KR100345825B1 - Method for Extraction, Isolation and Identification of Serotonins, Lignans and Flavonoids Improved Bone Formation from Safflower(Carthamus tinctorious L.) Seeds - Google Patents

Abstract

The present invention relates to a method for extracting serotonin, lignan and flavonoid components that promote bone formation from safflower seeds. The roasted safflower seeds are degreased with nucleic acid, and the residue is extracted with hot methanol by methanol, filtered and concentrated. Column chromatography with amide C-200 and column chromatography with Sephadex LH-20 - high performance liquid chromatography on 60%, 80% and 100% methanol extracts obtained by pure-liquid separation of serotonin, lignan and flavonoids Ingredients have excellent effects on the treatment of osteoporosis and fracture.

Description

[0001] The present invention relates to a method for extracting serotonin, lignan and flavonoid components from safflower seeds, which promotes bone formation. [0002] The present invention relates to a method for extracting serotonin, lignan and flavonoid components,

The present invention relates to a method for extracting serotonin, lignan and flavonoid components which promote bone formation from safflower seeds. More particularly, the present invention relates to a method for inhibiting osteoporosis by stimulating bone formation from roasted safflower seed to provide a serotonin component such as perroyl serotonin and coumaryl serotonin useful as a preventive and therapeutic agent for fracture and osteoporosis, a lignan component such as matairecinol and hydroxyactigencine, And a method of extracting flavonoid components such as acacetin

Safflower (Carthamus tinctorious L.) is a perennial plant belonging to the Compositae , and its origin is the mountainous region of Ethiopia or Ethiopia (Lee Chang-bok, Korean Botanical Garden, 1980). Safflower has been widely used as a dye (Yong Chang Soo, Korean Herbal Medicine, 1981), containing water-soluble yellow pigment and insoluble red pigment, and also has been used as a medicament for treatment of eosinophilic, hyperlipidemic and hyperlipidemia, Have been widely used as prescription drugs (Herbology, Lee, 1980).

On the other hand, safflower seeds have been used as calming and blood medicines in Korea, China and Japan (Haejakgwa, Han Dae Suk, 1995), and especially useful plants for the production of edible oils in the US and India (Ahn, Modern Hebrews, 1975). Safflower oil has been widely used as a health food material because it contains vitamin E tocopherol, vitamin F, linolenic acid, and natural minerals such as calcium, aluminum and bromine (Yoo Tae Jong, Health Food, 1995). In particular, safflower seeds contain a large amount of unsaturated fatty acids, and thus they are not only nutritionally important, but also have a high content of linolenic acid, thus being attracting attention as antithrombotic and antihypertensive new materials (Park, In, Kim, Thesis, 18: 81-87, 1929). In addition, as the administration of safflower seed powder has been reported to have an effect of improving fracture healing and lipid metabolism, functional foods using the safflower seeds have been actively developed (Kim, Jun-Hwan, et al., Korean Journal of Food Science and Nutrition, 27 (3): 698-704, 1998 ; Kim, Joon Hwan et al., The Korean Journal of Food and Nutrition, 28 (3): 625-631, 1999). Recently, the production and consumption of safflower seed has increased remarkably as it is known among the civilians that there is a remarkable effect on bone diseases such as osteoporosis and fracture. However, until now, there have been few studies that scientifically investigated the effect of safflower seeds on bone diseases.

On the other hand, safflower oil cake obtained as a by-product of safflower oil is important as a source of protein and dietary fiber, and also contains antioxidative polyphenol compounds such as serotonin, lignan and flavonoid, has attracted attention as a therapeutic agent (Sakamura S. et al, Agric Biol Chem, 44 (12):........ 2951-2954, 1980; Zhang, HL et al, Chem Pharm Bull, 44 (4) : 874-876, 1996; Zhang, HL et al ., Chem. Pharm. Bull . 45 (2): 1910-1914,1997). However, safflower oil contains lignans and steroids that exhibit bitter taste, diarrhea and toxicity, and so far it has been limited in part to use as food or feed (Palter R. Lundin, RE, Phytochem., 9: 2407-2409, 1970; Palter, R. et al, Phytochem, 11: 2871-2874, 1972; Palter, R. et al, Phytochem, 11:. 2327-2328 expression, 1972), safflower as due to the safflower visible... There was a difficulty in using by using. Therefore, safflower seeds are roasted to some extent to induce antioxidant activity and excellent aroma (Roh JS et al. , 8 (2): 88-92, 1999, Choi SW, J. Food Sci., Nutr. , 1999).

