KR20010034952A - Method for Extraction, Isolation and Identification of Serotonins, Lignans and Flavonoids Improved Bone Formation from Safflower(Carthamus tinctorious L.) Seeds - Google Patents

Abstract

PURPOSE: A method for extracting serotonin, lignan and flavonoid by extracting parched safflower seed in an aqueous methanol solution, concentrating the methanol extracts and performing column chromatography is provided, thereby producing raw material for functional foods and medicines used for the treatment of osteoporosis and a bone fracture. CONSTITUTION: The method for extracting a polyphenol compound from safflower comprises the steps of: grinding dried safflower seed and defatting; adding 50 to 80% aqueous methanol to the safflower powder, heating at 80deg.C, extracting and concentrating under pressure; performing column chromatography of the concentrate using dianion HP-20; and separating erotonin, lignan and flavonoid components using sephadex LH-20 column.

Description

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

The present invention relates to a method for extracting serotonin, lignan and flavonoid components that promote bone formation from safflower seeds. More specifically, serotonin components such as Peruroyl serotonin and coumaryl serotonin, which are useful for the prevention and treatment of fractures and osteoporosis by promoting bone formation from roasted safflower seeds, lignan components such as matyrethinol and hydroxyactinine and tilianin And a method for extracting flavonoid components such as acecetin

The safflower (Safflower, Carthamus tinctorious L.) is an annual vegetation belonging to the compositae and is native to Afghanistan's mountainous regions or Ethiopia (Lee Chang-bok, Korea Plant Book, 1980). Safflower flowers contain water-soluble yellow pigments and insoluble red pigments and have been widely used as dyes since ancient times (Yook Chang-soo, Korean Herbology, 1981). It has been widely used as a prescription (herbology, Lee Sang-in, 1980).

On the other hand, safflower seed has been used for soothing and bleeding in Korea, China, and Japan (biopharmaceutical, Han Dae-seok, 1995), and is a useful resource plant that is cultivated for cooking oil production, especially in the United States and India. Modern Herbology, 1975). Safflower seed oil contains abundant natural minerals such as calcium, aluminum and bromine, including vitamin E, tocopherol and vitamin F, linolenic acid (Ju et al., Health food, 1995). In particular, safflower seeds contain a large amount of unsaturated fatty acids, which are not only nutritionally important, but also have high linolenic acid content, which has attracted much attention as an anti-thrombotic and antihypertensive new material. Thesis, 18: 81-87, 1929). In addition, the recent discovery that the administration of safflower seed powder has the effect of improving the healing of bone and lipid metabolism, and the development of functional foods using the same has been actively conducted. Kim, Jun-Hwan et al., Korean Journal of Food and Nutrition, 28 (3): 625-631, 1999). Recently, the production and consumption of safflower seeds have been known to be surprisingly effective for bone diseases such as osteoporosis, fractures, and so on. However, few studies have scientifically examined the effects of safflower seed treatment for bone diseases.

Safflower oil cake, obtained by squeezing safflower seed oil and being a by-product, is not only important as a source of protein and dietary fiber, but also contains antioxidant polyphenol compounds such as serotonin, lignans, and flavonoids, resulting in hypertension, hyperlipidemia, and arteriosclerosis. It is attracting 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, which have bitterness, diarrhea and toxicity, so that their use as food or feed has been partially restricted (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, 1972) Difficult to use for diet. Therefore, safflower seeds are currently roasted to alleviate the above side effects, and at the same time, some safflower seeds are roasted and processed to induce antioxidant activity and the production of excellent fragrances (Roh JS et al., 8 (2): 88-92, 1999; Choi SW, J. Food Sci. & Nutr., 1999).

As described above, studies on the separation of bioactive substances from safflower flowers and seeds and their physiological and pharmacological effects, as well as the development of natural pigments and processed foods using safflower, have been conducted. There is little research on the effects and separation of active ingredients. Therefore, there is an urgent need to scientifically prove the effects of safflower seed on bone diseases.

