WO2013091070A1 - Mélanges de peptides cycliques provenant d'une graine de lin et leurs utilisations - Google Patents

Mélanges de peptides cycliques provenant d'une graine de lin et leurs utilisations Download PDF

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WO2013091070A1
WO2013091070A1 PCT/CA2012/001165 CA2012001165W WO2013091070A1 WO 2013091070 A1 WO2013091070 A1 WO 2013091070A1 CA 2012001165 W CA2012001165 W CA 2012001165W WO 2013091070 A1 WO2013091070 A1 WO 2013091070A1
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Prior art keywords
lcp
mso
mixture
methionine
msn
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PCT/CA2012/001165
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English (en)
Inventor
Martin John Reaney
Peta-Gaye Gillian BURNETT
Pramodkumar Dinkar JADHAV
Denis Paskal OKINYO-OWITI
Jianheng Shen
Youn Young Shim
Original Assignee
Martin John Reaney
Burnett Peta-Gaye Gillian
Jadhav Pramodkumar Dinkar
Okinyo-Owiti Denis Paskal
Jianheng Shen
Youn Young Shim
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Application filed by Martin John Reaney, Burnett Peta-Gaye Gillian, Jadhav Pramodkumar Dinkar, Okinyo-Owiti Denis Paskal, Jianheng Shen, Youn Young Shim filed Critical Martin John Reaney
Publication of WO2013091070A1 publication Critical patent/WO2013091070A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/55—Linaceae (Flax family), e.g. Linum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/64—Cyclic peptides containing only normal peptide links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
    • A61K2236/30—Extraction of the material
    • A61K2236/39—Complex extraction schemes, e.g. fractionation or repeated extraction steps

Definitions

  • Flax Lium usitatissimum, also known as common flax or linseed
  • the edible oil, linseed oil or flaxseed oil is obtained by extraction of flaxseed. Consumption of flaxseed oil increases concentration of a-linolenic acid (ALA) in blood plasma (2, 3, 4).
  • ALA is a precursor of the polyunsaturated fatty acids (PUFA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) that reduce the risks of cardiovascular diseases (2, 3, 5).
  • Flaxseed contains secoisolariciresinol diglucoside (SDG), a phytochemical antioxidant that acts as a precursor of mammalian lignans and a phytoestrogen (6, 7, 8):
  • Flaxseed oil also contains natural hydrophobic cyclic peptides comprising eight or nine amino acid residues.
  • the cyclopeptides were often referred to as cyclolinopeptides or CLs (A to K, Table 1), a designation that was associated with their discovery and isolation from Linum usitatissimum.
  • CLs cyclolinopeptides
  • a new designation is proposed that includes both the genus (3 letters - lin) and species (two letters - us) names. Therefore, the term Linus cyclopeptide or LCP has been used hereafter (Tables 1 and 2). This naming is consistent with that used in international protein databases. The new designation allows for expanded-naming of LCPs derived from the same mother compound, whereas the previous naming system was inconsistent and created considerable confusion.
  • Linus cyclopeptide amino acids were previously numbered arbitrarily or based on mass spectrometry fragmentation patterns. Thus many cyclic peptides are numbered with a prolyl residue as the first amino acid in the sequence.
  • Convention in numbering of protein amino acids begins from the N-terminal containing the free alpha amino group and proceeds to the C- terminal containing the free carboxyl group.
  • isoleucine (lie) is designated as the first amino acid residue in LCP-9
  • methionine (Met) is the first amino acid residue in sequences of other LCPs as shown in Table 2.
  • LCP-9 The amino acid sequences and structures of LCPs are shown in Table 2 and Figure 1 , respectively.
  • the first discovered linseed cyclopeptide, LCP-9 was isolated in 1959 (21 ) and later was shown to have immunosuppressant activity (22, 23). Since the original discovery of LCP-9, eight other LCPs have been isolated from flaxseed oil (14, 24, 25, 26).
