting bone resorption in post-menopausal animal models of osteoporosis (Arjmandi et al., J. Nutr ., 126: 161-167, 1996; Draper et al., J. Nutr ., 127: 1795-1799, 1997 Potter et al., Am. J. Clin. Nutr ., 68 (suppl): 1375S-1379S, 1998; Alekel Am. J. Clin.Nutr . 72: 844-852, 2000), and soybean isoflavone component, zenithine, like estrogen, promoted bone formation and differentiation (Anderson and Garner, Baillieres Clin Endocrinol Metab ., 12: 543-557, 1998). Coumestrol inhibited bone resorption and promoted bone calcification in tissue culture of chicken thigh (Tsutsumi, Biol. Pharm. Bull . 18: 1012-1015, 1995). 098 has also been reported to promote bone differentiation (Kawashima et al., Biochem. Pharmacol ., 26: 133-139, 1996).

Recently, the present inventors have revealed for the first time that matairesinol, a lignan component present in safflower seed, not only promotes bone formation and differentiation, but also strengthens calcium deposition in bone nodules to treat osteoporosis. , Patent Application No. 00-0003048; Patent Application No. 00-0017617 et al. As such, the phytoestrogen compound is a substitute for female hormone estrogen, and it can be seen that it is very effective in treating bone diseases such as fractures, osteoporosis, and bone dysplasia that appear in women after menopause.

Resveratrol (3,4'5-trihydroxystilbene) is a phytoalexin compound found in grapes and various plants (Langcake and Pryce, Physiol.Planta Pathol ., 9: 77-86, 1976). Langcake and Pryce, Experientia 33: 151-152, 1977; Sanders et al., J. Agric. Food Chem ., 48: 1243-1246, 2000), and several physiological and pharmacological activities including anticancer, antithrombotic, and anti-inflammatory activity. Action (Jang et al., Sci ., 275: 218-220, 1997). In particular, resveratrol has recently been identified as a phytoestrogen compound with a female hormone estrogen-like action (Gehm et al., Proc. Natl. Acad. Sci . 94: 14138-14143, 1997), and has shown postmenopausal women's heart disease. , As a bioactive substance that suppresses osteoporosis, dementia and many menopausal symptoms (Goldberg et al., Am. J. Enol. Vitic ., 46: 159-165, 1995; Jeandet et al., J. Phytopathology 143:..... 135-139, 1995; Mizutani, K. et al, Biochem Biophys Res Commun 253:.... 859-863, 1998; Mizutani, K. et al, J. Nutr Sci Vitaminol 46 : 78-83, 2000). However, unlike the above report on the resveratrol's estrogen agonist action (Turner et al., Endocrinology 140: 50-54, 1999), a recent study on the role of resveratrol's estrogen antagonist As reported, more detailed research on the estrogen action of resveratrol is needed.

On the other hand, several plants ( Dipterocarpaceae , Vitaceae , Cyperaceae , Gnetaceae and Leguminosae ) have found various components of oligostilbenes produced by oxidation and polymerization of resveratrol components (Kawabata et al., Tetrahedron Letters). 30: 3785-3788, 1989; Kurihara et al., Phytochem ., 30: 649-653, 1991), which are attracting attention as bioactive substances with anti-inflammatory, hepatoprotective and anti-hormonal effects as well as antibacterial activity. (Kawabata et al., Tetrahedron Letters 30: 3785-3788, 1989; Lee et al., Planta Med ., 64: 204-207, 1998; Oshima et al., Experientia 51: 63-66, 1995; Sarker et al , Tetrahedron 55: 513-524, 1999). However, until now, there have been few studies on the osteoblast-promoting action, the estrogen action, and the resulting osteoporosis treatment effect of the resveratrol derivative.

