PL217522B1 - Method of isolation of cyclopeptides from flax (linseed) - Google Patents

Description

Description of the invention

The subject of the invention is a method of separating cyclopeptides from flax, also from waste material such as linseed cake and chaff.

Linseed cyclopeptides are a group of cyclic peptides composed of 8 or more amino acid molecules with immunosuppressive activity.

Immunosuppressants are drugs that suppress or weaken immune responses; the indications for their use are organ and tissue transplants as well as immunological diseases; immunosuppressive effects include: adrenal cortex hormones, antimetabolites that inhibit biosynthesis processes, alkylating drugs that inhibit the production of antibodies, anti-lymphocyte globulin containing anti-lymphocyte antibodies, cyclosporine. Immunosuppressors inhibit the production of antibodies and immune cells in the body, i.e. immunogenesis. In transplantology, they are used to weaken the recipient's body's response to the transplanted tissue that is its antigen. Administration of immunosuppressants immediately after transplantation reduces the risk of transplant rejection.

A known and used immunosuppressant is cyclosporine. Its mechanism of action is based on binding to cyclophilin, a cytosolic protein, in immunocompetent lymphocytes, mainly T-type. The complex of these two proteins inhibits calcineurin, which is responsible for the activation of interleukin-2 transcription. They also inhibit the production of lymphokines and the release of interleukins, which leads to a reduction in the function of T-cell effectors.

However, a number of side effects have been observed in patients taking ciclosporin, which are especially seen in patients treated for transplantation indications and are usually more severe and more frequent. The most common of these are renal dysfunction, hypertension, tremors, headaches, and hyperlipidaemia. Therefore, new and better immunosuppressants are constantly being searched for.

The presence of cyclic peptides with a structure similar to cyclosporine was found in common flax seeds, which could suggest a potential immunosuppressive effect. When using them, no side effects on the human body were found. So far, the structures of the following cyclic peptides from flaxseed have been separated and determined:

- Nonapeptides: CLA (PPFFLIILV), CLB (PPFFVIMLI), CLC (PPFFVIMoLI),

- Octapeptides: CLD (PFFWIMoLL), CLE (PLFIMoLVF), CLF (PFFWVMoLMo), CLG (PFFWIMoLMo), CLH (PFFWIMoLM), CLI (PFFWVFFMLMo), CLJ (PLFIMLVF), CLMW (PMLM) (PMLM) PFFWIMLM), CLN (PFFWIMLMo), CLX (PPFFILLX)

- bicyclic decapeptide (BCD).

The most interesting for its action is the flax nonacyclopeptide A (cyclinopeptide A) of formula (1) defined below, also often abbreviated CLA. The main natural source of cyclopeptides is flax. Linseed cyclopeptides are nonpolar, hydrophobic and oxidation resistant. The cyclopeptides A and X are the only ones in the group that do not contain sulfur atoms. The structure of cyclopeptide A consists of 57 carbon atoms.

The CLA cyclo (PPFFLIlLV) nonapeptide molecule contains successively two proline amino acid residues, two phenylalanine, leucine, two isoleucine, leucine and one valine residue.

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The similarity of the amino acid sequence between cyclosporin and CLA suggested an immunosuppressive effect of the cyclopeptide, which has been confirmed by numerous medical studies. The mechanism of action of CLA on the body's immune system was found to be analogous to that of cyclosporin.

On the basis of preliminary medical studies, it was also found that, compared to cyclosporin, cyclopeptides exhibit a similar immunosuppressive effect at lower doses, have much lower toxicity and do not cause side effects. In addition, cyclopeptides also show anti-malarial activity.

The method of separating cyclopeptide A (CLA) from flax is known, presented in the patent application WO 2009/079792 A1, consisting in solvent extraction combined with adsorption on a bed of silica gel. Linseed oil was mixed with the adsorbent for 2 hours at the temperature of 60 ° C. After filtration, the adsorbent was placed in a chromatography column and de-oiled, washing with hexane and 20% ethyl acetate in hexane. The adsorbed peptides were eluted from the bed with a 10% solution of ethyl alcohol in ethyl acetate. Using this method, from 100 g of linseed oil, 146 mg of the fraction containing cyclopeptides was obtained, which is 0.146%. After chromatographic separation and purification of the end products, the following were obtained: 20.3 mg of pure CLA; 16.3 mg of CLC; 3.3 mg CLD; 10.7 mg CLE, 1.0 mg CLF; 6.1 mg CLG. The chromatographically separated CLA was purified by recrystallization.

