HK1063804A - Process for isolating and purifying secoisolariciresinol diglycoside (sdg) from flaxseed - Google Patents

Description

Method for separating and purifying secoisolariciresinol diglycoside from flaxseed

The invention relates to a method for isolating flax lignans (flax lignans). Specifically, the present invention provides a method for separating and purifying Secoisolariciresinol Diglycoside (SDG) from crushed flaxseed by supercritical carbon dioxide extraction and chromatographic separation.

Lignans are the hormones phytoestrogens (phytoestrogens) present in plants and are the defense mechanisms of plants against plant diseases and pests. They are also involved in the regulation of plant growth. Lignans belong to the group of phenolic compounds, having the structure of dibenzylbutane. Lignans exist in nature in the form of monomers or glycosides.

Lignans are commonly found in the plant kingdom, and more than 200 different lignans are now known. Flax (Linum usittissimum) is a very good source of lignans; flaxseed contains much more lignans than any other plant derived food product. The most common secoisolariciresinol is secoisolariciresinol, which is reported to be present in flaxseed at a concentration of 675. mu.g/g (wet weight) (Cassidy et al, 2000).

The lignans in flax, secoisolariciresinol and matairesinol (matairesinol), are precursors of the human lignans enterolactone (enterolacton) and enterodiol (enterodiol) synthesized by bacteria. Intestinal microbes metabolize/convert secoisolariciresinol (SECO and its glycosides, SDG) to mammalian lignans. The conversion of SDG in the gastrointestinal tract begins with the release of sugar moieties by gastric juice, enzymes in the gastrointestinal tract, or microorganisms, whereby SDG is first converted to the aglycone (aglycone) form corresponding to SECO. The microorganisms then metabolize SECO to enterodiol, which is oxidized to enterolactone, which is no longer metabolized by the microflora in the gastrointestinal tract (Borriello et al, 1985). Conversion of SDG to the biologically active form requires removal of the sugar moiety, two methyl groups and two hydroxyl groups. The intestinal microbiota varies from person to person, probably due to the fact that the production of biologically active lignans varies in each individual (McBurney & Thompson, 1989; Zhang et al, 1999).

Phytoestrogens have physiological effects, according to experimental and epidemiological studies. Lignans have anticancer effects in cell culture in vitro and in vivo in animals (Cassidy et al, 2000). Lignans are also antioxidants and, therefore, can prevent, for example, lipid peroxidation, and have positive effects on cardiovascular diseases (Prasad, 1997). This idea is also supported by epidemiological studies (Vanharanta et al, 1999). Lignans have also been reported to have antiviral and bactericidal activity (Adlercreutz, 1991).

Although lignans are common in nature, they have been studied very little. This is due in part to the difficulty of measuring and isolating these compounds. For the isolation of lignans on a laboratory scale, methods based on extraction (solvent/supercritical) and chromatographic separation have been used (Harris & Hagerty, 1993; Lojkov et al, 1997; Muir & Wescott, 1998). A further problem arising in the isolation process is that the yield of lignans is low and time consuming.

By using the method developed for isolating secoisolariciresinol diglucoside disclosed by the present invention, secoisolariciresinol diglucoside can be produced more efficiently than before. The method is based on supercritical extraction and chromatographic separation. Potential targets for use of SDG are, for example, functional foods.

It is therefore an object of the present invention to provide a process for the isolation of lignans, particularly SDG, from flaxseed, which comprises: first, fat is removed from crushed flaxseed by supercritical carbon dioxide extraction, the resulting crushed flaxseed substantially free of fat is ground to a granular powder, from which SDG is extracted into an alkaline lower alcohol. The alcoholic solution is centrifuged and the supernatant neutralized, concentrated and fractionated chromatographically. The SDG-rich fraction is recovered from the eluate and, if desired, further purified.

The invention is further described with reference to the accompanying drawings, in which:

fig. 1 shows a flow chart of the SDG separation and purification method according to the present invention.

FIG. 2 shows an HPLC chromatogram of SDG isolated and purified according to the present invention at a wavelength of 280 nm.

FIG. 3 shows the UV spectrum of SDG separated and purified according to the present invention at a wavelength of 200-400 nm.