Meanwhile, several in vitro screening methods are currently being developed to develop new phytoestrogens from plants (Diel et al., Planta Medica 65: 197-203, 1999; Liu et al., J. Agric Food Chem . , 2001), among which methods for measuring bone formation promotion and differentiation using osteoblastic cells in mice (Mizutani et al., Biochem. Biophys. Res. Commun . 253: 859-863, 1998) and estrogens Methods for measuring estrogen activity using dependent human breast cancer cells (MCF-7 or T47-D) (Mayr et al., Toxicology 74: 135-149, 1992; Gehm et al., Proc. Natl. Acad. Sci . 94: 14138 -14143, 1997; Tamir et al., Cancer Research 60: 3704-3709, 2000; Chansakaow et al., Planta Medica 66: 572-575, 2000; Yoshikawa et al., Chem. Pharm. Bull . 48: 1039- 1044, 2000; Riby et al., Biochem. Pharmacol 60: 167-177, 2000) is the simplest search method currently used most widely.

Recently, the inventors have isolated and identified for the first time anticancer and antimutagenic resveratrol and its derivatives which are not present in the root of the peony from unused peony seeds (Choi et al., J. Food Sci. & Nutr ., 4). : 163-166, 1998) In addition, the content of these compounds has been found to be significantly different depending on the variety and maturity (Choi et al., J. Food Sci. & Nutr ., 4: 163-166, 1998; Choi et al., J. Agric.Food Chem ., 2001, accepted).

Accordingly, the present inventors measured the method of promoting bone formation and estrogen action of resveratrol and its derivatives isolated from peony seeds by cell culture.Resveratrol and its derivatives contained in peony seeds 17beta-estradiol, known as a phytoestrogen compound, has been found to have an osteoporosis treatment similar to that of the genistein component of soybean.

The present invention aims to separate four stilbene compounds, resveratrol and its derivatives from peony seed which promotes bone formation and has estrogen activity, which can be used as an estrogen replacement agent which is useful for the treatment of bone disease in the future. It is done.

1 is a high performance liquid chromatogram of four stilbene compounds isolated from peony seeds.

The extraction method of trans-resveratrol and its derivatives from peony seed is characterized by separating the four stilbene compounds from the peony seed, evenly crushing the dried peony seed and then degreasing it by adding normal-nucleic acid, Methanol was added to the mixture, and the mixture was repeatedly extracted at room temperature, followed by filtration and concentration to obtain a methanol extract. The extract was solubilized with methanol and charged into a DIION HP-20 column previously equilibrated with an aqueous methanol solution. Eluting and then depressurizing and concentrating the methanol extract, dissolving the concentrated extract with methanol and adsorbing to a Sephadex LH-20 column previously equilibrated with methanol, eluting fractions with methanol, Two UV absorption fractions separated sequentially through the detector filter (Ⅰ, Ⅱ) , III) under reduced pressure and concentration, and the previous fractions were dissolved in methanol, and preparative liquid chromatography was repeated to carry out trans-resveratrol-4'- O -beta-D-glucoside from fractions I and II. Separating trans-resveratrol and separating trans-viniferin and cis-viniferin from fraction III in the same manner.

Another method for extracting trans-resveratrol and its derivatives from the peony seed of the present invention is to separate four stilbene compounds from the peony seed, and the methanol mixture obtained by pulverizing the dry peony seed and extracting and filtering at room temperature by adding methanol Depressurizing and concentrating the extract to obtain a methanol extract, adding methanol solution to the methanol extract to dissolve it, adding normal-nucleic acid to degrease, and depressurizing and condensing the lower layer of the degreased extract to ether and ethyl. Adding acetate and fractionating sequentially to obtain an ether extract and ethyl acetate extract, and transferring the ether extract to a column filled with silica gel by suspending it in chloroform / methanol and diluting the fraction by chloroform / methanol for elution. Fractions of are divided into six fractions, fraction 3 of which is Concentration under reduced pressure and recrystallization again with methanol to obtain trans-resveratrol, fractions 4 and 5 under reduced pressure and concentration, dissolved in methanol, transferred to a column packed with Sephadex LH-20, and eluted with methanol. Obtaining a cis-viniferin and trans-viniferin mixture sequentially separated through an ultraviolet detector filter, and dissolving the above mixture with water-soluble methanol and repeating preparative liquid chromatography to perform trans-viniferin and cis- Obtaining Viniferin, and ethyl acetate extract was dissolved in methanol in the same manner as before, Sepadex LH-20 column chromatography fractionated and eluted into 6 fractions, and then the third fraction was concentrated and trans-reds. Obtaining veratrol-4'- 0 -beta-D-glucoside.