  • Other cyclopeptides, LCP-1 and LCP-3 that do not contain oxidized methionine
  • CID-MS CID-MS, but there are no reports of extensive spectroscopic characterization (15). Note that methionine is often oxidized to methionine sulfoxide (Mso) after synthesis or isolation.
  • Cyclopeptides LCP-7, LCP-9 and (Mso 1 )-LCP-5 were previously shown to exhibit immunosuppressive activity (22, 23, 24) and induce apoptosis in human lung epithelial cancer cell lines (27). Also note that methionine residues may be further oxidized to the methionine sulfone (Msn).
  • Msn methionine sulfone
  • a recent review showcased two new potent immunosuppressant LCPs, (Msn )-LCP-5 and (Msn )-LCP-7 (28). Of all the studied LCPs, LCP-9 displayed the best immunosuppressive activity, followed by (Msn )-LCP-5 and (Msn 1 )-l_CP-7 (28). Cyclopeptides (Msn 1 )-LCP-5 and (Msn )-LCP-7 were P T/CA2012/001165
  • Stefanowicz also disclosed a recovery of hydrophobic peptides from flaxseed, although this process does not allow for the separation and isolation of each hydrophobic peptide.
  • the present application relates to methods for isolating cyclic peptides from flaxseed by simplifying the peptide extracts to increase the efficiency of chromatography for recovery of specific peptide(s).
  • the application also includes novel simplified mixtures of Linus cyclopeptides from flaxseed.
  • the present application includes a method of simplifying a mixture of cyclic peptides from flaxseed comprising: (a) treating a mixture of cyclic peptides from flaxseed oil with a chemical reagent that converts one or more functional groups in the cyclic peptides to a functional group that is common to one or more other cyclic peptides in the mixture to provide a simplified mixture; and, optionally,
  • the chemical reagent that converts the one or more functional groups in the cyclic peptides is selected from an oxidizing agent, a reducing agent and a desulfurization reagent.
  • the functional group in the cyclic peptides that is converted is a methionine and the chemical reagent converts all methionine moieties (Met) to methionine sulfoxides (Mso) or methionine sulfones (Msn).
  • the functional group is an oxidized form of methionine, such as methionine sulfoxide and/or methionine sulfone, and the chemical reagent converts all of these oxidized forms to methionine.
  • the functional group is a methionine, methionine sulfoxide and/or methionine sulfone, and the chemical reagent converts all of these functional groups to the corresponding desulfurized compound.
  • the mixture of cyclic peptides extracted from flaxseed oil comprises (Mso 1 )-LCP-5, (Msn )-L.CP-5, (Mso 1 )-LCP-7, (Msn )-LCP-7, and LCP-9.
  • cyclopeptides comprising methionine are treated with, for example, iodoacetonitrile in the presence of an amine base in an inert solvent at a temperature of about 50 °C to about 150 °C, or about 100 °C, for about 1 hour to about 48 hours to provide the corresponding "-SCH 2 CN" derivative.
  • novel derivatives may be treated with an oxidizing agent to provide the corresponding sulfoxide or sulfone nitrile.
  • Figure 1 shows the structures of LCPs that have been described in previous literature.
  • Figure 2 shows the structures of LCP-5 and modified cyclopeptides (Abu 1 )-LCP-5 and (Abu )-LCP-7.
  • Figure 3a shows an HPLC-DAD chromatogram of crude Linus cyclopeptide extract from fresh flax oil.
  • 1 (Mso 1 3 )-LCP-3; 2, (Mso 3 )-LCP-1 ; 3, (Mso )-LCP-7; 4, (Mso )-LCP-5; 5, (Mso 3 )-LCP-3; 6, (Mso 1 )-LCP-2; 7, (Mso )-LCP-1 ; 8, LCP-7; 9, LCP-3; 10, LCP-9; 11 , LCP-5; 12, LCP-1 ; 13, LCP-2.
  • Figure 3b shows an HPLC-DAD chromatogram of the crude
  • Linus cyclopeptide extract from trysil cake showing (Mso 1 )-LCP-7, (Msn 1 )- LCP-7, (Mso 1 )-LCP-5, (Msn )-LCP-5, and LCP-9 (from left to right).