Removing oil from crushed flaxseed

For the extraction of lignans, the cold-pressed crushed flaxseed is degreased by means of a supercritical carbon dioxide extraction apparatus. Firstly, supercritical carbon dioxide is used for removing easily-extracted grease in crushed flaxseed. For example, suitable extraction conditions are: e.g. 1-5 hours, a pressure of 300-450atm, a temperature of 50-80 ℃. The crushed flaxseed may be re-extracted with supercritical carbon dioxide modified with a lower alcohol such as ethanol, for example, under the conditions of re-extraction: 1-4 hours, a pressure of 300-450atm and a temperature of 50-80 ℃. The lower alcohol is suitably used in an amount of 5 to 10%. This additional re-extraction enables further removal of more polar lipid components and other non-fat soluble organic compounds from the flaxseed.

Hydrolytic extraction of SDG

Secoisolariciresinol diglycoside is tightly bound or complexed in the linseed matrix and therefore it is difficult to obtain large amounts of secoisolariciresinol diglycoside if extracted with e.g. pure methanol. Thus, in the process of the present invention, alkaline lower alcohol, preferably methanol, is used for extraction, but ethanol may also be used, and the crushed flaxseed is ground into a granular powder before extraction.

Thus, the substantially fat-free crushed flaxseed obtained from the supercritical chromatography column is ground to particles as small as possible. A suitable particle size is less than 0.55 mm. The powder is extracted with a basic lower alcohol such as sodium hydroxide-methanol in a conventional mixer or magnetic stirrer for 24 hours. Preference is given to using 0.05-1M sodium hydroxide-methanol, which is prepared by dissolving sodium hydroxide in anhydrous methanol, for example in a ratio of 1: 20 (w/v). The extraction is preferably carried out in an argon atmosphere for 16 to 24 hours.

Hydrolysis occurs when extraction is performed. Then carrying out process step (i) or (ii):

(i) the alkaline lower alcohol is centrifuged from the precipitate formed during hydrolysis. The supernatant is carefully separated, for example, into a volumetric flask and then neutralized by adjusting its pH to 6-7 with an acid, for example, concentrated hydrochloric acid. The precipitated salt was allowed to settle to the bottom of the bottle. The extract was carefully decanted from the top of the precipitated salt. The salt is washed several times with the lower alcohol, and the combined alcohol extracts are evaporated to almost dryness, for example with a rotary evaporator. To the concentrated solution is added a preparative C18 material (e.g., Waters C18125 ) in a ratio of, for example, 4: 1(w/w), and the sample is evaporated to maximum dryness using a rotary evaporator.

(ii) The pH of the eluate is adjusted to 6-7 with concentrated acid. The solid and the extract were separated from each other by centrifugation and the supernatant was then carefully poured into a volumetric flask or directly into a round-bottomed flask. The supernatant was evaporated to almost dryness, then preparative C18 material was added to the solution and the sample was evaporated to maximum dryness using a rotary evaporator.

Chromatographic enrichment of SDG

The mixture of samples mixed with C18 material was loaded into a flash chromatography system. The column is finally equilibrated with water-methanol or water-ethanol as eluent. SDG is eluted from the sample cartridge (sample cartridge) to the purification column with water-methanol or water-ethanol mixture. The eluate flowing through the column was collected. Finally, the purification column is washed with water-methanol or water-ethanol before the next operation.

The sample-C18 material mixture can also be packed into an open C18-chromatographic column from which SDG is eluted accordingly with an aqueous lower alcohol such as methanol or ethanol.

Analysis of SDG

The extracts were analyzed for SDG by High Performance Liquid Chromatography (HPLC). As analytical column, a reverse phase column is preferably used, and as eluent, a phosphate buffer and methanol having a concentration gradient are preferably used. The compound was identified based on retention time and UV spectrum.

Storage and further purification of SDG

After analysis, the SDG-rich fractions were pooled and evaporated to maximum dryness. The samples were quantitatively transferred to a refrigerator with a small amount of water, deep frozen and lyophilized. Lyophilized SDG is a light yellow powder with a purity of at least 80%.

If desired, the separated SDG can be further purified using an open C18 column. The lyophilized SDG was dissolved in a small amount of water and then added to the column. The desalted and other unidentified materials are washed with water, and then SDG is eluted from the column with a lower alcohol such as methanol or ethanol. The alcohol was evaporated in a rotary evaporator to obtain SDG in the form of crystals. The purity is at least 90%.