Recent epidemiologic and clinical studies report that antioxidant phytochemicals in cereals and fruit vegetables are the next generation of medicines to outperform vitamins, including cancer, as well as angina, high blood pressure, diabetes and dementia. It has been shown to have an excellent effect in preventing and treating degenerative chronic diseases (Hasler, CM, Food Technology, 52 (11): 63-70, 1998). In particular, the most attention of these plant chemicals is the isoflavonoids and lignans components (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 estrogen activity, which has been shown to prevent hormone-dependent breast, prostate and colon cancers, as well as to prevent heart disease, hypertension and osteoporosis, which occur in postmenopausal women. (Dwyer, JT et al., J. Am. Diet.Assoc., 94: 739-743, 1994; Tang, BY and Adams, NRJ Endocrinol., 85: 291-297, 1980; Aldercreutz, H. et al. , J. Steroid Biochem.Mol. Biol., 44: 147-153, 1993). In addition, phytoestrogens have been reported to have antioxidant effects, hyperlipidemia, and atherosclerosis treatment effects (Franke, A. A. et al., J. Agric. Food Chem., 42: 1905-1913, 1994). To date, the best known phytoestrogens are the soy isoflavones, genistein, and the lignan compounds of daidzein and flaxseed, secoisolariciresinol, 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 the role of estrogens and anti-estrogens by being transformed into mammalian isoflavones and lignans by human intestinal microorganisms to occupy estrogen binding sites competitively with 17β-estradiol in vivo (Miksikek , RJ, J. Steroid Biochem.Mol. Biol., 49: 153-160, 1994). Epidemiological studies have shown that Asians who consume more soybeans have lower incidence of estrogen-dependent cancer than Westerners, and Japanese who consume more processed foods made from soybeans have lower incidence of uterine and breast cancer than other developed countries. The fact is revealed (Adlercreutz, H. et al., Lancet, 342: 1209-1210, 1993; Kirkman, LM et al., Nutr. Cancer 24: 1-12, 1995). Soybean isoflavone compounds have been shown to be effective in preventing fractures and osteoporosis caused by estrogen deficiency 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 an important source of oilseed seeds, has been shown to not only prevent female hormone-dependent cancer, but also affect the metabolism of female hormones and delay menopause (Orcheson, L. et al., Fed). Am. Soc.Exp. Biol. J., 7: 291-296, 1993; Phipps, WR et al., J. Clin. Endocrinol. Metab., 77: 1215-1219, 1993).

As such, plants containing phytoestrogens, such as isoflavonoids and lignans, can be widely spotted as useful medicines for treating osteoporosis and cardiovascular diseases as well as cancers such as breast cancer, prostate cancer and colon cancer. In particular, plant seeds, such as soybean and flax seed, have been reported to contain a lot of phytoestrogens (Adlercreutz H., Boca Raton, Fla: CRC Press Inc., 137-196, 1991; Thompson, LU et al., Nutr). Cancer 16: 43-52, 1991).

Therefore, the effects of osteoporosis and fracture treatment and menstrual period prolongation of safflower seed, which are known in the public, may be due to phytoestrogens. Surprisingly, roasted safflower seeds contain lignan, a materinolol known as an antioxidant phytoestrogen compound, hydroxyarctigenin, a similar substance, and thylli, a flavonoid component similar to genistein in soybeans. Tilianine and acecetin were found (Choi, SW et al., J. Food Sci. And Nutr., 4 (4): 25-29, 1999). In addition, the antioxidant serotonin component present in large amounts in safflower seeds was expected to have a significant effect 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 is characterized by water and ethyl acetate in methanol extracts of defatted safflower seeds. The ethyl acetate soluble portion obtained by adding silica gel chromatography was subjected to silica gel chromatography to separate the above three substances sequentially. As the above three mixtures not dissolved in ethyl acetate were excluded, the extraction effect was low, and silica gel column chromatography alone. Since the method cannot effectively separate each component of the serotonin, lignan and flavonoid compounds, there is a disadvantage that it is difficult to use practically. Moreover, the effect of each component on skeletal formation is not known at all.