  • Figure 3c shows an LC-MS chromatogram of the crude Linus cyclopeptide-enriched extract from trysil cake showing (Mso )-LCP-7, (Msn 1 )- LCP-7, (Mso 1 )-LCP-5, (Msn 1 )-LCP-5, and LCP-9 (from left to right).
  • Figure 3d shows HPLC-DAD chromatograms of isolated Linus cyclopeptides (Mso 1 )-LCP-7, a mixture of (Msn 1 )-LCP-7 and (Mso 1 )-LCP-5, (Msn 1 )-l_CP-5, and LCP-9, respectively (from top to bottom).
  • Figure 4 shows an HPLC-DAD chromatogram of (Mso 1 3 )-LCP-1
  • Figure 5 shows an LC-MS spectrum of the oxidized crude Linus cyclopeptide extract showing (Msn 1 )-LCP-7, (Msn )-l_CP-5, and LCP-9 (from left to right).
  • Figure 6 shows an LC-MS spectrum of the reduced crude Linus cyclopeptide extract showing (Msn 1 )-LCP-7, (Msn )-IXP-5, LCP-7, and LCP-5 (from left to right). Note that LCP-5 and LCP-9 co-elute.
  • Figure 7a shows an ESI-MS spectrum of (Abu 1 )-LCP-7 obtained from desulfurization of (Mso 1 )-LCP-7.
  • Figure 7b shows an ESI-MS spectrum of (Abu )-IXP-5 obtained from desulfurization of (Mso 1 )-LCP-5.
  • Figure 7c shows an ESI-MS spectrum of (Abu )-LCP-1 obtained from desulfurization of (Mso 3 )-LCP-1.
  • Figure 7d shows an ESI-MS spectrum of (Abu 1 Cy 4 )-LCP-5 obtained from desulfurization and reduction of phenylalanine 4 of (Msn 1 )-LCP- 5.
  • Figure 8a shows an LC-MS spectrum of the olefin (Dhb 1 )-LCP-7 obtained from desulfurization of (Mso )-LCP-7.
  • Figure 8b shows an LC-MS spectrum of the olefin (Dhb 1 )-l_CP-5 obtained from desulfurization of (Mso 1 )-LCP-5.
  • Figure 9a shows an H-NMR of the nitrile (MetCN 1 )-LCP-7 obtained from (Mso )-LCP-7.
  • Figure 9b shows an 13 C-NMR of the nitrile (MetCN 1 )-LCP-7 obtained from (Mso )-LCP-7.
  • Figure 9c shows an ESI-MS spectrum of the nitrile (MetCN 1 )- LCP-7 obtained from (Mso 1 )-L.CP-7.
  • Figure 9d shows an ESI-MS spectrum of the nitrile (MsoCN )-
  • Figure 9e shows an ESI-MS spectrum of the nitrile (MsnCN )-
  • Figure 10a shows an ESI-MS/MS spectrum of the methionine sulfonyl ethoxycarbonyl (MetEOC 1 )-LCP-7.
  • Figure 10b shows an ESI-MS/MS spectrum of the methionine sulfonyl ethoxycarbonyl (MsnEOC 1 )-LCP-7.
  • Figure 11 a shows dose response curves of LCP-9 binding to
  • Figure 11 b shows SPR sensorgram responses for Linus cyclopeptides (100pm) binding to HSA
  • 1 (Msn 1 )-LCP-5, (Msn )-LCP- 7, LCP-9
  • 2 (Abu ' 3 )-LCP-1 , (Abu 1 )-l_CP-5, (Abu 1 )-LCP-7, LCP-9
  • 3 (Abu 1 )-LCP-5, (Abu 1 )-LCP-7, LCP-9
  • 4 (Mso 1 )-l_CP-5, (Mso 1 )-LCP-7, LCP-9
  • 5 (Mso 3 )-LCP-1 , (Mso 1 )-LCP-5, (Mso )-LCP-7, LCP-9.