Preferred concentrations of methanol used in the present invention are 40% to 100% aqueous solution, most preferably 80% to 100% aqueous solution. In this case, ethanol may be used instead of methanol as the extraction solvent. Preferred temperature in the extraction step in the present invention is 20 ℃ to 60 ℃ and most preferably 50 ℃. In addition, it is advisable to block the sunlight at all stages of extraction.

The four stilbene compounds extracted from the peony seed by the above extraction method were trans- Resveratrol of formula I, and trans-resveratrol-4'-glucoside of formula II. O -β-D-glucopyranoside), trans - ε- Viniferin of formula III, and cis - ε- Viniferin of formula IV.

(Formula I) (Formula II)

(Structure III) (Structure IV)

Bone disease therapeutic agent containing any single compound selected from trans-resveratrol, trans-resveratrol-4'-glucoside, trans-viniferin or cis-viniferin as an active ingredient extracted from peony seed according to the present invention These bone disease treatment agents can be used in the form of transfusions, pills, tablets or capsules containing trans-resveratrol, trans-resveratrol-4'-glucoside, trans-viniferin or cis-viniferin as an active ingredient. Can be used as

Hereinafter, the present invention will be described in more detail with reference to the following examples, which do not limit the technical scope of the present invention.

<Example 1>

The dried peony seed was crushed evenly and then degreased by adding normal-nucleic acid, 100% methanol was added to the obtained skim foil, and extracted three times at room temperature for one week, followed by filtration and concentration to obtain methanol extract (25.5g). It was solubilized by adding 20% water-soluble methanol solution and packed into a DIION HP-20 column (6cm in diameter x 60cm in length) which had been equilibrated with 20% water-soluble methanol solution before adsorbing 20% water-soluble methanol (5L), 40% Aqueous methanol (10 L) and 80% aqueous methanol (10 L) solutions were eluted sequentially. Sepadex LH-20 (pama), which was dissolved in methanol (30 mL) by dissolving the dried 80% aqueous methanol extract obtained under reduced pressure and concentration in methanol (30 mL), and then equilibrating with methanol (30 mL). Sia, Sweden) was transferred to a column (diameter 2.5 cm x length 1 m) and eluted by dividing it into 150 fractions of 3 ml per tube with 1 liter of the same solvent. At this time, three ultraviolet absorption fractions (I, II & III) which are separated sequentially through the UV detector filter (280 nm) are collected and decompressed and concentrated. Among them, fraction I (fraction 20-45) and fraction II (fraction 71-) 93) as a white powder from the trans - reds vera trawl -4'- O-beta -D- glucoside (trans -resveratrol-4'- O -β- D-glucoside, molecular weight 390), 0.24g of amorphous trans and light brown - 0.18 g of resveratrol ( trans -resveratrol, molecular weight 228) was obtained, respectively.

Subsequently, fraction III (fraction 112-135) was dissolved in methanol again, and preparative high-performance liquid chromatography was repeated to give light brown amorphous trans- viniferin (molecular weight 484, separation time 22.5 minutes) 2.23 g And 1.25 g of cis -viniferin ( cis -viniferin, molecular weight 484, separation time 27.9 minutes) were obtained, respectively.

At this time, the separation conditions of the preparative liquid chromatography used are as follows:

Waters Delta Prep-4000 Fast Liquid Chromatography;

Column, RCM Novapak C ₁₈ (2.5 cm × 10 cm);

Ultraviolet detector (UV _{280 nm} );

Flow rate, 5.0 ml / min;

Solvent (A, 25% aqueous acetonitrile solution containing 0.1% trifluoroacetic acid.