Thus, studies on the separation of physiologically active substances from safflower flowers and seeds and their physiological and pharmacological actions have been carried out, and studies on the development of natural pigments and processed foods using safflower have been carried out. However, There have been few studies on the effect and the separation of active ingredients. Therefore, it is urgent to scientifically prove the effect of safflower seeds on bone diseases.

Recent epidemiological reports and clinical studies report that antioxidant phytochemicals contained in various cereals and fruit and vegetables are the next-generation therapies that will surpass vitamins, as well as cancer, as well as cancer (including angina, hypertension, diabetes, and dementia) (Hasler, CM, Food Technology, 52 (11): 63-70, 1998). It has been shown to be effective in preventing and treating degenerative chronic diseases. Among these phytochemicals, in particular, isoflavonoids and lignans are the most recently attracted attention (Adlercreutz H., Bailliers Clin. Endocrinol. Metab. 12 (4): 605-623, 1998). These components are diphenol-based compounds, whose structure and molecular weight are similar to steroid estrogen, so-called phytoestrogen. Phytoestrogens have antiestrogen and estrogenic activity and prevent hormone-dependent breast cancer, prostate cancer and colon cancer as well as preventing heart disease, hypertension and osteoporosis, which are common in postmenopausal women (Dwyer, JT et al., J. Am. Diet Assoc., 94: 739-743, 1994. Tang, BY and Adams, NR J. Endocrinol. , 85: 291-297, 1980; et al., J. Steroid Biochem. Mol. Biol. , 44: 147-153, 1993). In addition, phytoestrogens have been reported to have antioxidant effects, hyperlipemia and atherosclerosis (Franke, AA et al., J. Agric. Food Chem. , 42: 1905-1913, 1994). To date, the most well-known phytoestrogen compounds are the isoflavones compounds genistein, daidzein and flaxseed lignan compounds secoisolariciresinol, Mathai is Lecinol (matairesinol) (Lundh, TJ et al, J. Assoc Off Anal Chem, 71:........ 938-941, 1988; Aldercreutz, H. et al, J. Steroid Biochem Mol Biol ., 44: 147-153, 1993; Hutchins, AM et al, J Am Diet Assoc, 95: 769-774, 1995; Hutchins, AM et al, J. Am Diet Assoc, 95:...... 545 -551, 1995). These compounds are known to play an estrogenic and antiestrogenic role as they are transformed into mammalian isoflavones and lignan components by human intestinal microorganisms and occupy estrogen binding sites competitively with the 17β-estradiol in vivo (Miksikek , RJ, J. Steroid Biochem. Mol. Biol. , 49: 153-160, 1994). According to epidemiological studies, Asians who consume a lot of soybeans are less likely to develop estrogen-dependent cancers than those of Westerners, and the Japanese who consume a lot of processed foods made from soybeans have a lower incidence of uterine cancer and breast cancer than other advanced countries (Adlercreutz, H. et al., Lancet, 342: 1209-1210, 1993; Kirkman, LM et al., Nutr. Cancer 24: 1-12, 1995). And soybean isoflavone compounds have the effect of preventing fractures and osteoporosis caused by a lack of estrogen after menopause (Draper, CR et al., J. Nutr. , 127: 1795-1799, 1997; Anderson, JJB and Garner, SC Nutr Res ., 17 (10): 1617-1632, 1997).

On the other hand, flaxseed, which is important as a flow seed resource, has been shown to not only prevent female hormone-dependent cancers, but also to affect the metabolism of female hormones to delay postmenopause (Orcheson, L. et al., Fed J. Am. Soc. Exp. Biol., J., 7: 291-296, 1993; Phipps, WR et al., J. Clin. Endocrinol. Metab., 77: 1215-1219, 1993).