  • Figure 12 shows the mass spectrograph of (Msn 1 )-LCP-5.
  • Figure 13 shows over-oxidated cyclopeptide products.
  • Figure 14 shows stability of various cyclopeptides after addition of a quenching agent.
  • Figure 15 shows a correlation in protein levels of two different flax lines.
  • Figure 16 shows an HPLC chromatogram of an oxidized extract of flax line CDC Bethune.
  • Figure 17 shows an HPLC chromatogram of flax lines Hollandia and Z1 1637 in comparison to CDC Bethune.
  • Figure 18a shows a chromatogram of Hollandia and Z1 1637 (Figure 18a) flax lines from different years.
  • Figure 18b shows a chromatogram of Saskatoon and Morden flax lines from different years.
  • Figure 19 shows the structure of LCP-N.
  • Figure 20 shows a mass spectrograph and chromatogram of
  • Figure 21 shows the structure of (Mso )-LCP-N.
  • Figure 22 shows a mass spectrograph and chromatogram of (Mso )-LCP-N.
  • Figure 23 shows the structure of (Msn 1 )-LCP-N.
  • Figure 24 shows a mass spectrograph and chromatogram of
  • Figure 25 shows the amino acid sequence of various cyclopeptides.
  • Figure 26 shows the amino acid sequence of LCP-N.
  • the second component as used herein is chemically different from the other components or first component.
  • a “third” component is different from the other, first, and second components, and further enumerated or “additional” components are similarly different.
  • the term "simplified mixture” as used herein refers to a mixture of Linus cyclopeptides from flaxseed, in particular flaxseed oil extract that has been treated with a chemical reagent and treatment with the reagent serves to reduce the number of different cyclopetides in the mixture and/or simplify the separation of the different cyclopeptides in the mixture.
  • reducing agent means any compound or combination of compounds that reduces a desired functional group.
  • a reducing agent results in the overall addition of electrons, or in the case of organic chemistry, hydrogen atoms to the functional group.
  • oxidizing agent means any compound or combination of compounds that oxidizes a desired functional group. An oxidizing agent results in the overall loss of electrons, or in the case of organic chemistry, hydrogen atoms from the functional group.
  • the term "desulfurization agent” as used herein means any compound of combination of compounds that removes a sulfur-containing functional group from a molecule.
  • alkylating agent means any compound or combination of compounds that results in the addition of an alkyl group or substituted alkyl group to a molecule, typically via the reaction of a nucleophilic atom in the molecule (such as S, N or O) with the alkylating agent under nucleophilic substitution reaction conditions.
  • quenching agent means any compound or combination of compounds that prevents or diminishes the over- oxidation of a desired functional group.
  • alkyl group as used herein means a functional group comprising saturated, unsaturated, branched chain, unbranched chain and cyclic hydrocarbons.
  • An alkyl group may comprise from 1 to 20 carbon atoms.
  • substituted alkyl group as used herein means an alkyl group comprising one or more substituent groups, such as esters, amides, carboxylic acids, amines, alcohols and thiols.
  • the expression "proceed to a sufficient extent” as used herein with reference to the method or process steps disclosed herein means that the reaction or process steps proceed to an extent that conversion of the starting material or substrate to product is maximized. Conversion is maximized when greater than about 5, 10, 15, 20, 25, 30, 35, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100% of the starting material or substrate is converted to product.
  • subject as used herein includes all members of the animal kingdom including mammals, and suitably refers to humans.
  • pharmaceutically acceptable salt means an acid addition salt or a base addition salt, which is suitable for, or compatible with, the treatment of patients.
  • pharmaceutically acceptable acid addition salt means any non-toxic organic or inorganic salt of any basic compound.
  • Basic compounds that form acid addition salts include, for example, compounds comprising a thiol group.
  • Illustrative inorganic acids that form suitable salts include hydrochloric, hydrobromic, sulfuric and phosphoric acids, as well as metal salts such as sodium monohydrogen, orthophosphate and potassium hydrogen sulfate.