According to the present invention, SDG can be isolated and purified from flaxseed in high purity and yield. The fact that no organic solvent is used to remove oil from the crushed flaxseed may for example be considered as an advantage of the process of the invention over the known art. In addition, its direct alkaline decomposition in lower alcohols can efficiently release the SDG in the flax matrix, while also being able to degrade the so-called flax resin (flaxgum) which affects the SDG separation. The eluate is anhydrous and therefore evaporates more easily and rapidly, for example in a rotary evaporator, than in aqueous eluate systems. In the process according to WO96/30468, SDG is extracted with 50-70% methanol, the extract is then evaporated to a viscous liquid and then decomposed with a base. Aqueous alcoholic extracts evaporate much more slowly than methanol or ethanol alone. In addition, the fractionation of the compounds is fast and efficient based on Flash chromatography (Flash chromatography) used in the present invention. The process of the invention is also easily scalable to industrial scale.

Examples

Removing oil from crushed flaxseed by supercritical extraction

Extracting cold-pressed crushed flaxseed of 1-2kg with supercritical carbon dioxide under a pressure of 450atm and a temperature of 70 deg.C. The extraction time was about 5 hours. Extraction of the material with ethanol modified supercritical carbon dioxide was continued for about 2 hours. After extraction, the fat-free crushed flaxseed is ground to a granular powder with a particle size of < 0.55 mm.

Hydrolytic extraction of Secoisolariciresinol Diglycoside (SDG)

100g of linseed powder without fat were extracted for 24 hours in a magnetic stirrer under argon atmosphere with 2000 ml of 1M sodium hydroxide-methanol (1: 20, w/v).

After extraction and hydrolysis, alkaline methanol was centrifuged (1500rpm, 10 minutes). The supernatant was carefully separated into a volumetric flask and its pH adjusted to 6-7 with concentrated HCl. The precipitated salt was allowed to settle to the bottom of the bottle. The extract was carefully decanted from the top of the precipitated salt. The salt was washed several times with methanol and the combined methanol extracts were evaporated to almost dryness using a rotary evaporator. To the concentrated solution was added preparative C18 material (Waters C18125 ), sample: the ratio of C18 was 4: 1(w/w), and the solution was evaporated to maximum dryness using a rotary evaporator.

Flash chromatography for enrichment of SDG

A15 g sample: the mixture of C18 was loaded into the sample barrel of the flash system. The flash 40C18 column (Biotage) was activated with 300 ml 80% methanol, 300 ml 50% methanol and finally 300 ml 40% methanol. The sample cartridge was attached to the activated column. SDG was eluted from the sample cartridge with 650 ml of 40% methanol. After removing the sample cartridge, the column was additionally rinsed with 350 ml of 40% methanol. The 40% methanol that passed through the column was collected in tubes with 50 ml fractions in each tube. The SDG eluted from the flash 40C18 column was 250-450 ml. Finally, the column was purified with 80% methanol before the next run.

Analysis of SDG

The eluate fraction was analyzed for SDG by high performance liquid chromatography. As analytical column, a reverse phase column (Waters Nova Pak C18, 3.9X 150mm) was used, and as eluent, sodium dihydrogen phosphate buffer (pH 2.9) having a concentration gradient of 0.05M and methanol were used. The compounds were identified based on retention time and UV spectroscopy (200-400nm) (FIGS. 2 and 3).

Storage and further purification

After analysis, the SDG-rich fractions were pooled and evaporated to maximum dryness. The samples were quantitatively transferred to a refrigerator with a small amount of water, deep frozen and lyophilized. Lyophilized SDG is a light yellow powder with a purity of at least 80%.

Isolated aliquots of SDG were further purified using an open C18 column (Waters, preparative C18 column). The lyophilized SDG was dissolved in a small amount of water and then added to the column. Salts and other unidentified materials were eluted from the column with water, and then SDG was eluted from the column with methanol. The methanol was evaporated in a rotary evaporator to give crystalline SDG with a purity of about 90%.

