KR20100004492A - Separation method of sesamin from magnolia kobus - Google Patents

Abstract

PURPOSE: A method for isolating sesamin from Magnolia kobus is provided to obtain sesamin and simplify isolation process. CONSTITUTION: A method for isolating sesamin from Magnolia kobus comprises: a step of dipping Magnolia kobus powder in methanol to obtain methanol crude extract; a step of extracting methanol crude extract with hexane to obtain hexane-soluble part and hexane-insoluble part; a step of extracting hexane-insoluble part with toluene to obtain toluene-soluble part and toluene-insoluble part; a step of performing silica gel column chromatography of toluene-soluble part(MKB); a step of performing thin-layer chromatography and detecting with UV ray lamp and coloring agent to obtain MKB-1 to MKB-9 fractions; a step of obtaining sesamin compound from fraction of MKB-2. The health food is used in the form of beverage, capsule, tablet, pill or powder.

Description

Separation method of sesamin from Magnolia kobus

The present invention relates to a method for separating sesamine from magnolia, and more particularly, to obtain a crude methanol extract by immersing the magnolia powder in methanol, extracting the crude methanol extract with hexane to obtain a hexane soluble portion and an hexane insoluble portion. Extracting the hexane insoluble part with toluene to obtain a toluene soluble part and a toluene insoluble part, obtaining nine primary aliquots using silica gel column chromatography for the toluene soluble part (MKB), and preparing the first aliquots. Separation of sesamin from magnolia, which comprises the steps of obtaining thin secondary chromatography and obtaining nine secondary fractions of MKB-1 to MKB-9 by searching with an ultraviolet lamp and a coloring agent, and obtaining a sesamin compound from the fraction of MKB-2. It was on the way.

Magnolia kobus is a species belonging to the Magnoliaceae family. It is a deciduous broad-leaved arboreous tree that lives in Korea and Japan, and is mainly used as garden water and buds are used for the treatment of rhinitis. Extraction components for magnolia include α-pinene, β-pinene, cineol, limonene, etc. Among the components, the action is very diverse, such as aesthetic, aromatic, and analgesic. The pharmacological action known to date has a component that lowers blood pressure, and among the volatile components, there is also the effect of local convergence.

M. kobus DC, a northern variant of magnolia ( M. kobus ). var. The ethanol extracts of borealis were sequentially extracted and fractionated and searched for diethyl ether solubles. 12 species including magnostellin A, 3 species including aschantin, yangambin and syringaresinol in the neck, 5 species including aschantin, yangambin, syringaresinol and kobusinol B in bark, and 3 species of lignan compounds such as kobusin, aschantin and yangambin in buds (Kim et al ., 1996, 1998).

Extracts, the secondary metabolites of magnolia and trees, which are typically represented by magnolia, are used in medicines, biopesticides, natural pigments or natural flavors, food additives and dietary supplements. . From magnolia and trees, substances have been separated and purified and their physiological activity is known.

Sesamine (sesamin) represented by the following structural formula (1) is a fat-soluble lignan component contained in white sesame seeds, has a function of promoting the detoxification of alcohol in the liver, has an antioxidant effect, and also inhibits the production of lipid peroxide and carcinogenesis It has been reported to have an inhibitory effect.

...구조식(1)

... Formula (1)

Currently, most of the prior art related to sesamin is separated from sesame, sesame gourd, sesame oil, especially when it is separated from sesame, the content is known to average 30%.

The patent application for Sesamin is currently reported to be used as a functional material by separating sesame seeds from sesame seeds and is used in combination with other herbal preparations. Research on how to separate the antioxidant components contained in sesame is being actively conducted, and these studies are focused on the mass- ification of the separation process itself in order to use the antioxidant components as food or medicine. These findings have been reported, for example by Kamal and Fukuda, both of which start sesame in the form of oil. This is intended to supply large amounts of raw materials at the beginning of the separation process by reducing the vast volume by taking sesame seeds in the form of oil.

First, Kamal et al. Proposed a method of separating sesamin and sesamoline by conducting silica gel column 4-5 times after squeezing sesame oil to obtain saponified oil. This oil has to accumulate, and the process of obtaining oil, which is negligible and complex, and the oil is a residue that must be removed again during the separation process. It must pass through, which in turn serves as a limiting factor for the single pass through the column.