  • Illustrative organic acids that form suitable salts include mono-, di-, and tricarboxylic acids such as glycolic, lactic, pyruvic, malonic, succinic, glutaric, fumaric, malic, tartaric, citric, ascorbic, maleic, benzoic, phenylacetic, cinnamic and salicylic acids, as well as sulfonic acids such as p-toluene sulfonic and methanesulfonic acids.
  • Either the mono- or di-acid salts can be formed, and such salts may exist in either hydrated, solvated or substantially anhydrous forms.
  • acid addition salts are more soluble in water and various hydrophilic organic solvents, and generally demonstrate higher melting points in comparison to their free base forms. The selection of the appropriate salt will be known to one skilled in the art.
  • compositions that form a basic addition salt include, for example, compounds comprising a carboxylic acid group.
  • Illustrative inorganic bases that form suitable salts include lithium, sodium, potassium, calcium, magnesium or barium hydroxide.
  • Illustrative organic bases which form suitable salts include aliphatic, alicyclic or aromatic organic amines such as methylamine, trimethylamine and picoline, alkylammonias or ammonia. The selection of the appropriate salt will be known to a person skilled in the art.
  • a desired compound salt is achieved using standard techniques. For example, the neutral compound is treated with an acid or base in a suitable solvent and the formed salt is isolated by filtration, extraction or any other suitable method.
  • solvate means a compound or its pharmaceutically acceptable salt, wherein molecules of a suitable solvent are incorporated in the crystal lattice.
  • a suitable solvent is physiologically tolerable at the dosage administered. Examples of suitable solvents are ethanol, water and the like. When water is the solvent, the molecule is referred to as a "hydrate”.
  • solvates will vary depending on the compound and the solvate. In general, solvates are formed by dissolving the compound in the appropriate solvent and isolating the solvate by cooling or using an antisolvent. The solvate is typically dried or azeotroped under ambient conditions.
  • the compounds described herein have at least one asymmetric centre. Where compounds possess more than one asymmetric centre, they may exist as diastereomers. It is to be understood that all such isomers and mixtures thereof in any proportion are encompassed within the scope of the present application. It is to be further understood that while the stereochemistry of the compounds may be as shown in any given compound listed herein, such compounds may also contain certain amounts (e.g. less than 50%, suitably less than 20%, more suitably less than 10%, more suitably less than 5%) of compounds of the application having alternate stereochemistry.
  • treating means an approach for obtaining beneficial or desired results, including clinical results.
  • beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, diminishment of the reoccurrence of disease, and remission (whether partial or total), whether detectable or undetectable.
  • Treating and “Treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment.
  • Treating” and “treatment” as used herein also include prophylactic treatment.
  • a subject with early stage cancer can be treated to prevent progression or metastases, or alternatively a subject in remission can be treated with a compound of the application to prevent recurrence.
  • Treatment methods comprise administering to a subject a therapeutically effective amount of a compound of the application and optionally consist of a single administration, or alternatively comprise a series of applications.
  • the compounds of the application may be administered at least once a week.
  • the compounds may be administered to the subject from about one time per three weeks, or about one time per week to about once daily for a given treatment.
  • the compound is administered 2, 3, 4, 5, or 6 times daily.
  • the length of the treatment period depends on a variety of factors, such as the severity of the disease, the age of the patient, the concentration, the activity of the compounds of the application, and/or a combination thereof. It will also be appreciated that the effective dosage of the compound used for the treatment or prophylaxis may increase or decrease over the course of a particular treatment or prophylaxis regime. Changes in dosage may result and become apparent by standard diagnostic assays known in the art. In some instances, chronic administration may be required. For example, the compounds are administered to the subject in an amount and for a duration sufficient to treat the patient.
  • the term "effective amount” or “therapeutically effective amount” means an amount effective, at dosages and for periods of time necessary to achieve the desired result. Effective amounts may vary according to factors such as the disease state, age, sex, and weight of the subject. The amount of a given compound that will correspond to such an amount will vary depending upon various factors, such as the given drug or compound, the pharmaceutical formulation, the route of administration, the type of disease or disorder, the identity of the subject or host being treated, and the like, but can nevertheless be routinely determined by one skilled in the art.