Based on the information contained in the cited document, the performance of the described process cannot be determined.

The cyclopeptide content depends on the type of flax and the place of cultivation. Two types of commercial linseed oils were analyzed, showing the content of cyclopeptide A 0.001% and 0.05%.

The separation of oil from flax seeds using the supercritical carbon dioxide extraction method has been described in the patent literature and in publications in specialist journals. The use of liquid or supercritical CO2 to separate from flax for example: diglycosides, α-linoleic acid and a mixture of ω-3 linolenic acids has also been described.

The available literature does not describe the method of separating cyclopeptides from flax using the extraction process with liquid or supercritical CO2, and its use for the concentration or isolation of cyclopeptides derived from flax.

The object of the present invention was the isolation of the cyclopeptide mixture from flax by extraction with carbon dioxide in the liquid or supercritical state. For this purpose, liquid or supercritical carbon dioxide fractionating extraction can also be used. Cyclopeptides thus isolated can be purified by chromatography.

Supercritical extraction is a mass transfer process that uses compressed gases in supercritical conditions instead of classic solvents. This form of extraction uses the low viscosity at relatively high density, which is a positive feature of gases and liquids from the point of view of mass exchange processes. As a result, the extractant has a high dissolving power and good penetration properties. The dissolving power of the same solvent varies widely and depends on the temperature and pressure of the process. It is possible to carry out extraction with liquid gas at high pressure and at a temperature lower than its critical temperature.

In some cases, a stream of liquid solvent called an "entrainer" is added continuously to a liquid or supercritical gas stream. Proper selection of the entrainer can significantly increase the speed and selectivity of extraction.

Carbon dioxide is physiologically neutral and does not react with most substances of natural origin and easily separates from the extracted product. It has bacteriostatic properties, is non-flammable, cheap and readily available. Processes on an industrial scale are carried out with its full recirculation.

The source of cyclopeptides obtained by the method according to the invention may be the whole flax plant, its seeds, linseed oil and waste materials after flax processing, such as: cake, chaff, roots.

Common flax seeds are extracted or pressed to obtain oil. The residues (cake) still contain approx. 10% of oil and, inter alia, cyclopeptides. Linseed oil contains cyclopeptides in the amount from 0.01 to 0.05% (depending on the variety of the flax it is obtained from and the processing process). The content of cyclopeptides varies depending on the variety of flax and amounts to: the Modron variety - 0.05% and the Sapphire variety - 0.04%.

PL 217 522 B1

Waste materials from flax processing are an equally good source of these valuable products. They contain cyclopeptides in the amount of about 0.01 - 0.05%. In addition to cyclopeptide A, there are also other cyclopeptides, mainly CLE and CLC.

The method of separating cyclopeptides from flax by extraction, according to the invention, consists in that the flax, preferably dried and crushed, or flaxseed oil or a waste product from flax processing, such as flax cake or chaff, preferably crushed, is extracted with liquid or supercritical carbon dioxide and optionally, the extract is separated into fractions enriched in the desired cyclopeptides.

Preferably, extraction with liquid or supercritical carbon dioxide is carried out at a temperature of 15 ° C to 80 ° C, under a pressure of 7.3 MPa to 50 MPa.

Preferably, the extraction is carried out with the addition of an entrainer, which is a polar or non-polar substance.

The polar entrainer used is preferably a C1-C8 aliphatic alcohol, most preferably methanol, ethanol or propan-2-ol.

Ethyl acetate is preferably used as the non-polar entrainer.

Preferably, the extract is fractionated by supercritical fractionating extraction.

Preferably the extract is purified by chromatography.