Thus, plants containing phytoestrogen, such as isoflavonoids and lignans, can be greatly appreciated as useful medicines for treating osteoporosis and cardiovascular diseases as well as cancers such as breast cancer, prostate cancer and colon cancer, In particular, it has been reported that phytoestrogen compounds are abundantly contained in plant seeds such as soybean and flax (Adlercreutz H., Boca Raton, Fla: CRC Press Inc., 137-196, 1991; Thompson, LU et al., Nutr Cancer 16: 43-52, 1991).

Therefore, it is possible that the effect of treatment of osteoporosis and fracture of safflower seed, which is known in the private sector, and the lengthening of menstrual period of women are attributable to phytoestrogen compounds. Surprisingly, surprisingly, roasted safflower seeds contain mallowiresin, a lignan ingredient known as an antioxidant phytoestrogen compound, hydroxyarctigenin, a similar ingredient, and tylli, a flavonoid component similar to soybean genistein, Tilianine and acacetin (Choi et al., J. Food Sci. And Nutr. , 4 (4): 25-29, 1999). In addition, antioxidant serotonin, which is abundant in safflower seed, is expected to have a significant impact on bone formation.

Separation of serotonin, lignans and flavonoids from safflower seeds has been reported by Sakamura Sadao ( Agric. Biol. Chem ., 44 (12): 2951-2954, 1980), but this method uses water and ethyl acetate The ethyl acetate-soluble fraction obtained was subjected to silica gel chromatography to sequentially separate the above three substances. As a result, the above three compounds not dissolved in ethyl acetate were excluded, thereby lowering the extraction effect. Further, the silica gel column chromatography alone There is a disadvantage in that it is difficult to effectively utilize each component of serotonin, lignan and flavonoid compound because it can not be effectively separated. Also, the effect of each component on skeletal formation is not known at all.

Therefore, the present inventors isolated high purity serotonin, lignan and flavonoid components from roasted safflower seeds and cultured them by cell culture. Of the six polyphenols contained in roasted safflower seeds, lignans and serotonin components are known to be 17 beta - promoting bone formation similar to estradiol and genistein components.

Accordingly, an object of the present invention is to provide a method for extracting serotonin, lignan and flavonoid components, which is useful for treating bone formation from roasted safflower seeds and treating fractures and osteoporosis.

The above object of the present invention is achieved by extracting 60%, 80% and 100% water-soluble methanol solution from roasted safflower seeds and then concentrating the 60%, 80% and 100% methanol extracts and further adding them to Sephadex LH-20 column and fraction- The serotonin, lignan and flavonoid components were separated by liquid chromatography, and the effects of the components isolated from the safflower seeds on bone formation promoting action were investigated.

The configuration and operation of the present invention will be described below.

1 is a graph showing an aliquot-high-speed liquid chromatogram of six phenolic compounds isolated from roasted safflower of the present invention.

In the present invention, fat is removed from roasted safflower seeds, and then the mixture is extracted with hot water, followed by solubilization with 60% water-soluble methanol, adsorbed on a Diaion HP-20 column, extracted with 60%, 80% and 100% aqueous methanol solutions, Followed by Fusepadex LH-20 column chromatography to isolate the polyphenolic compounds serotonin, lignan and flavonoid components; The polyphenol compound is extracted from safflower seed in the same manner as described above, followed by solubilization by adding 80% water-soluble methanol and adsorbing it on a polyamide C-200 column to purely isolate the polyphenol compound; In the same manner as above, the polyphenol compound was extracted from safflower seed, solubilized by adding 80% water-soluble methanol, passed through a Sep-Pak C ₁₈ column and purified by preparative-high performance liquid chromatography to purely isolate the polyphenol compound. And examining the effect of the purely isolated component on bone formation promoting action.

The preferred concentration of methanol used in the present invention is 50-120% aqueous solution, most preferably 60-100% aqueous solution. In this case, ethanol may be used as an extraction solvent instead of methanol.