Therefore, the present inventors have isolated high-purity serotonin, lignan and flavonoid components from roasted safflower seeds and established them by cell culture. Among the six polyphenols contained in roasted safflower seeds, lignan and serotonin components are known as phytoestrogens. The present invention was completed by confirming that it promotes bone formation similarly to estradiol and genistein components.

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

The object of the present invention is extracted from roasted safflower seed with 60%, 80% and 100% aqueous methanol solution, and then concentrated to 60%, 80% and 100% methanol extract, and then Sephadex LH-20 column and preparative-fast Liquid chromatography was used to separate the serotonin, lignan and flavonoid components and was accomplished by investigating the effects of the components isolated from safflower seed on promoting bone formation.

Hereinafter, the configuration and operation of the present invention.

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

The present invention removes fat from roasted safflower seed, extracts with hot water, solubilizes by adding 60% water-soluble methanol, adsorbs to DIION HP-20 column, extracts with 60%, 80% and 100% water-soluble methanol solution, and concentrates the extract. Sephadex LH-20 column chromatography to separate the polyphenol compounds serotonin, lignan and flavonoid components; Extracting the polyphenolic compound from safflower seed in the same manner as described above, solubilizing by adding 80% water-soluble methanol and adsorbing the polyamide C-200 column to purely separating the polyphenolic compound; Extracting the polyphenolic compound from safflower seed in the same manner as described above, solubilizing by adding 80% water-soluble methanol, passing through Sep-Pak C ₁₈ cartridge, and preparatively separating the polyphenolic compound by preparative high-speed liquid chromatography; Investigating the effect on the bone formation promoting action of the purely separated components.

Preferred concentrations of methanol used in the present invention are 50 to 120% aqueous solution, most preferably 60 to 100% aqueous solution. In this case, ethanol may be used instead of methanol as the extraction solvent.

Preferred temperature in the hot water extraction step in the present invention is 70 to 90 ℃ and most preferably 80 ℃.

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

Example 1

In this example, 100 g of dry safflower seed was roasted at 200-250 ° C. for 5 minutes, pulverized, and then 1 L of 100% normal nucleic acid was added thereto, followed by extraction in a boiling water for 2 hours to remove oil. The extraction operation was repeated twice, and fat was removed. Then, 1 L of 60% aqueous methanol was added to the remaining residue, followed by extraction and filtration for 2 hours in a boiling water. The extraction procedure was repeated twice, and the methanol extract obtained under reduced pressure was concentrated to 200 mL, and then 500 mL of 100% normal nucleic acid was added thereto to remove the oil rising to the nucleic acid layer. Then, 100 ml of 60% aqueous methanol was added to the methanol extract, mixed, filtered, and the filtrate was transferred to a column of 5 cm diameter filled with 500 g of Diaion HP-20 (Mitsubishi Chemical, Japan), and water, 20% Water-soluble methanol, 40% water-soluble methanol, 60% water-soluble methanol, 80% water-soluble methanol and 1 L of 100% methanol solution were each eluted in sequence. Here, 0.2 g of a dry substance obtained by depressurizing and concentrating a 60% aqueous methanol extract was dissolved in methanol, and then transferred to a 2 cm diameter column filled with 100 g of Sephadex LH-20 (Pamacia, Sweden) and tubed with 1 L of methanol. Elution was carried out in 100 fractions of 5 mL per sugar, eluted with N-feruloylserotonin (N-feruloylserotonin, molecular weight 352, melting point 118 ° C) and N- (para-coumaroyl) serotonin (N- (p-coumaroyl). ) serotonin, molecular weight 322, melting point 194 ° C.) 73 mg (yield 86 and 73 mg%, respectively, based on dry safflower seed). Subsequently, 45 mg of 80% methanol extract was subjected to Sephadex LH-20 column chromatography in the same manner as described above to obtain 7 mg of matairesinol (molecular weight 358, melting point 94 ° C.) and 8′-hydroxyactinin. 25 mg (8'-hydroxyarctigenin, molecular weight 388, melting point 168 DEG C) were obtained (yield 7 and 25 mg%, respectively, based on dry safflower seed). Sepadex LH-20 column chromatography was performed on 76 mg of 100% methanol extract in the same manner as above to obtain 11 mg of tilianine (molecular weight 446, melting point of 125 ° C) and acecetin (molecular weight of 284, melting point of 255). 52 mg) were obtained (yields 11 and 52 mg%, respectively, on dry safflower seed).