By the method according to the invention, thanks to the extraction with carbon dioxide in the liquid or supercritical state, with or without the addition of entrainer, it is possible to isolate cyclopeptides from flax in a short time (10 hours) with high efficiency, free from solvent residues, free from solvent residues, using as a material starting linseed oil, chaff and raw linseed cakes. The process also enables the multiple concentration of cyclopeptides in the samples that can be directly used as food or medicinal products. The use of an entrainer for fractionating extraction increases the efficiency and significantly shortens the process time due to the presence of cyclopeptides in the first fractions. Cyclopeptides show a biological effect already at a concentration of 1 μΜ, as stated in the patent description WO 2009/079792, and the effect on cancer cells was obtained using a concentration of 0.1-100 μΜ. In the extracts obtained by the method according to the invention, the concentration of cyclopeptide A ranges from 1-125 μΜ, so, depending on the need, the extracts can be used directly in pharmaceutical preparations. Supercritical fractionation extraction can produce a product containing more than 3% of cyclopeptides that can be used directly as an active ingredient. This is a sufficient therapeutic dose for many applications. The purified cyclopeptides are white or almost white sediments, while the extracts obtained with the use of liquid or supercritical carbon dioxide are in the form of oil ₃ with a density of 810 to 950 kg / dm ³ . The identification of the cyclopeptides isolated by the method of the invention was carried out by means of HPLC-MS / ESI. The chromatogram made in the SIM mode shows the signals from the cyclopepliids: CLA (1040.65); CLE (977.52), CLC (1074.57); Na + CLL (1075.40); CLI (1068.50), CLC sulfone (1090.52) (Fig. 1). The process according to the invention is illustrated by the following examples.

P r z x l a d I.

100 g of comminuted and homogenized linseed cakes were placed in a 150 ml tubular extractor, which were extracted with a stream of liquid carbon dioxide at a pressure of 7.3 - 8 MPa and a temperature of 25 ° C. The carbon dioxide flowed through the extractor at a rate of 50 l / h, based on normal conditions. After depressurizing the carbon dioxide stream to atmospheric pressure, it was directed to a receiver, maintained at -30 ° C, where the liquid extract was collected. After 6.5 hours of extraction, 2.28 g of extract containing 0.154% ClA, 0.116% CLE and 0.079% CLC were collected as an oily liquid. After another 15 hours of extraction, 4.38 g of oil containing 0.088% CLA, 0.096% CLE and 0.049% CLC of light yellow color were collected, and after another 14 hours, 1.87 g of extract containing 0.148% CLA, 0.158% CLE and 0.076% CLC as a yellow oil.

The extracts were purified by column chromatography on silica gel, pre-eluted with toluene (200 ml) followed by 8: 1 toluene: methanol. In total, the obtained fraction was 5.5 mg with 74% CLA (HPLC) as a white solid, and 10.6 mg with 48.5% CLA (HPLC) as an off-white solid, and also a fraction with 11 mg containing 80% CLE cyclopeptide as a white solid with an m.p. and a fraction with a weight of 7.8 mg containing 75% CLC as a white solid.

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Pure cyclopeptide samples were used as standards for the quantification of flax cyclopeptides by HPLC.

The analyzes were performed on a Merck-Hitachi HPLC gradient apparatus equipped with a L-7400 UV-VIS spectrophotometric detector, a pump and a L-7100 type four-solvent low-pressure gradient system. The system was controlled by the HPLC System Menager Model D-7000 program via the D-7000 interface. The separation of the products was performed on a Zorbax 300SB column, 150 mm long and 4.6 mm in diameter, filled with Agilent C-18 chamfer. The mobile phase flow was 1 ml / min, wavelength λ = 210 nm. Mobile phase A: 0.1% TFA / H ₂ O, Mobile phase B: ACN, run time: 75 min. The analysis was performed in the time program: gradient from 10% B to 85% B in 65 min.

The CLA retention time is 54.5 min.

CLC retention time is 44.0 min.

The CLE retention time is 47.22 min.

P r z x l a d II.

Extraction from the linseed cakes was carried out in the extractor as in example 1, with carbon dioxide under supercritical conditions, at the temperature of 40 ° C and the pressure of 20 MPa.

After 7.5 hours of extraction, 3.11 g of an oil extract containing 0.210% CLA, 0.158% CLE and 0.098% CLC were collected, after another 15 hours of extraction, 3.18 g of an oil extract containing 0.116% CLA, 0.091% CLE and 0.055% CLC were collected and after a further 13 hours 0.49 g of an oil extract containing 0.098% CLA, 0.098% CLE and 0.097% CLC.

The extracts were purified by column chromatography. In total, there was obtained a solid fraction of 5.9 mg with 71% CLA content (HPLC) and a solid fraction of 6.6 mg, containing 46.6% of CLA (HPLC), and a solid fraction of 12.5 mg, containing 62% CLE cyclopeptide and a solid fraction weighing 10.5 mg containing 43% CLC.

HPLC analyzes were performed under the conditions set out in Example I.

P r x l a d III.

Extraction from the linseed cakes was carried out in the extractor as in Example 1, with supercritical carbon dioxide at 35 ° C, pressure 21 MPa, and methanol (polar entrainer) in the amount of 2 ml / h was passed through the feed of carbon dioxide through the batch bed.