In the present invention, the temperature is preferably 70 to 90 ° C, and most preferably 80 ° C.

Hereinafter, specific methods of the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited to these Examples.

Example 1

In this Example, 100 g of dried safflower seed was roasted at 200-250 ° C for 5 minutes and then pulverized. Then, 1 L of 100% normal nucleic acid was added thereto, and the oil was removed by extraction in hot water for 2 hours. The extraction operation is repeated twice to remove the fat, and 1 L of 60% water-soluble methanol is added to the remaining residue, followed by extraction and filtration for 2 hours in a hot water bath. The above extraction operation was repeated twice. The methanol extract was concentrated to 200 mL, and then 500 mL of 100% normal nucleic acid was added thereto to remove oil floating in the nucleic acid layer. Then, 100 mL of 60% water-soluble methanol was added to the methanol extract, followed by filtration. The filtrate was transferred to a 5 cm diameter column packed with 500 g of Diaion HP-20 (Mitsubishi Chemical, Japan) Water-soluble methanol, 40% water-soluble methanol, 60% water-soluble methanol, 80% water-soluble methanol and 1 L of a 100% methanol solution were respectively eluted. Here, 0.2 g of a dry substance obtained by reducing and concentrating the 60% water-soluble methanol extract was dissolved in methanol, transferred to a column having a diameter of 2 cm filled with 100 g of Sephadex LH-20 (Parmasia, Sweden) the eluted sugar divided into 100 fractions by 5 mL serotonin reel in Peru N- (N-feruloylserotonin, molecular weight 352, melting point 118 ℃) and 86 mg N- (para-one as circles) serotonin (N- (p -coumaroyl ) serotonin, molecular weight 322, melting point 194 ° C), respectively (yield: 86 and 73 mg%, respectively). Then, 45 mg of 80% methanol extract was subjected to Sephadex LH-20 column chromatography to obtain 7 mg of matairesinol (molecular weight 358, melting point 94 ° C) and 8 mg of 8'-hydroxyactyzine (8'-hydroxyarctigenin, molecular weight 388, melting point 168 ° C) (yield: 7 and 25 mg%, respectively). 76 mg of 100% methanol extract was subjected to Sephadex LH-20 column chromatography in the same manner as described above to give 11 mg of tilianine (molecular weight 446, melting point 125 ° C) and acacetin (molecular weight 284, melting point 255 ℃) (yield: 11 and 52 mg%, respectively, based on dried safflower seed).

Example 2

100 g of roasted safflower seed was pulverized, and 1 L of 80% water-soluble methanol was added thereto, followed by extraction and filtration for 2 hours in a hot water bath. The extraction procedure was repeated three times. The methanol extract was concentrated to 200 mL, and then 500 mL of 100% normal nucleic acid was added to separate the nucleic acid layer and the methanol layer to remove the oil extracted in the nucleic acid. Then, the methanol extract was reduced to 100 mL, concentrated, filtered, and transferred to a column having a diameter of 5 cm filled with 100 g of Polyamide C-200 (Hikyu Pure Chemical Industries, Japan) Methanol, 1% aqueous solution of 60% water-soluble methanol and 100% methanol, respectively. In the 20% methanol fraction, 54 mg of the flavonoid mixture, 38 mg of the lignan mixture in the 60% methanol fraction and 0.15 g of the serotonin mixture in the 100% methanol fraction were obtained, respectively. Then 54 mg of the flavonoid fraction was dissolved in methanol and transferred to a column of 2 cm in diameter packed with 50 g of Sephadex LH-20 (Parmasia, Sweden). Each fraction was divided into 100 fractions , 7 mg of tilianine (molecular weight 446, melting point 125 ° C) and 37 mg of acacetin (acacetin, molecular weight 284, melting point 255 ° C) were obtained, respectively. 38 mg of the lignan mixture was subjected to Sephadex LH-20 column chromatography to obtain 5 mg of matairesinol (molecular weight 358, melting point 94 ° C) and 8'-hydroxyarctigenin , Molecular weight: 388, melting point: 168 캜) (yield: 5 and 20 mg%, respectively). Finally, 0.15 g of the serotonin mixture was subjected to Sephadex LH-20 column chromatography in the same manner as described above to obtain 76 mg of N-feruloylserotonin (molecular weight 352, melting point 118 캜) one to Kumar) serotonin (N- (p -coumaroyl) serotonin, molecular weight 322, respectively, to give the 59 mg melting point 194 ℃) (yield based on each of 76 and 59 mg%, dry safflower).