Example 2

After grinding 100 g of roasted safflower seed, 1 L of 80% water-soluble methanol was added thereto, followed by extraction and filtration in hot water for 2 hours. After the extraction operation was repeated three times, the methanol extract obtained under reduced pressure and concentration was concentrated to 200 mL, and then, 500 mL of 100% normal nucleic acid was added thereto, and the oil extract was extracted into the nucleic acid layer and the methanol layer. After depressurizing and concentrating the methanol extract to 100 mL, it was filtered and the filtrate was transferred to a column of 5 cm diameter filled with 100 g of polyamide C-200 (Hwagwang Pure Chemical Co., Ltd.), 20% aqueous methanol, Elution of 60% water soluble methanol and 1 L of 100% methanol solution was in turn. 54 mg of the flavonoid mixture in 20% methanol fraction, 38 mg of the lignan mixture in 60% methanol fraction, and 0.15 g of the serotonin mixture in 100% methanol fraction were obtained, respectively. Then, 54 mg of the flavonoid fractions were dissolved in methanol, and then transferred to a 2 cm diameter column filled with 50 g of Sephadex LH-20 (Pamacia, Sweden) and eluted with 100 ml of 2 mL per tube with 1 L of methanol. To obtain 7 mg of tilianine (molecular weight 446, melting point 125 ℃) and acecetin (acacetin, molecular weight 284, melting point 255 ℃) 37 mg (yield 7 and 37 mg%, respectively, based on dry safflower seed). Sepadex LH-20 column chromatography was performed on 38 mg of the lignan mixture in the same manner as described above to obtain 5 mg of matairesinol (molecular weight 358, melting point 94 ° C) and 8'-hydroxyarctigenin , Molecular weight 388, melting point 168 ° C.), 20 mg were obtained (yield 5 and 20 mg%, respectively, based on dry safflower seed). Finally, 0.15 g of the serotonin mixture was subjected to Sephadex LH-20 column chromatography in the same manner to prepare 76 mg of N-feruloylserotonin (molecular weight 352, melting point 118 ° C) and N- (para- 59 mg of comaroyl) serotonin (N- (p-coumaroyl) serotonin, molecular weight 322, melting point 194 ° C.) were obtained, respectively (yield 76 and 59 mg%, based on dry safflower seed).

Example 3

After grinding 100 g of roasted safflower seed, 1 L of 100% normal nucleic acid was added thereto, followed by extraction in boiling water for 3 hours to remove oil. 80% water-soluble methanol was added to the residue obtained by repeating the extraction operation twice, and extraction and filtration were performed for 2 hours in a boiling water. The methanol extraction liquid obtained by repeating the above extraction operation three times was concentrated under reduced pressure to 100 mL. 500 mL of ethyl acetate was added thereto, and the resultant was partitioned into an ethyl acetate layer and an aqueous layer to obtain 1.7 g of an ethyl acetate extract. This solution was dissolved in 10 mL 80% aqueous methanol and passed through Sep-Pak C ₁₈ Katriz (Waters, USA). The extract was diluted with methanol and then separated and purified by preparative high-performance liquid chromatography. N-feruloylserotonin (molecular weight 352, melting point 118 ° C) 78 mg, N- (para-coumaroyl) serotonin (Np-coumaroylserotonin, molecular weight 322, melting point 194 ° C) 67 mg (yields 78 and 67, respectively) mg%, dry safflower seed), 5 mg of Matyr Resinol (molecular weight 358, melting point 94 ° C) and 24 mg of 8'-hydroxyarctigenin (8'-hydroxyarctigenin, molecular weight 388, melting point 168 ° C) (yield 5 each) And 24 mg% based on dry safflower seed), and 10 mg of tilianine (molecular weight 446, melting point 125 ° C.) and 47 mg of acecetin (molecular weight 284, melting point 255 ° C.), respectively (yield 10 and 47 mg% based on dry safflower seed).