The present invention is to provide a method for separating sesamin from magnolia.

The present invention can be provided as a dietary supplement containing sesamine separated from magnolia, the dietary supplement is any one selected from beverages, capsules, tablets, tablets, powders (powder) Can be formulated.

According to the present invention, nine primary aliquots are obtained by using silica gel column chromatography for toluene soluble part (MKB) obtained by sequentially extracting magnolia powder with methanol, hexane and toluene, and the primary aliquots are thin layers. Separation method of sesamin from magnolia, which comprises the steps of chromatographing to obtain nine secondary fractions of MKB-1 to MKB-9 by searching with ultraviolet lamp and color developer, and then obtaining sesamine compound from the fraction of MKB-2. Can provide.

Sesamin isolated from magnolia by the present invention may be provided as a dietary supplement of any one selected from beverages, capsules (tablets), tablets (tablets), pills, powders (powder).

According to the present invention, sesamine obtained mainly from sesame seeds, sesame gourd, and sesame oil can be obtained from magnolia, providing a new source of sesamin as well as providing a new separation method of sesamin by the present invention.

The present invention shows a method for separating sesamin from magnolia.

The present invention is to obtain a crude methanol extract by immersing magnolia powder in methanol; Extracting the crude methanol extract with hexane to obtain a hexane soluble part and a hexane insoluble part; Extracting the hexane insoluble part with toluene to obtain a toluene soluble part and a toluene insoluble part; Obtaining nine primary aliquots using silica gel column chromatography for the toluene soluble portion (MKB); Developing the first aliquots with thin layer chromatography to search for an ultraviolet lamp and a colorant to obtain nine secondary fractions of MKB-1 to MKB-9; A method of separating sesamine from magnolia, comprising the step of obtaining a sesamin compound from the fraction of MKB-2.

The magnolia powder may be a powder obtained from any one or more selected from the leaves, the neck, and the bark of the magnolia. Preferably, the powder obtained from the magnolia bark is preferably used in terms of yield of sesamin.

When obtaining the crude methanol extract from the magnolia powder as described above, the magnolia powder was immersed in methanol for 1 to 5 days at room temperature to obtain a methanol crude extract, and the crude crude extract was subjected to 1 to 5 times to obtain a methanol crude extract from the magnolia powder. Extracts can be obtained.

When obtaining the crude methanol extract from the magnolia powder in the above, the magnolia powder was immersed in methanol for 3 days at room temperature to obtain the crude methanol extract, and the methanol crude extract could be obtained from the magnolia powder by performing the operation three times to obtain the crude methanol extract. have.

In the above, silica gel column chromatography for toluene soluble part of magnolia powder is silica gel using n- hexane: EtOAc (85: 15v / v ~ 0: 100v / v) as elution solvent for toluene soluble part (MKB). (silica gel) was fractionated using column chromatography, and the content of n- hexane in the composition of the eluting solvent was decreased by 10 (v / v) and the content of EtOAc was increased by 10 (v / v). Nine primary aliquots can be obtained. That is, toluene soluble part (MKB) was subjected to silica gel column chromatography using n- hexane: EtOAc (85:15, v / v) as an elution solvent to obtain an aliquot, wherein silica gel column chromatography was used as an elution solvent. The composition was n- hexane: EtOAc (85: 15, v / v), n- hexane: EtOAc (75: 25, v / v), n- hexane: EtOAc (65: 35, v / v), n- hexane: EtOAc (55: 45, v / v), n- hexane: EtOAc (45: 55, v / v), n- hexane: EtOAc (35: 65, v / v), n- hexane: EtOAc (25: 75 , v / v), n- hexane: EtOAc (15: 85, v / v), n- hexane: EtOAc (0: 100, v / v) for each aliquot obtained from thin layer chromatography ( TLC, silica gel 60 F ₂₅₄ , developing solvent: toluene-ethyl formate-formic acid = 5: 4: 1, v / v / v), followed by UV (254 nm) lamp and colorant (50% H ₂ SO ₄ ) Was divided into nine primary fractions of MKB-1 to MKB-9, and sesamin, a crystalline substance, is separated from the MKB-2 fraction.

The present invention provides a dietary supplement comprising sesamin isolated from magnolia by the above-mentioned method.