<Example 2>

After grinding 300 g of dry peony seeds, 2 L of methanol was added thereto, followed by extraction and filtration at room temperature for 24 hours. Methanol extract (37.5 g) was obtained by depressurizing and concentrating the methanol mixture extract obtained by repeating the above extraction operation three times. Next, 500 ml of 80% water-soluble methanol solution was added to the methanol extract to dissolve it, and 500 ml of normal-nucleic acid was added thereto, followed by repeated degreasing twice. The resulting lower layer was concentrated under reduced pressure to 200 ml, and then 1 liter of ether and ethyl acetate were added thereto. Each was added sequentially and repeatedly fractionated twice to obtain an ether extract (7.88g) and ethyl acetate extract (0.54g), respectively. The total ether extract was suspended in 30 ml of a chloroform / methanol (3: 1, v / v) mixture, and then transferred to a column of 5 cm diameter, filled with 700 grams of silica gel 60 (70-130 mesh, Merck, Germany), followed by chloroform. 2 ml of methanol (3: 1, v / v) mixture was eluted in 200 fractions, 5 ml per tube. These fractions were divided into six fractions (fraction 1, 5-15, 0.1 g; fraction 2, 16-45, 0.59 g; fraction 3,46-78, 0.81 g; fraction 4, 79-91, 0.14 g; Fraction 5, 92-123, 0.62 g; Fraction 6, No. 124-167, 3.2 g), collected fraction 3, reduced pressure and concentration, and recrystallized with methanol again to give a light brown trans-resveratrol ( trans 0.18 g of resveratrol, molecular weight 228) was obtained. Then, fractions 4 and 5 were collected, decompressed and concentrated, and then dissolved in methanol. A predetermined amount was charged in a column of 2 cm diameter filled with 100 g of Sephadex LH-20 (Pamacia, Sweden), followed by 1 liter of methanol per tube. Eluate the solution into 200 fractions of 3 ml each. At this time, 4.02 g of a mixture of viiniferin (cis- and trans-viniperin), which were sequentially separated through an ultraviolet detector filter (280 nm), was obtained. Next, the mixture was dissolved in methanol, and preparative liquid chromatography was repeated. 2.21 g of light brown amorphous trans- viniferin (molecular weight 484, separation time, 17.7 minutes) and cis-viniferin ( cis- viniferin, molecular weight 454, separation time 22.5 minutes), 1.27 g were obtained, respectively. Preparative-Liquid Chromatography (Waters Delta Prep-4000 Fast Liquid Chromatography; Column, RCM Novapak C ₁₈ (2.5 cm × 10 cm); Ultraviolet Detector (UV _{280 & 308} nm); Flow, 5.0 mL / min; Solvent, 0.1 30% acetnitrim solution with% trifluoroacetic acid).

In the same manner as above, the ethyl acetate extract was dissolved in methanol, and then eluted with 6 fractions by Sepadex LH-20 column chromatography. The third fraction was concentrated to give a trans-resveratrol-4'- white powder. O - beta -D- glucoside (trans -resvera- trol-4'- O -β -D-glucoside, molecular weight 390) was obtained 0.23g.

<Example 3>

5 g of dry peony seeds were pulverized, and 300 ml of methanol was added thereto, followed by repeated extraction for 24 hours in a magnetic stirrer adjusted to 50 ° C., followed by filtration, reduced pressure, and concentration. Next, methanol extract (0.83 g) was dissolved in 100 ml of 80% aqueous methanol, and thereafter, 300 ml of normal-nucleic acid was added thereto, followed by repeated degreasing twice, and the lower layer was depressurized and concentrated. This extract (0.7 g) was dissolved in 10 ml of methanol, 1 ml of which was diluted 10-fold with distilled water, and then adsorbed onto a Sep-pak C18 cartridge (Waters, USA) previously activated with methanol and distilled water, followed by distilled water. After washing, eluting with 5 ml of 40% and 60% aqueous acetonitrile, respectively, and using trans-resveratrol-4'- O -beta-D-glucoside from 40% aqueous methanol extract using preparative-high-performance liquid chromatography ( 11.8 mg of trans -resveratrol-4'- O -β-D-glucoside (separation time, 16.8 minutes) was isolated. Then from 60% aqueous methanol extracts in the same way trans- resveratrol (trans -resveratrol; separation time, 24.2 minutes) and 7.6mg trans- beanie Perrin (trans -viniferin, molecular weight 484, separation time, 23.5 minutes] 45mg and cis-beanie 39 mg of perrin ( cis- viniferin; molecular weight 484, separation time 32.6 minutes) were separated, respectively, using preparative high performance liquid chromatography (Waters Delta Prep 4000 high performance liquid chromatography; column, RCM Novapak C ₁₈ (2.5 cm × 10 cm); UV detector (UV _{280 & 308} nm); speed, 5.0 ml / min; solvent (solvent A, 10% water soluble acetonitrile solution containing 0.1% triproroacetic acid; solvent B, 50% water soluble acetonitrile solution, solvent A-> Concentration gradient elution with solvent B), and the chemical structures of the four compounds separated at this time are the same as those of the above-mentioned invention, and the graph of the high-speed liquid chromatography for analysis is shown in FIG. And these compounds Detailed ultraviolet, infrared, nuclear magnetic resonance and mass spectroscopy spectrum data were as shown in Table 1 and Table 2 below.