Reference to the literature

Adlercreutz，H.1991.Diet and Sex Hormone Metabolism.In：Rowland，I.R.(ed.)Nutrition，Toxicity and Cancer.CRC Press.p.137-195.ISBN0-8493-8812-0。

Borriello，S.P.，Setchell，K.D.R.，Axelson，M.&Lawson，A.M.1985.Production and metabolism of lignans by the human faecal flora.Joumal ofApplied Bacteriology 58：37-43。

Cassidy，A.C.，Hanley，B.&Lamuela-Raventos，M.2000.Isoflavones，lignans and stilbenes-origins，metabolism and potential importance to humanhealth.Joumal of the Science of Food and Agriculture 80：1044-1062。

Harris，R.K.&Hagerty，W. J.1003.Assays of potentially anticarcinogenicphytochemicals in flaxseed.Cereal Foods World 38(3)，147-151。

McBumey，M.I.&Thompson，L.U.1987.Effect of human faecalinoculum on in vitro fermentation variables.British Joumal of Nutrition 58：233-243。

Lojkova，L.，Slanina，J.，Mikesova，M.，Taborska，E.&Vejrosta，J.1997.Supercritical fluid extraction of lignans from seeds and leaves of Schizandrachinesis.Phytochemical analysis 8：261-265。

Muir，A.&Wescott，N.D.1998.Process for extracting lignans fromflaxseed.Patent application WO 96/30468。

Prasad，K.1997.Hydroxyl radical-scavenging property ofsecoisolariciresinol diglucoside(SDG)isolated from flaxseed.Molecular andcellular biochemistry 168(1/2)：117-123。

Vanharanta，M.，Voutilainen，S.，Lakka，T.A.，van der Lee，M.，Adlercreutz，H.&Salonen，J.T.1999.Risk of acute coronary events according to serumconcentrations of enterolactone：a prospective population-based case controlstudy.Lancet 354：2112-2115。

Zhang，Y，Wang，G.-J.，Song，T.T.，Murphy，P.A.&Hendrich，S.1999.Urinary disposition of the soybean isoflavones daidzein，genistein and glyciteindiffers among humans with moderate fecal isoflavone degration activit y.Journalof Nutrition 129：957-962。

Claims (10)

1. A method for isolating Secoisolariciresinol Diglycoside (SDG) from flaxseed, characterized in that:

a) removing oil and fat from cold-pressed and pulverized semen Lini by supercritical carbon dioxide extraction,

b) grinding the resulting substantially fat-free crushed flaxseed into a granular powder,

c) extracting SDG from the obtained powder into alkaline lower alcohol,

d) centrifuging the obtained lower alcohol solution, neutralizing the supernatant,

e) recovering the supernatant, concentrating, mixing with C18 material, and evaporating the solvent to almost dry

The degree of dryness is such that,

f) the resulting mixture was fractionated by flash chromatography,

g) recovering a fraction enriched in SDG, and

h) the fraction enriched in SDG was purified in an open C18 column.

2. A method according to claim 1, characterized in that: in the final stage of the extraction, a lower alcohol is used as an adjuvant for the supercritical carbon dioxide.

3. A method according to claim 2, characterized in that: the lower alcohol is ethanol.

4. A method according to claim 1, characterized in that: in step b), the crushed flaxseed is ground to a powder having a particle size of 0.55 mm.

5. A method according to claim 1, characterized in that: the alkaline lower alcohol is anhydrous methanol or ethanol in which sodium hydroxide is dissolved.

6. The method according to claim 5, characterized in that: the concentration of sodium hydroxide in the lower alcohol is 0.05-1M.

7. A method according to claim 1, characterized in that: step d) is carried out by any of the following methods: (i) centrifuging the lower alcohol solution and then neutralizing the supernatant by adjusting its pH to 6-7 with a concentrated acid, or (ii) neutralizing the alcohol solution by adjusting its pH to 6-7 with a concentrated acid and then centrifuging.

8. A method according to claim 1, characterized in that: in step f), the mixture is fractionated by flash chromatography in a C18 column using water-methanol or water-ethanol as eluent.

9. A method according to claim 1, characterized in that: in step h), the mixture is chromatographed on an open C18 column using a lower alcohol as eluent, using C18.

10. A method according to claim 9, characterized in that: the lower alcohol is methanol.