According to the present invention, sesamine separated from magnolia by the above-mentioned method is 0.1 to 10% by weight, preferably 0.1 to 5% by weight, more preferably 0.1 to 1% by weight, based on the total weight of the health food. Provide food.

The dietary supplement may be formulated into any one selected from beverages, capsules, tablets, pills, powders.

In the dietary supplement containing sesamin isolated from the magnolia, each supplement in addition to sesamin may include a known subsidiary material, the content of such subsidiary materials and subsidiary materials are those skilled in the art Since it can be selected by the enemy, the description of the subsidiary materials in the dietary supplement containing sesamin separated from magnolia will be omitted.

Investigation of the sesamin separation method from the magnolia of the present invention, in order to achieve the object of the present invention, it is preferable to provide a separation method of sesamin from magnolia under the conditions mentioned above.

Hereinafter, the content of the present invention will be described in detail through examples and test examples. However, these are intended to explain the present invention in more detail, and the scope of the present invention is not limited thereto.

<Example>

Magnolia bark obtained from 30-year-old magnolia was dried in Gajwa Experiment Forest (Gaju-dong, Jinju-si, Gyeongsangnam-do), and then pulverized and milled into a grinder (20.0 Kg). The magnolia bark powder was added to methanol (MeOH), immersed and extracted for 3 days at room temperature, and this operation was performed three times to obtain a crude extract of magnolia bark methanol. The methanol extract of magnolia bark (994.30 kg) was sequentially extracted and fractionated using hexane ( n- hexane) and toluene (toluene, 123.16 g) to obtain a soluble part of magnolia bark toluene (see FIG. 1).

Silica gel column chromatography (40) using n- hexane: EtOAc (85: 15-0: 100, v / v) as an eluting solvent for the above-mentioned magnolia bark toluene soluble part (MKB, 123.16 g) 500 primary aliquots were obtained using -120 mesh, Japan Kanto Chemical Co., Ltd.).

Each of the primary aliquots obtained above was developed on thin layer chromatography (TLC, silica gel 60 F ₂₅₄ , developing solvent: toluene-ethyl formate-formic acid = 5: 4: 1, v / v / v), followed by UV Nine fractions (MKB-1 to MKB-9) were obtained by searching by (254 nm) lamp and color developer (50% H ₂ SO ₄ ). The crystalline sesamin compound (17.0 g) was isolated from the fraction of MKB-2 (81.15 g).

<Test Example>

In the above example, the characteristics of the structural identification of the sesamin compound isolated from the bark of magnolia were measured as follows (1), and the results are shown in the following (2).

(1) Instrumental analysis for structural identification of sesamin compounds

Mass spectrometry of the sesamin compound isolated from the magnolia bark in the above example was measured using an electron impact mass spectrometer (EI-MS) spectrum using JEOL JMS-600W.

¹ H-, and NMR, as well as the ¹³ C-NMR 2D NMR of ^{1 H- 1 H Correlation Spectroscopy (COSY} ), Nuclear overhauser and exchange spectroscopy (NOESY), Distortionless enhancement by polarization transfer (DEPT), Heteronuclear multiple quantum coherence (HMQC ) And nuclear magnetic resonance (NMR) spectra such as heteronuclear multiple bond coherence (HMBC) were measured using the Varian Unity-Inova 500 of the Seoul Branch of the Korea Research Institute of Basic Science. Optical rotation was measured using a JASCO P-1020 polarimeter. TMS (tetramethylsilance) was used as an internal standard, and CDCl ₃ (chloroform- d ) was used as a solvent.

(2) Structural Identification of Sesamin Compound

White crystal. mp 123-124 ° C. [a] ^20.4 _D + 61.5 ° ( c = 0.58, CH ₂ Cl ₂ ), lit + 63.2 °.

EI-MS m / z : 354 (M ⁺ ), 323, 203, 178, 161, 149 (base ion), 135.

¹ H-NMR (500 MHz, chloroforum- d ): δ 3.05 (2H, br.s , H-8, H-8 '), 3.86 (2H, dd , J = 2.8, 9.2 Hz, H-9b, H -9b '), 4.23 (2H, dd , J = 6.6, 8.5 Hz, H-9a, H-9a'), 4.68 (2H, d , J = 3.7 Hz, H-7, H-7 '), 5.95 (4H, s , OCH ₂ O × 2), 6.77 (2H, d , J = 8.0, H-5, H-5 '), 6.80 (2H, dd , J = 2.0, 8.5 Hz, H-6, H -6 '), 6.84 (2H, d , J = 2.0 Hz, H-2, H-2').