  • administered means administration of a therapeutically effective dose of a compound or composition of the application to a cell either in cell culture or in a patient.
  • the present application includes a method of simplifying a mixture of cyclic peptides from flaxseed comprising:
  • the mixture of cyclic peptides is obtained by combining flaxseed oil with an adsorbent that binds the hydrophobic peptides from the oil, followed by separation of the adsorbent and extraction of the hydrophobic peptides from the adsorbent using suitable solvents.
  • the combining of the adsorbent with the flaxseed oil is done by combining about 0.5% (w/v) to about 10% (w/v), or about 1 % (w/v) to about 5% (w/v), adsorbent with flaxseed oil and mixing at a temperature of about 20 °C to about 100 °C, or about 50 °C to about 70 °C, for about 30 minutes to 12 hours, or about 1 hour to about 5 hours, after which the adsorbent is separated from the oil, for example, by filtration.
  • the adsorbent is silica.
  • the collected silica may be stored for any amount of time prior to further manipulation.
  • the longer the adsorbent is stored the greater the amount of methionine sulfoxide and methionine sulfone containing peptides in the peptide mixture.
  • the peptide-laden adsorbent is stored at about room temperature for 1 day to about 1 year. In a further embodiment, peptide-laden adsorbent is stored at about room temperature for less than 1 day.
  • the peptide-laden adsorbent is extracted with suitable solvents to obtain the mixture of cyclic peptides used in the methods of the present application. It is an embodiment that the peptide-laden adsorbent is extracted with a range of solvent systems starting with non-polar solvent systems (such as hexane), and moving toward more polar solvent systems (such as combinations of alcohols in non-polar solvents).
  • non-polar solvent systems such as hexane
  • the mixture of cyclic peptides is first extracted or washed with a non-polar solvent such as hexane, followed by extraction or washing with a combination of a polar solvent and a non-polar solvent, such as ethyl acetate in hexane, followed by extraction with an alcoholic solvent optionally in combination with a polar or non-polar solvent, such as about 5% (v/v) to about 20% (v/v) methanol in dichloromethane.
  • a non-polar solvent such as hexane
  • a non-polar solvent such as ethyl acetate in hexane
  • an alcoholic solvent optionally in combination with a polar or non-polar solvent, such as about 5% (v/v) to about 20% (v/v) methanol in dichloromethane.
  • the mixture of cyclic peptides used in the methods of the application is extracted from the peptide-laden adsorbent in the more polar solvent fractions, including, for example, methano!/dichloromethane extracted fractions.
  • the mixture of cyclic peptides extracted from the peptide-laden adsorbent is further treated to remove materials, such as other non-cyclic peptides, that are less soluble in an alcoholic solvent like methanol.
  • the materials that are less soluble in methanol are removed by dissolving the mixture of cyclic peptides in methanol and cooling to a temperature of about -10 °C to about - 30 °C, or about -20 °C, and separating, for example by filtration or decanting, the non-dissolved residues. In an embodiment, this procedure is repeated one or more times.
  • the mixture of cyclic peptides from flaxseed oil is obtained by combining flaxseed oil with an immiscible solvent that extracts hydrophobic peptides from the oil, followed by separation of the immiscible solvent and removal of that solvent to recover the hydrophobic peptides.
  • immiscible solvent it is meant any solvent that does not combine with flaxseed oil to make a homogeneous solution. Examples of such immiscible solvents are ethanol, methanol, and isopropanol. One skilled in the art would know that there are several other polar solvents that will not mix with flax oil. The hydrophobic peptides are then recovered via solvent removal.
  • the mixture of cyclic peptides from flaxseed oil is obtained by combining flaxseed oil with an acidic solution that extracts hydrophobic peptides from the oil, followed by separation of the acid and removal of the hydrophobic peptides from the solvent using suitable adsorbents and solvents.