At this time, the analytical HPLC conditions are as follows.

Column, μBondapak C18 (3.9 cm × 30 cm); Gradient gradient elution with solvent A (10% water soluble acetonitrile solution containing 0.1% triproroacetic acid) & solvent B (50% water soluble acetonitrile solution), solvent A-> solvent B for 50 minutes; Ultraviolet detector (UV ₃₀₈ nm); Speed, 1.0 ml / min. 1 , trans-resveratrol-4′- O -beta-D-glucoside; 2 , trans-resveratrol; 3 , trans-viniferin; 4 , cis-viniferin .

Table 1: Spectral data of ultraviolet and infrared spectroscopy of resveratrol and its derivatives isolated from peony seeds

Instrumental analysis UV _λmax nm (log ε ) IR _νmax (cm ^-1 ) trans -ResveratrolResveratrol-4'- O -β-D-glucopyranoside trans - ε -Viniferin cis - ε -ViniferinSuffruticosol ASuffruticosol B 219 (4.28), 308 (4.02), 320 (3.34) 219 (4.28), 308 (4.02), 320 (3.34) 218 (4.52), 312 (4.29), 324 (4.32) 218 (s), 287 (s ), 308 (s), 325 (4.38) 225 (4.08), 282 (3.42) 225 (4.10), 282 (3.45) 3200-3300, 1587, 1512,1149, 9643200-3300, 1589, 1507,1150, 9663230, 1594, 1510, 1440,1002, 9643230, 1594, 1510, 1440,1002, 9643000-3600, 1600, 1500,1440, 1330, 1230,1160, 1000, 8303000-3600, 1600, 1500,1440, 1330, 1230,1160, 1000, 830

^* UV absorption spectrum of the sample is measured after melting with methanol solution.

^* The infrared absorption spectrum of the sample is measured by using KBr plate.

Table 2: Nuclear Magnetic Resonance (in CD ₃ OD) and Mass Spectrometer Spectrum Data of Resveratrol and Its Derivatives Isolated from Peony Seeds