Example 3

After roasting 100 g of roasted safflower seeds, 1 L of 100% normal nucleic acid was added thereto, and the oil was extracted by extraction from hot water for 3 hours. The above extraction operation was repeated twice, and 80% water-soluble methanol was added to the residue, followed by extraction and filtration for 2 hours in a hot water bath. The above extraction procedure was repeated three times and the methanol extract solution was reduced to 100 mL and concentrated. 500 mL of ethyl acetate was added thereto, and the mixture was divided into an ethyl acetate layer and an aqueous layer, and 1.7 g of an ethyl acetate extract was obtained. This was dissolved in 10 mL of 80% water-soluble methanol, passed through Sep-Pak C ₁₈ cartridges (Waters, USA), and the resulting extract was diluted with methanol and then separated and purified by preparative high performance liquid chromatography. 78 mg of N-feruloylserotonin (molecular weight 352, melting point 118 캜), 67 mg of N- (para-coumaroyl) serotonin (N- p -coumaroylserotonin, molecular weight 322, melting point 194 캜) 5 mg of matairecinol (molecular weight 358, melting point 94 ° C) and 24 mg of 8'-hydroxyarctigenin (molecular weight 388, melting point 168 ° C) (yield: 67 mg, based on dried safflower seed) 10 mg of tilianine (molecular weight 446, melting point 125 ° C) and 47 mg of acacetin (molecular weight 284, melting point 255 ° C) were obtained respectively (yield: 5 and 24 mg%, based on dried safflower seed) 10 and 47 mg%, based on dried safflower seed).

The conditions for preparative-high performance liquid chromatography were as follows. Waters Delta prep 4000 high performance liquid chromatography; Column, RCM Novapak C ₁₈ (25 mm x 100 mm x 2 cartridges); Ultraviolet detector (concentration gradient elution for 60 min with UV ₃₀₀ solvent B). The preparative-high-speed liquid chromatograms of the six compounds thus separated were as shown in Fig. 1, and their detailed ultraviolet, infrared and mass spectral data were as shown in Table 1. Hydrogen-nuclear magnetic resonance data and carbon-nuclear magnetic resonance data were as shown in Tables 2a and 2b and Tables 3a and 3b, respectively.

Serotonin is (a) N-perroyl serotonin or (b) N- (para-coumaroyl) serotonin depending on the substituent, and the general formula is the same as the following general formula (I);

(a) N-perroyl serotonin (S1): wherein R ₁ = H, R ₂ = OCH ₃ , R ₃ = H.

(b) N- (para-coumaroyl) serotonin (S2): wherein R ₁ = H, R ₂ = H, R ₃ = H.

The lignan is (a) matairecinol or (b) 8'-hydroxyactigene according to the substituent, and the general formula is as shown in the following general formula (II);

(a) mathiasecinol (L1): wherein R ₁ = H, R ₂ = H, and R ₃ = H.

(b) 8'-hydroxyactigene (L2): wherein R ₁ = CH ₃ , R ₂ = H, R ₃ = OH.

Depending on the substituent, the flavonoid is either (a) tilylene or (b) acacetin and the general formula is as in formula (III) below;

(a) Tilylene (F1): wherein R ₁ = glucose and R ₂ = CH ₃ .

(b) Acacetin (F2): wherein R ₁ = H and R ₂ = CH ₃ .