After 5.5 hours of extraction, 3.24 g of oil extract containing 0.011% CLA, 0.001% CLE and 0.005% CLC were collected, after another 5 hours of extraction, 3.04 g of oil extract containing 0.340% CLA, 0.24% CLE and 0.16% CLC and after a further 18 hours 3.48 g of oil extract containing 0.012% CLA, 0.011% CLE and 0.006% CLC.

The extracts were purified by column chromatography.

In total, there was obtained a solid fraction with a weight of 6.9 mg with 80% CLA content (HPLC) and a solid fraction with a weight of 14.4 mg containing 38% CLA (HPLC) and a solid fraction with a weight of 11.2 mg containing 72% CLE cyclopeptide and a solid fraction of 9.5 mg containing 55% CLC.

P r x l a d IV.

Extraction was performed as in Example 3 using a nonpolar entrainer ethyl acetate which was dosed at 2 ml / h. After 5.5 hours of extraction, 2.99 g of oil extract containing 0.052% CLA, 0.038% CLE and 0.025% CLC were collected, after another 13 hours of extraction, 2.96 g of oil extract containing 0.274% CLA, 0.190% CLE and 0.130% CLC were collected. and after a further 6 hours, 0.12 g of an oil extract containing 1.258% CLA, 1.160% CLE and 0.600% CLC.

The extracts were purified by column chromatography.

In total, the obtained solid fraction was 8.5 mg with CLA content 73% (HPLC) and a solid fraction of 11 mg containing 36% CLA (HPLC), as well as a solid fraction of 11.7 mg containing 68% CLE cyclopeptide and the fraction a solid of 8.2 mg containing 62% CLC.

P r z k ł a d V.

Extraction was carried out in the extractor as in example 1, but instead of linseed cakes, oil in the amount of 100 g was used, obtained by extraction with toluene of linseed cakes, containing 0.1278% CLA. The extraction temperature was 22 ° C and the pressure was 20 MPa. After 18 hours of extraction, 17.0 g of oil extract containing 0.110% CLA, 0.037% CLE and 0.098% CLC were collected, after another 15 hours of extraction, 14.39 g of oil extract were collected with 0.58% CLA, 0.30% CLE and 0 , 47% CLC and after a further 10 hours 9.28 g of oil extract containing 0.13% CLA, 0.012% CLE and 0.15% CLC.

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The extracts were purified by column chromatography.

In total, there was obtained a solid fraction of 95.5 mg with the content of CLA 84% (HPLC) and a solid fraction of 56 mg containing 61% of CLA (HPLC), a solid fraction of 63 mg containing 72% of CLE cyclopeptide and a solid fraction of 118 mg containing 76% CLC.

P r x l a d VI.

Extraction was carried out as in example 1, with carbon dioxide under supercritical conditions, at a temperature of 40 ° C, under a pressure of 20 MPa, with linseed chaff being used instead of linseed cakes. After 25.5 hours of extraction, 2.27 g of an oil extract containing 0.063% ClA and 0.062% CLC were collected.

The extracts were purified using column chromatography. In total, there was obtained a solid fraction of 3.1 mg with 48% CLA content (HPLC) and a solid fraction of 3.2 mg containing 45% CLC.

Claims (7)

Patent claims A method of separating cyclopeptides from flax, characterized in that the flax, preferably dried and ground, or linseed oil, or a waste product from the processing of flax, such as linseed cake or chaff, preferably ground, is extracted with liquid or supercritical carbon dioxide and, if necessary, the extract is separated into fractions enriched in the desired cyclopeptides. 2. The method according to p. The process of claim 1, wherein the extraction is carried out at a temperature of 15 ° C to 80 ° C, under a pressure of 7.3 MPa to 50 MPa. 3. The method according to p. The method of claim 1, characterized in that the extraction is carried out with the addition of an entrainer, which is a polar or non-polar substance. 4. The method according to p. The process of claim 3, characterized in that a C1-C8 aliphatic alcohol, in particular methanol, ethanol or propan-2-ol, is used as the polar entrainer. 5. The method according to p. The process of claim 3, wherein the non-polar entrainer is ethyl acetate. 6. The method according to p. The process of claim 1, wherein the extract is fractionated by supercritical fractionating extraction. 7. The method according to p. The process of claim 1, wherein the extracts are purified by chromatography.