Instrumental analysis trans -Resveratrol Resveratrol-4'- O -β-D-glucopyranoside ¹ H-NMRH-2H-4H-6H-2'H-3'H-5'H-6'HαHβSugar moietyH-1H-2H-3H-4H-5H-6aH-6b 6.43 (d, J = 2.5 Hz ) 6.14 (t, J = 2.5 Hz) 6.43 (d, J = 2.5 Hz) 7.33 (d, J = 8.5 Hz) 6.75 (d, J = 8.5 Hz) 6.75 (d, J = 8.5 Hz) 7.33 ( d , J = 8.5 Hz) 6.69 ( d , J = 16.5 Hz) 6.14 ( d , J = 16.5 Hz) 6.46 (d, J = 2.0 Hz ) 6.18 (t, J = 2.0 Hz) 6.46 (d, J = 2.0 Hz) 7.43 (d, J = 8.5 Hz) 7.07 (d, J = 8.5 Hz) 7.07 (d, J = 8.5 Hz) 7.43 ( d , J = 8.5 Hz) 6.07 ( d , J = 16.0 Hz) 6.98 ( d , J = 16.0 Hz) 4.92 ( d , J _1,2 = 7.5 Hz) 3.41 ( t , J _{2, 3} = 7.8 Hz) 3.44 ( m , J _3,4 = 8.4 Hz) 3.30 ( t , J _4,5 = 8.4 Hz) 3.47 ( m , J _5,6a = 2.6 Hz, J _5,6b = 5.7 Hz) ( dd , J _{6a, 6b} = 12.5 Hz) 3.71 ( dd , J _{6a, 6b} = 12.5 Hz) ¹³ C-NMRC-1C-2C-3C-4C-5C-6CαCβC-1'C-2'C-3'C-4'C-5'C-6'Sugar moietyG-1G-2G-3G-4G- 5G-6 141.31105.76159.37102.64159.37108.20127.02130.42131.40129.38115.84158.37116.48128.79 141.01105.95159.66102.95159.66105.95128.52128.88133.18128.58117.90158.65117.90128.58102.2174.9078.1371.3677.9762.50 FABMS ( m / z ) 228 [M ⁺ ] 390 [M ⁺ ]

Coupling constants ( J in Hz) in parentheses. d , doublet; dd , double doublet; t , triplet; m , multiplet.

trans - ε -Viniferin cis - ε -Viniferin ¹ H-NMRH-2 & H-6 7.14 ( d , J = 8.5) H-3 & H-5 6.76 ( d , J = 8.5) H-7 5.36 ( d , J = 5.5) H-8 4.34 ( d , J = 5.5) H-10 6.24 ( d , J = 2.0) H-12 6.16 ( t , J = 2.0) H-14 6.24 ( d , J = 2.0) H-2 '7.03 ( d , J = 8.5) H-3 '6.64 ( d , J = 8.5) H-5' 6.64 ( d , J = 8.5) H-6 '7.03 ( d , J = 8.5) H-7' 6.81 ( d , J = 16.5) H- 8 '6.56 ( d , J = 16.5) H-12' 6.24 ( d , J = 2.0) H-14 '6.17 ( d , J = 2.0 6.93 ( d , J = 8.5) 6.70 ( d , J = 8.5) 5.17 ( d , J = 6.0) 3.77 ( d , J = 2.0) 5.92 ( d , J = 2.0) 6.08 ( t , J = 2.0) 5.92 ( d , J = 2.0) 6.92 ( d , J = 8.5) 6.57 ( d , J = 8.5) 6.57 ( d , J = 8.5) 6.92 ( d , J = 8.5) 6.19 ( d , J = 12.0) 6.02 ( d , J = 12.0) 6.21 ( d , J = 2.0) 6.23 ( d , J = 2.0) ¹³ C-NMRC-1 133.79C-2 & C-6 128.21C-3 & C-5 116.31C-4 158.55C-7 94.71C-8 57.72C-9 148.25C-10 109.34C-11 159.77C-12 101.76C-13 159.83C-14 109.12C-1 '130.71C-2' 128.78C-3 '116.39C-4' 158.55C-5 '116.39C-6' 128.78C-7 '130.22C-8' 123.72C -9 '136.93C-10' 119.31C-11 '163.01C-12' 96.86C-13 '160.06C-14' 104.35 133.84128.50116.20157.8294.9157.73147.33107.24159.53101.82159.53107.24130.07131.13115.88158.39115.88131.13131.63126.63137.81120.37162.7896.58159.53108.91 FABMS ( m / z ) 454 [M ⁺ ] 454 [M ⁺ ]

d , doublet; t , triplet.