Ultraviolet, infrared and mass spectrometric data of six phenolic compounds isolated from roasted safflower seeds S1 S2 L1 L2 F1 F2 UV _{? Max} 288, 315 290, 318 230, 280 228, 278 268, 324 270, 303 (s), 328 IR? _Max (cm ^-1 ) 3400, 3180, 1662, 1600, 1589, 1529 3380, 3378, 1650, 1600, 1580, 1530 3412, 1760, 1620, 1520 3400, 1780, 1600, 1520, 1470 3180, 1670, 1620, 1520, 840, 824 3170, 1660, 1610, 1520, 840, 820 EI-MS (m / z ) 352 [M < + >] 322 [M < + >] 358 [M < + >] 388 [M < + >] 3446 [M +] < 284 [M +] [week] S1: N-perrolyl serotonin, S2: N- (p-Coumaryl) serotonin, L1: mathiresicinol, L2: 8'-hydroxyactigensin, F1: tilylene, F2: acacetin.

Hydrogen-nuclear magnetic resonance data of six phenolic compounds isolated from roasted safflower seeds S1 S2 L1 L2 F1 F2 2 7.02 (s) 7.02 (s) 6.55 (d, J = 2.0) 6.69 (d, J = 3.0) 3 6.67 (s) 6.54 (s) 4 6.95 (d, J = 2.0) 6.95 (d, J = 1.5) 5 6.68 (d, J = 8.0) 6.85 (d, J = 7.5) 6 6.66 (dd, J = 2.5, 8.5) 6.65 (dd, J = 2.5, 7.5) 6.51 (dd, J = 8.0, 2.0) 6.56 (dd, J = 8.0, 2.0) 6.33 (d, J = 2.0) 6.10 (d, J = 2.0) 7 7.15 (dd, J = 8.5) 7.15 (dd, J = 2.0, 6.5) 2.50 (m) 3.11 (d, J = 14.0) 2.85 (d, J = 14.0) 8 2.66 (m) 2.48 (m) 6.65 (d, J = 2.0) 6.32 (d, J = 2.0) 9 3.92 (dd, J = 8.3, 7.5) 4.15 (dd, J = 8.8, 7.5) 3.84 (d, J = 1.5) 3.97 (dd, J = 8.0, 1.5) 10 2.92 (t) 2.92 (t) 11 3.57 (t) 3.56 (t) NOTE The coupling constant ( J ) in parentheses is in Hz units s; Singlet d; Doublet dd; Double doublet t; Triplet m; Multiplet ^* Determined in DMSO

Hydrogen-nuclear magnetic resonance data of six phenolic compounds isolated from roasted safflower seeds S1 S2 L1 L2 F1 F2 2' 7.11 (d, J = 2.0) 7.38 (dd, J = 2.5, 8.5) 6.66 (d, J = 2.0) 6.85 (d, J = 2.0) 7.92 (d, J = 9.0) 7.91 (d, J = 9.0) 3 ' 6.78 (dd, J = 2.5, 8.5) 7.08 (d, J = 9.0) 7.07 (d, J = 9.0) 5 ' 6.78 (d, J = 8.0) 6.78 (dd, J = 2.5, 8.5) 6.70 (d, J = 8.0) 6.85 (d, J = 8.0) 7.08 (d, J = 9.0) 7.07 (d, J = 9.0) 6 ' 7.01 (d, J = 2.0) 7.38 (dd, J = 2.5, 8.5) 6.57 (dd, J = 8.0, 2.0) 6.69 (dd, J = 4.0, 1.5) 7.92 (d, J = 9.0) 7.91 (d, J = 9.0) 7 ' 7.43 (d, J = 16.0) 7.44 (d, J = 16) 2.81 (dd, J = 14.0, 7.0) 2.88 (dd, J = 14.0, 5.5) 2.80 (dd, J = 14.0, 5.0) 8' 6.41 (d, J = 15.5) 6.39 (d, J = 16) 2.66 (m) NH ^* 9.75 (s) 9.78 (s) CONH ^* 7.26 (t) 7.43 (t) OCH ₃ 3.87 3.78, 3.77 3.78, 3.77, 3.76 3.88 3.87 Glu-1 4.78 (d, J = 7.34) Glu 2-6 3.18-3.77 (m) NOTE The coupling constant ( J ) in parentheses is in Hz units s; Singlet d; Doublet dd; Double doublet t; Triplet m; Multiplet ^* Determined in DMSO