¹³ C-NMR (125 MHz, chloroforum- d ): δ 54.34 (C-8, C-8 '), 71.71 (C-9, C-9'), 85.79 (C-7, C-7 '), 101.05 (C-OCH ₂ O), 106.48 (C-2, C-2 '), 108.18 (C-5, C-5'), 119.33 (C-6, C-6 '), 135.08 (C-1 , C-1 '), 147.10 (C-4, C-4'). 147.97 (C-3, C-3 ').

¹ H- ¹ H COZY: H-8'↔H-9'a / H-9'b / H-7 ', H-8↔H-9a / H-9b / H-7, H-3'↔ H-2 ', H-3↔H-2.

NOESY: H-7'↔H-9'a / H-9b, H-7↔H-9b / H-9'b.

HMBC: H-8 '→ C-7' / C-8 / C-7 / C-1 ', H-7' → C-8 '/ C-9 / C-8 / C-9' / C- 1 '/ C-6' / C-2 ', H-9 → C-7' / C-7 / C-8, H-8 → C-8 '/ C-7' / C-7 / C- 9 '/ C-1, H-7 → C-8' / C-9 / C-8 / C-9 '/ C-1 / C-6 / C-2, H-9' → C-8 ' / C-7 '/ C-7, H-6' → C-2 '/ C-1' / C-4 '/ C-7', H-3 '→ C-2' / C-4 '/ C-5 '/ C-1', H-2 '→ C-6' / C-4 '/ C-1' / C-7 ', H-6 → C-2 / C-4 / C-1 / C-7, H-3 → C-5 / C-2 / C-4, H-2 → C-1 / C-2 / C-4 / C-6, OCH ₂ O → C-5 '/ C-4 ′, OCH ₂ O → C-5 / C-4.

Sesamin isolated from the toluene soluble part of magnolia bark was obtained as white amorphous crystals. In the EI-MS spectrum of sesamin (see Figure 2), molecular ion (M ⁺ ) The peak showed m / z 354 (M ⁺ ), and the main ion peaks showed m / z 323, 203, 178, 161, 149, 135 and the like. The fragment ion peak of m / z 135 corresponds to 3, 4-dioxymethylenephenyl ion.

4H min of δ 3.05 (2H, H-8, H-8 ′), δ 4.68 (2H, J = 3.7 Hz, H-7, H-7 ′) in the ¹ H-NMR spectrum of sesamin (see FIG. 3). Br corresponding to. Signals of singlet and doublet are due to the methane protons of H-8, H-8 'and H-7, H-7' appearing in the furofuran substructure. Also shown are two doublets of δ 3.86 (2H, J = 2.8, 9.2 Hz, H-9b, H-9b ') and δ 4.23 (2H, J = 6.6, 8.5 Hz, H-9a, H-9a'). The signals belonged to the methylene protons present in the furofuran structure. And the singlet at δ 5.95 corresponds to two dioxymethylene protons. The double doublet corresponding to 2H min at δ 6.80 (2H, dd , J = 2.0, 8.5 Hz, H-6, H-6 ') appearing in the hypolonial field is defined as 1, 3, 4-3 substituted nuclei. The signal present was attributed to H-6 and H-6 ′ protons due to ortho ( J = 8.5 Hz), meta ( J = 2.0 Hz) binding.

Sesamine's ¹³ C-NMR spectrum (see Fig. 4) was able to observe all 20 carbon signals. The signal of δ 101.05 was attributed to the characteristic carbon derived from dioxymethylene. The signal at δ 135.08 is due to the C-1 and C-1 'carbons of the aromatic nucleus bound to furofuran. Other δ 54.34 is C-8, C-8 'carbon, δ 71.71 is C-9, C-9' , δ 85.79 were attributed to each of the C-7, C-7 'carbon. In the DEPT spectrum (see FIG. 5), three quaternary carbons, two secondary carbons, and five primary carbon signals were identified. In the ¹ H- ¹ H COSY spectrum (see FIG. 6) and the NOESY spectrum (see FIG. 8) of sesamin, correlated peaks between H-7, H-8, and H-9 were identified. Also in the HMBC spectrum of sesamin (see Figure 7) dioxymethylene protons and δ 147.10 (C-4, 4 '). A cross peak of 147.97 (C-3, 3 ') was confirmed. In the sesamin HSQC spectrum (see FIG. 9), the number of carbons was found to be 20. As a result of comparison with the above instrumental analysis and literature (Rana et al ., 2000, Atal et al ., 1967), it was identified as (+)-sesamin having 20 hydrogen 18 carbons.