  • the mixture of cyclic peptides from flaxseed oil comprises, consists essentially of or consists of (Mso 1 )-LCP-5, (Msn )-LCP-5, (Mso 1 )-LCP-7, (Msn )-LCP-7, and LCP-9.
  • the mixture of cyclic peptides from flaxseed oil comprises, consists essentially of or consists of (Mso 1 )-LCP-5, (Mso )-l_CP-7, and LCP-9.
  • the mixture of cyclic peptides from flaxseed oil comprises, consists essentially of or consists of (Mso 1 )-l_CP-5 a nd (Mso 1 )-LCP-7.
  • the mixture of cyclic peptides from flaxseed oil comprises, consists essentially of or consists of (Msn 1 )-LCP-5, (Msn )-LCP-7, and LCP-9.
  • the mixture of cyclic peptides from flaxseed oil comprises, consists essentially of or consists of (Mso 3 )-LCP-1 , (Mso 1 )-LCP-2, (Mso 3 )-LCP-3, (Mso )-LCP-5, (Mso 1 )-LCP-7, and LCP-9.
  • freshly pressed flaxseed oil comprises fifteen distinct cyclopeptides
  • the mixture of cyclic peptides from flaxseed oil comprises, consists essentially of or consists of: (Mso 1 )-LCP-1 , (Mso 1 )-LCP-1 , (Mso 3 )-LCP-1 , LCP-1 , (Mso 1 )- LCP-2, LCP-2, (Mso 3 )-LCP-3, (Mso 3 )-LCP-3, (Mso 1 )-LCP-3, LCP-3, (Mso 1 )- LCP-5, LCP-5, (Mso 1 )-LCP-7, LCP-7, and LCP-9.
  • the chemical reagent that converts the one or more functional groups in the cyclic peptides is selected from an oxidizing agent, a reducing agent, an alkylating agent and a desulfurization reagent.
  • the method further optionally comprises a quenching agent which prevents the over-oxidation of the functional group.
  • the functional group in the cyclic peptides that is converted is a methionine and the chemical reagent converts all methionine residues to methionine sulfoxides or methionine sulfones.
  • the functional group is an oxidized form of methionine, such as methionine sulfoxide and/or methionine sulfone, and the reagent converts all of these oxidized forms to methionine.
  • the functional group is a methionine, methionine sulfoxide and/or methionine sulfone and the reagent converts all of these functional groups to corresponding desulfurized compounds.
  • Products of desulfurization include the alkane/alkene amino acid (S)-2-amino-butanoic acid (Abu), (S)- homoserine (Hse), (S)-2-amino-3-butenoic acid (Abe), and (S)-2-amino-2- butenoic acid (Dhb).
  • the chemical reagent that converts the one or more functional groups in the cyclic peptides is an oxidizing agent.
  • the oxidizing agent is any oxidizing agent that oxidizes sulfur atoms.
  • the oxidizing agent is an inorganic or organic peroxide (i.e. a compound comprising an "O-O" functionality), sodium periodate, oxygen, singlet oxygen, ozone and/or OxoneTM.
  • the oxidizing agent is m-chloroperbenzoic acid.
  • the oxidizing agent is optionally used in combination with a quenching agent to prevent or diminish the over-oxidation of the functional group.
  • the quenching agent is sodium thiosulfate (Na 2 S 2 03), which, for example, prevents the uneven conversion of Mso-containing peptides to Msn-containing peptides.
  • the functional group in the cyclic peptides that is converted is a methionine and the chemical reagent is an oxidizing agent that converts all of the methionine residues to methionine sulfoxides or methionine sulfones. It is an embodiment that the methionine residues are converted to methionine sulfoxides by treating the mixture of cyclic peptides with about 1 equivalent of the oxidizing agent in an inert solvent, at a suitable temperature and time for the reaction to proceed to a sufficient extent, for example at about -20 °C to about 100 °C, for 10 minutes to about 24 hours.