At this time, the conditions of preparative high-speed liquid chromatography were as follows. Waters Delta Prep 4000 Fast Liquid Chromatography; Column, RCM Novapak C ₁₈ (25 mm × 100 mm × 2 cartridge); Ultraviolet detector (UV ₃₀₀ ); Rate, 5.0 mL / min; Solvent (solvent A, 20% water soluble methanol containing 0.5% triproroacetic acid; solvent B, 80% water soluble methanol, solvent A-> concentration gradient elution with solvent B for 60 minutes). At this time, the preparative high-speed liquid chromatograms of the six compounds separated were as shown in FIG. 1, and their detailed ultraviolet, infrared and mass spectrometry data were as shown in Table 1. Hydrogen-nuclear magnetic resonance data and carbon-nuclear magnetic resonance data were as shown in Tables 2a, 2b and 3a, 3b, respectively.

Serotonin is either (a) N-ferroylserotonin or (b) N- (para-coumaroyl) serotonin depending on the substituent, and the general formula is as follows:

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

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

Lignan is either (a) matairesinol or (b) 8'-hydroxyactinin depending on the substituent and the general formula is as shown in formula (II) below;

(a) Matyr Resinol (L1) where R ₁ = H, R ₂ = H, R ₃ = H.

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

The flavonoids are either (a) tilanine or (b) acecetin, depending on the substituent, and the general formula is as shown in general formula (III) below;

(a) tilianin (F1) where R ₁ = glucose, R ₂ = CH ₃ .

(b) Acecetin (F2), where R ₁ = H, 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 _λmax 288, 315 290, 318 230, 280 228, 278 268, 324 270, 303 (s), 328 IR _νmax (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 +] [Note] S1: N-ferrolyl serotonin, S2: N- (p-coumaryl) serotonin, L1: matyrethinol, L2: 8'-hydroxyactinine, F1: tilyanine, F2: acecetin.

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; Singlet d; Doubletdd; Double doublet t; Triplet m; Multiplet ^* Determined by 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; Singlet d; Doubletdd; Double doublet t; Triplet m; Multiplet ^* Determined by DMSO

Carbon-nuclear magnetic resonance data of six phenolic compounds isolated from roasted safflower seed (methanol- _d6 ) 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; N-ferroyl serotonin, S2; N- (para-kuromaroyl) serotonin, L1; Matyr Resinol, L2; 8'-hydroxyactinine, F1; Tilianin F2; Axetine

Carbon-nuclear magnetic resonance data of six phenolic compounds isolated from roasted safflower seed (methanol- _d6 ) 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; N-ferroyl serotonin, S2; N- (para-kuromaroyl) serotonin, L1; Matyr Resinol, L2; 8'-hydroxyactinine, F1; Tilianin F2; Axetine

Experimental Example 1: Effect of Isolated Polyphenol Compounds on the Promoting Bone Formation

In the present experimental example, the polyphenol compounds obtained in Examples 1, 2, and 3, namely, Peruloryl serotonin and coumaryl serotonin, which are serotonin components, matareresinol and hydroxyactinine, which are lignan components, and tiliananin and aca, which are flavonoid components, In order to investigate the effect of cetin on bone formation promoting effect, it was measured using ROS 17 / 2.8, an osteoblast cell line, bone cells that form bone. ROS 17 / 2.8 cells were treated with 10% fetal bovine serum and 1% antibiotic-antibacterial mixture (100 IU penicillin, 100 mg streptomycin, 250 ng amphotericin, Gibco BRL) without phenol red. Cell proliferation was inoculated with added Dulbecco's Modified Eagle medium and cultured in a 5% C0 ₂ incubator maintained at 37 ° C for MTT, 3- (4,5-dimethyl- Thiazol-2-yl) -2,5-diphenyltetrazolin bromide) was measured by MTT assay based on metabolic reduction. At this time, to determine whether safflower seed promotes bone formation, 17β-estradiol, which is known to promote bone cell proliferation, and genistein isolated from soybean are set as a positive reference. The sample-free addition was set as a control and compared. The MTT assay method was as follows. After ROS cells were planted in 96 wells so that the number of cells was 5 × 10 ³ , the medium composition was changed to 0.5% estrogen deficient-fetal bovine serum for 2.5 hours to exclude the effect of fetal bovine serum, and 2.5% estrogen deficient. In fetal bovine serum, 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 formazan crystal, a purple precipitate formed by empty, 150 μl 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 were as shown in Table 4 below.