From the results of the structural identification of the sesamin compound, it was confirmed that the compound isolated from the magnolia bark in the above example was the sesamin compound of the following structural formula (1).

...구조식(1)

... Formula (1)

As described above, although described with reference to a preferred embodiment of the present invention, those skilled in the art will be variously modified and modified within the scope of the present invention without departing from the spirit and scope of the invention described in the claims below. It will be appreciated that it can be changed.

According to the present invention, sesamin can be separated from magnolia, and sesamin which has been mainly separated from sesame, sesame gourd, and sesame oil can be obtained by various methods.

1 is a process chart showing a step of sequentially extracting methanol, hexane and toluene from magnolia to obtain a toluene soluble portion and a toluene insoluble portion of magnolia.

2 is an EI-MS spectrum of the sesamine compound prepared in Example.

3 is a ¹ H-NMR spectrum (CDCl ₃ ) of the sesamine compound prepared in Example.

4 is a ¹³ C-NMR spectrum (CDCl ₃ ) of the sesamine compound prepared in Example.

5 is a DEPT spectrum (CDCl ₃ ) of the sesamine compound prepared in Example.

6 is a ¹ H- ¹ H COSY spectrum (CDCl ₃ ) of the sesamine compound prepared in Example.

7 is HMBC spectrum (CDCl ₃ ) of the sesamine compound prepared in Example.

8 is a NOESY spectrum (CDCl ₃ ) of the sesamine compound prepared in Example.

9 is an HSQC spectrum (CDCl ₃ ) of the sesamine compound prepared in Example.

Claims (8)

(1) distilling magnolia powder into methanol to obtain a crude methanol extract, (2) extracting the crude methanol extract with hexane to obtain a hexane soluble portion and a hexane insoluble portion, (3) extracting the hexane insoluble portion with toluene to obtain a toluene soluble portion and a toluene insoluble portion, (3) obtaining nine primary aliquots using silica gel column chromatography for the toluene soluble part (MKB), (4) obtaining the nine fractions of MKB-1 to MKB-9 by developing the first aliquots with thin layer chromatography and searching with an ultraviolet lamp and a coloring agent; (5) A method for separating sesamine from magnolia comprising the step of obtaining a sesamin compound from the fraction of MKB-2. The method for separating sesamin from magnolia according to claim 1, wherein the magnolia powder is a powder obtained from at least one selected from the leaves, the neck and the bark of the magnolia. The method for separating sesamine from magnolia according to claim 1, wherein the magnolia powder is immersed in methanol for 3 days at room temperature to obtain a crude methanol extract, and the methanol crude extract is obtained three times. The silica gel column chromatography according to claim 1, wherein n- hexane: EtOAc (85: 15 v / v-0: 100 v / v) is used as the elution solvent for the toluene soluble part (MKB). Preparative, N- hexane content in the composition of the eluting solvent was reduced by 10 (v / v) by the eluting solvent by increasing the content of 10 by 10 (v / v) to obtain nine primary fractions Separation method of sesamin from magnolia. The method of claim 1, wherein the nine primary aliquots are each subjected to thin layer chromatography (TLC, silica gel 60 F ₂₅₄ ) with a solvent of toluene-ethyl formate-formic acid = 5: 4: 1 (v / v / v). Separating the sesamin from magnolia, characterized in that after the development, to obtain nine secondary fractions of MKB-1 to MKB-9 by searching with a UV lamp and a colorant of 50% H ₂ SO ₄ . The method of claim 1, wherein the sesamin compound is obtained from the fraction of MKB-2 by chromatographic method. A health supplement comprising sesamin isolated by the method of claim 1. The dietary supplement of claim 7, wherein the dietary supplement is any one of a formulation selected from beverages, capsules, tablets, pills, and powders.

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