Carbon - nuclear magnetic resonance data (methanol - _d6 ) of six phenolic compounds isolated from roasted safflower seeds S1 S2 L1 L2 F1 F2 One 131.45 133.40 2 124.27 124.26 113.26 113.82 164.52 164.25 3 112.66 112.37 149.04 150.50 103.83 103.56 4 103.51 103.51 146.38 148.83 182.52 182.34 5 151.15 151.14 116.08 113.14 162.19 161.89 6 112.37 112.51 122.23 122.17 102.64 99.57 7 112.50 112.65 38.90 41.93 161.23 163.67 8 133.13 132.22 42.53 44.42 97.89 94.39 9 129.44 129.44 72.94 71.92 160.08 160.12 10 26.46 26.47 108.89 104.76 11 41.48 41.47 [Note] S1; Peruroyl serotonin, S2; N- (para-cumarnoyl) serotonin, L1; Matairecinol, L2; 8 ' -hydroxyactizenine, F1; Tilylene F2; Axessin

Carbon - nuclear magnetic resonance data (methanol - _d6 ) of six phenolic compounds isolated from roasted safflower seeds S1 S2 L1 L2 F1 F2 One' 128.32 127.76 130.77 128.20 125.38 124.98 2' 111.52 130.55 113.89 114.92 129.45 129.11 3 ' 149.27 116.69 149.00 149.09 115.91 115.58 4' 149.80 160.09 146.19 146.69 162.87 162.40 5 ' 116.43 116.69 116.16 116.01 115.79 115.58 6 ' 123.21 130.55 123.04 124.06 129.41 129.12 7 ' 141.96 141.71 35.35 32.01 8' 118.88 118.56 47.75 77.39 9 ' 181.67 180.57 CO 169.22 169.28 OCH ₃ 56.37 56.33, 56.28 56.40, 56.52, 56.72 56.08 56.04 Glu-1 101.82 Glu-2 73.12 Glu-3 76.54 Glu-4 72.43 Glu-5 76.82 Glu-6 61.13 [Note] S1; Peruroyl serotonin, S2; N- (para-cumarnoyl) serotonin, L1; Matairecinol, L2; 8 ' -hydroxyactizenine, F1; Tilylene F2; Axessin

Experimental Example 1: Effect of the isolated polyphenol compound on bone formation promoting action

In the present experimental example, the polyphenol compounds obtained in Examples 1, 2 and 3, namely, perrolyl serotonin and coumaryl serotonin, which are serotonin components, matairecinol and hydroxyactigenein, lignan components, tyllian, In order to investigate the effect of cetin on the bone formation promoting action, the osteoblast cell line, bone cell forming bone, was measured using ROS 17 / 2.8. (100 IU penicillin, 100 mg streptomycin, 250 ng amphotericin, Gibco BRL) supplemented with 10% fetal bovine serum and 1% antibiotic-antimicrobial agent without phenol red (MTT, 3- (4,5-dimethyl-l, 3-dihydro-lH-pyrazol-3-yl) -ethanone was prepared by incubating in Dulbecco's Modified Eagle medium and incubating in a 5% CO ₂ incubator maintained at 37 & thiazol-2-yl) -2,5-diphenyltetrazolin bromide) by the MTT assay method based on the metabolic reduction. At this time, 17β-estradiol (17β-estradiol), which is known to promote osteocyte proliferation, and genistein isolated from soybean were set as a positive reference in order to determine whether the ingredient of safflower seed promotes bone formation. And control without addition of sample. The MTT assay method was as follows. ROS cells were seeded in 96 wells at a density of 5 × 10 ³ cells. To eliminate the effect of fetal bovine serum, the culture medium was changed with 0.5% estrogen deficiency-fetal bovine serum for 24 hours. 2.5% estrogen deficiency - Six phenolic compounds isolated from roasted safflower seeds in addition to estrogen and genistein under fetal serum conditions were stimulated for 48 hours at 10 ^-6 to 10 ^-15 molar concentrations. To each well, 50 μl of a solution of Melti (1.1 mg / ml) was added and cultured at 37 ° C for 4 hours. 150 μl of dimethylsulfoxide was added to dissolve the formazan crystal, which was a violet precipitate formed by the emittie, and the absorbance of each well was measured using a microplate reader to determine the absorbance of 540 nm. At this time, the bone formation promoting action of each component was expressed as a relative value to the control (control). The results are shown in Table 4 below.