Experimental Example 1 Experiment of Bone Formation Promotion of Resveratrol and Its Derivatives Isolated from Peony Seeds

In this experimental example, the bone formation promoting action of the resveratrol and its derivatives obtained in Examples 1, 2, and 3 was measured using ROS 17 / 2.8, which is an osteoblast cell line, which is bone-forming osteoblasts. It was. That is, ROS 17 / 2.8 cells were mixed with 10% fetal bovine serum and 1% antibiotic-antibacterial mixture (100 IU penicillin, 100 mg streptomycin, 250 nanogram amphotericin, Gibco BRL) without phenol red. Cell proliferation was inoculated in Dulbecco's Modified Eagle medium added to the culture medium in a 5% CO ₂ incubator maintained at 37 ° C. (MTT, 3- (4,5-dimethyl). It was measured by an MTT assay based on metabolic reduction of -thiazol-2-yl) -2,5-diphenyltetrazolin bromide).

At this time, 17beta-estradiol, which is known to promote bone cell proliferation, and genistein isolated from soybean were used as a positive reference to determine whether peony seeds promote bone formation. And, the sample-free addition was set as a control and compared. The MTT assay method is as follows. After transplanting ROS cells into 96 wells with 5 × 10 ³ cells, the medium composition was changed to 0.5% estrogen deficient-fetal bovine serum and 2.5% estrogen deficient-for 24 hours to rule out the effects of fetal bovine serum. Under fetal bovine serum conditions, six phenolic compounds isolated from roasted safflower seeds in addition to estrogen and genistein were stimulated for 48 hours at 10 ^-6 to 10 ^-15 molar concentrations. 50 μl of an empty (1.1 mg / ml) solution was added to each well and incubated at 37 ° C. for 4 hours.

To dissolve the formazan crystal, a purple precipitate formed by the Mt, 150 microliters of dimethylsulfoxide was added and dissolved, and the absorbance of each well was 540 using a microplate reader. Measured at nm. At this time, the bone formation promoting action of each component is expressed as a relative value to the control (control). The results are shown in Table 3.

Table 3: Effect of four stilbene compounds isolated from peony seed on proliferation of osteoblast-like cells (ROS 17 / 2.8)

compound Concentration (m) Control 10 ^-15 10 ^-12 10 ^-9 10 ^-6 Trans- Reds vera trawl (trans -Resveratrol) 100 120 122 120 132 Trans- Reds vera trawl -4'-O- beta -D- glucoside (trans -Resveratrol 4'-O-β -D-glucoside) 100 110 91 85 92 Cis-beanie Perrin (cis -Viniferin) 100 117 120 124 120 Trans- beanie Perrin (trans -Viniferin) 100 113 122 125 120 17beta-estradiol ^* (17β-Estradiol, E ₂ ) 100 128 126 138 128 Genistein ^* (Genistein) 100 110 113 111 117

As shown in Table 3 above, (1) 17 beta-estradiol (E ₂ ) had a cell proliferation effect of about 130% at a low concentration of 10 ^-15 M, and a physiological concentration of about 10 ^-9 M. It increased to 138% and promoted the proliferation of bone cells in a concentration-dependent manner. (2) Genistein of soybean also increased ROS osteoblast proliferation by up to 117%, but showed lower effect than estradiol (E ₂₎ . (3) All four stilbene compounds isolated from peony seeds tended to promote the proliferation of ROS osteoblasts in a concentration-dependent manner. In particular, trans-resveratrol and viniferin compounds were as much as estradiol (E ₂ ) and genistein. High cell proliferation rate.

Experimental Example 2 Experiment of Estrogen Activity of Resveratrol and Its Derivatives Isolated from Peony Seeds

In this experimental example, the estrogen activity of resveratrol and its derivatives obtained in Examples 1, 2, and 3 was measured using MCF-7, an estrogen-sensitive breast cancer cell line.

That is, MCF-7 cells were treated with 10% fetal calf serum (FCS, Biowhittaker) without phenol red and 1% antibiotic-antibacterial mixture (100 IU penicillin, 100 mg streptomycin, 250 nm amphotericin, Gibco BRL). Cell proliferation was performed by inoculating Dulbecco's Modified Eagle medium (D-MEM) added to the cell proliferation while incubating in a 5% CO ₂ incubator maintained at 37 ° C. It was measured by the (MTT assay) method.

At this time, 17beta-estradiol and genistein isolated from soybean were used as a positive reference and sample-free as a control to measure the estrogen activity of the peony seed. Set and compare.