Effects of Six Polyphenolic Compounds Isolated from Roasted Safflower Seed on Proliferation of Osteoblast-like Cells (ROS 17 / 2.8) compound Concentration (m) Control 10 ^-15 10 ^-12 10 ^-9 10 ^-6 N-Feruloylserotonin 100 105 107 100 98 N- (para-coumaroyl) serotonin (N- (p-Coumaroyl) serotonin) 100 105 108 96 113 Matairesinol 100 103 116 130 158 8'-Hydroxyarctigenin 100 98 80 102 95 Tilianine 100 84 80 95 112 Acecetin 100 115 112 120 126 17beta-estradiol ^* (17β-Estradiol, E ₂ ) 100 128 126 138 128 Genistein ^* (Genistein) 100 110 113 111 117

As shown in Table 4, 17 beta-estradiol (E ₂ ) already exhibited about 130% cell proliferation effect at a low concentration of 10 ^-15 M, about 140 at a physiological concentration of 10 ^-9 M Increasing by% promotes proliferation of bone cells in a dose-dependent manner. Dozens of soybeans also dose-dependently increased ROS osteoblast proliferation by up to 115%, but showed lower effects than estradiol (E ₂ ). All six polyphenolic compounds isolated from roasted safflower seed also tended to promote the proliferation of ROS osteoblasts in a dose-dependent manner. In particular, the matyrethinol and acecetin compounds showed high cell proliferation rates as much as estradiol (E ₂ ) and genistein, respectively. Indicated.

From the results of Table 4, it was found that, among the six polyphenol components isolated from roasted safflower seeds, the matyrethinol and acecetin components may act as substitutes for 17β-estradiol and genistein, estrogen compounds that promote bone formation.

As described in the above Examples and Experimental Examples, the residue obtained by degreasing roasted safflower seed with nucleic acid was extracted by boiling water with methanol, filtered and concentrated, and then subjected to column chromatography with Diion HP-20 and polyamide C-200. The obtained 60%, 80% and 100% methanol extracts were subjected to column chromatography and preparative-high-performance liquid chromatography with Sephadex LH-20, and thus the pure serotonin, lignan and flavonoid components have excellent effects in treating osteoporosis and fractures. It is a very useful invention in the raw material industry of functional food and medicine for the treatment of osteoporosis and fracture.

Claims (4)

(1) crushing and drying the dried safflower seed; and (2) adding 50-80% aqueous solution of methanol to the safflower seed powder degreased in the first step, heating and extracting at 80 ° C., and decompressing and concentrating. (3) performing column chromatography on the concentrate prepared in the second step using Diion HP-20, and (4) extracting the extract prepared in the third step using a Sephadex LH-20 column. Method for extracting safflower seed polyphenolic compound comprising the step of pure separation of serotonin, lignan and flavonoid components using. The method for extracting safflower seed polyphenolic compound according to claim 1, wherein the concentrate is subjected to column chromatography using polyamide in the third step. The method of claim 1, wherein the ethyl acetate layer is added to the concentrate in the third step, and the ethyl acetate layer is separated into an aqueous layer, and the ethyl acetate layer is depressurized and concentrated. In the fourth step, the ethyl acetate layer is separated into high-performance liquid chromatography. Extraction method of safflower seed polyphenolic compound, characterized in that the pure separation by using a chromatography. Peruloryl serotonin and coumaryl serotonin, matyrresinol and hydroxyactinine, tiliananin and acecetin prepared by the method according to any one of claims 1 to 3.