Effects of six polyphenols isolated from roasted safflower seeds on the proliferation of osteoblast-like cells (ROS 17 / 2.8) compound Concentration (M) Control 10 ^-15 10 ^-12 10 ^-9 10 ^-6 N-Feruloylserotonin < / RTI > 100 105 107 100 98 N- ( p- coumaroyl) serotonin < / RTI > 100 105 108 96 113 Matairesinol 100 103 116 130 158 8'-Hydroxyarctigenin < / RTI > 100 98 80 102 95 Tilianine 100 84 80 95 112 Acacetin 100 115 112 120 126 17 beta-estradiol _{^{* (17β-Estradiol, E 2}} ) 100 128 126 138 128 Genistein ^* 100 110 113 111 117

As shown in the above Table 4, 17 beta-estradiol (E ₂ ) already exhibited a cell proliferation effect of about 130% at a low concentration of 10 ^-15 M, and a physiological concentration of about 10 ^-9 M to about 140 %, Which promoted proliferation of bone cells in a dose dependent manner. Soybean genistein also increased ROS osteoprotein proliferation by up to 115% in a dose-dependent manner, but showed lower efficacy than estradiol (E ₂ ). All six polyphenols isolated from roasted safflower seeds showed a dose - dependent tendency to promote the proliferation of ROS bone cells. In particular, matairecinol and acacetin compounds showed cell proliferation rates as high as those of estradiol (E ₂ ) and genistein Respectively.

From the results shown in Table 4, it was found that among the six polyphenols isolated from roasted safflower seeds, matairecinol and acacetin can act as substitutes for 17β-estradiol and genistein, which are estrogen compounds promoting bone formation.

The roasted safflower seeds obtained by defatting roasted safflower seeds with a nucleic acid were extracted with hot water with methanol, filtered and concentrated, and subjected to column chromatography using Diaion HP-20 and polyamide C-200 The serotonin, lignan and flavonoid components purified by column chromatography and fractionation-high performance liquid chromatography on the obtained 60%, 80% and 100% methanol extracts with Sephadex LH-20 were excellent in the treatment of osteoporosis and fracture It is a very useful invention in the raw material industry of functional foods and medicines for the treatment of osteoporosis and fracture.

Claims (5)

(1) a step of pulverizing and drying the dried safflower seeds, (2) a step of adding methanol 50% to 80% aqueous solution to the pulverized safflower obtained by degreasing in the first step, heating to 80 ° C and extracting, (3) carrying out column chromatography using Diaion HP-20 as the concentrate prepared in the second step, and (4) extracting the extract prepared in the third step using a Sephadex LH-20 column A method for extracting a polyphenol compound composed of perlouryl serotonin derived from safflower seed, coumaryl serotonin, mattiirencinol, hydroxyactigencine, tilianin, and acacetin. 2. The method according to claim 1, wherein the concentrate is subjected to column chromatography using polyamide in the third step, characterized in that perlouryl serotonin derived from safflower seed, coumaryl serotonin, mattiirencinol, , Tilianin, and acacetin. The method according to claim 1, wherein the third step is a step of adding ethyl acetate to the concentrate, fractionating the mixture into ethyl acetate and water, concentrating the ethyl acetate layer under reduced pressure, and concentrating the ethyl acetate layer in the fourth step, The method of extracting a polyphenol compound composed of perlouryl serotonin, coumaryl serotonin, mathiirencinol, hydroxyactigencine, tilianin, and acacetin derived from safflower seeds, which is characterized by separating pure water by using water. delete A composition for promoting bone formation, characterized in that it contains at least one compound selected from the group consisting of peruroyl serotonin, coumaryl serotonin, mathiirecinol, hydroxyactinen, tilianin and acacetin derived from safflower seeds