The MTT assay method is as follows. MCF-7 cells were exchanged with fresh medium 2-3 times a week and washed with PBS buffer (pH 7.0), followed by centrifugation by detachment of adherent cells using 0.05% tyrosine, 0.002% identity, followed by accumulation. The medium was added to the prepared cancer cells, the cancer cells were evenly mixed with a pipette so that the cells were evenly distributed, divided into a predetermined amount by 10ml in a 75cm cell culture flask, and used in the experiment while subcultured every 4 to 5 days. 1 ml of MCF-7 cell line (1 × 10 ⁵ cells / ml) was dispensed in a 24-well plate and incubated for 24 hours (37 ° C., 5% CO ₂ ). Toxicity experiments were conducted with sulfoxide (dimethylsulfoxide, DMSO), and each sample was dissolved in DMSO and added at concentrations of 100, 200, 300, 400 and 500 µg / well and incubated for 48 hours. 100 microliters of empty solution was added to the culture solution, followed by incubation for 4 hours, and the supernatant was removed with aspirate by carefully treating the formazan formed at the bottom of the well so as not to scatter.

1 ml of mixed solution of DMSO and EtOH (1: 1, v / v) was added to the formed formazan to be sufficiently dissolved, followed by 570 nm using an UV-vis spectrophotometer (Pharmacia Biotech 80-1205-20, Sweden). Absorbance was measured at. At this time, the estrogen activity of each component is expressed as a relative value to the control. The results are shown in Table 4.

Table 4. Estrogen Activity of Four Stilbenes Isolated from Peony Seeds

compound Concentration (m) Control 10 ^-7 10 ^-6 10 ^-5 Trans- Reds vera trawl (trans -Resveratrol) 100 133 175 198 Trans- Reds vera trawl -4'- O-beta -D- glucoside (trans -Resveratrol 4'-O- β- D -glucoside) 100 121 143 164 Cis-beanie Perrin (cis -Viniferin) 100 126 161 185 Trans- beanie Perrin (trans -Viniferin) 100 128 169 189 17beta-estradiol (17β-Estradiol, E ₂ ) 100 146 178 201 Genistein 100 131 167 187

As shown in Table 4, (1) 17 beta-estradiol (E ₂ ) already exhibited a 146% cell proliferation effect at a low concentration of 10 ^-7 M, about 201% at a concentration of 10 ^-5 M It increased up to to promote cell growth in a concentration-dependent manner. (2) Genistein of soybean also increased MCF-7 cell proliferation up to 187% in concentration-dependent manner, but showed lower effect than estradiol (E ₂₎ . (3) All four stilbene compounds isolated from peony seeds tended to promote the proliferation of MCF-7 cells in a concentration-dependent manner. In particular, trans-resveratrol and viniferin compounds were treated with estradiol (E ₂ ) and genistein, respectively. As high as the cell proliferation rate.

These results suggest that the four stilbene compounds isolated from peony seeds not only promote bone formation but also act as replacements for estrogen compounds (17β-estradiol, genistein), which are widely used as phytoestrogen compounds with estrogen activity. Could know.

The components of four stilbene compounds, trans-resveratrol and derivatives thereof, isolated from the peony seed of the present invention can be provided as a new material of medicines and functional foods for the treatment of bone diseases (fracture and osteoporosis). In addition, the ingredients are prepared in the form of sap, pills, tablets, capsules with the usual excipients can be used for the prevention and treatment of bone diseases, as well as in the form of powder or granule tea and beverage with food additives. It can be used as a functional dietary supplement.

Claims (7)

delete delete delete delete delete An osteoblast proliferation promoter comprising trans-viniferin of the following structural formula (III) or cis-viniferin of the following structural formula (IV) extracted from peony seeds as an active ingredient. (Structure III) (Structure IV) 7. The osteoclast growth promoter according to claim 6, wherein the osteoclast growth promoter is provided in the form of a transfusion, pill, tablet, or capsule, which is trans-viniferin or cis-viniferin extracted from peony seed.