  • the methionine residues are converted to methionine sulfones by treating the mixture of cyclic peptides with greater than 2 equivalents of the oxidizing agent in an inert solvent, at a suitable temperature and time for the reaction to proceed to a sufficient extent, for example at about 0 °C to about 100 °C, for 10 minutes to about 24 hours.
  • the oxidation process is terminated by the addition of sufficient reducing agent to neutralize any further oxidation.
  • the reducing agent is sodium thiosulfate.
  • Other reducing agents known to those skilled in the art may also prevent further oxidation of the peptides.
  • a mixture of cyclic peptides extracted from flaxseed oil comprises, consists essentially of or consists of (Mso 1 )-LCP-5, (Msn 1 )-LCP-5, (Mso )-LCP-7, (Msn 1 )-l_CP-7, and LCP-9 and treatment of this mixture with an oxidizing agent provides a simplified mixture comprising, consisting essentially of or consisting of (Msn 1 )-L.CP-5, ( sn 1 )- LCP-7, and LCP-9.
  • the functional group in the cyclic peptides that is converted is a methionine sulfoxide and/or methionine sulfone
  • the chemical reagent is a reducing agent that converts all of these oxidized forms of methionine to methionine.
  • reducing agents include, but are not limited to, metal hydride reducing agents including lithium aluminum hydride, sodium borohydride, diisobutylammonium hydride and sodium bis(2-methoxyethoxy)aluminum hydride, sodium amalgam and zinc/mercury amalgam.
  • methionine sulfoxides and/or methionine sulfones are converted to methionine residues by treating the mixture of cyclic peptides with excess reducing agent, for example sodium borohydride and iodine, at a suitable temperature and time for the reaction to proceed to a sufficient extent, for example at about -20 °C to about 100 °C, for 10 minutes to about 24 hours.
  • excess reducing agent for example sodium borohydride and iodine
  • the mixture of cyclic peptides from flaxseed oil comprises, consists essentially of or consists of cyclopeptides (Mso 1 )-LCP-5, (Msn )-LCP-5, (Mso 1 )-LCP-7, (Msn )-LCP-7, and LCP-9, and treatment of this mixture with a reducing agent provides a mixture comprising, consisting essentially of or consisting of cyclopeptides LCP-5, (Msn 1 )-LCP-5, LCP-7, (Msn 1 )-LCP-7, and LCP-9.
  • the functional group in the cyclic peptides that is converted is a methionine, methionine sulfoxide, and/or methionine sulfone
  • the chemical reagent is a desulfurization agent that converts all of these forms of methionine to the desulfurized alkane form.
  • desulfurization agents include, but are not limited to, metals such as Raney nickel in the presence of hydrogen and sodium.
  • methionine, methionine sulfoxides, and/or methionine sulfones are converted to 2-amino butyrate by treating the mixture of cyclic peptides with excess desulfurization agent, for example Raney nickel, in an alcohol solvent, at a suitable temperature and time for the reaction to proceed to a sufficient extent, for example at about 20 °C to about 150 °C, for 30 minutes to about 72 hours.
  • desulfurization agent for example Raney nickel
  • a mixture of cyclic peptides extracted from flaxseed oil comprises (Mso 1 )-l_CP-5, (Msn 1 )-LCP-5, (Mso 1 )- LCP-7, (Msn 1 )-LCP-7, and LCP-9 and treatment of this mixture with a desulfurization agent provides a mixture comprising LCP-9, and desulfurized cyclopeptides (Abu )-LCP-5 and (Abu 1 )-LCP-7 (see Figure 2).
  • a mixture of cyclic peptides extracted from flaxseed oil comprises (Mso )-LCP-5, (Msn 1 )-l_CP-5, (Mso 1 )-L.CP-7, (Msn 1 )-LCP-7, and LCP-9 and treatment of this mixture with a desulfurization agent provides a mixture comprising LCP-9, and desulfurized cyclopeptides (Abe 1 )-l_CP-5 and (Abe 1 )-LCP-7.
  • a mixture of cyclic peptides extracted from flaxseed oil comprises (Mso 1 )-IXP-5, (Msn )-LCP-5, (Mso 1 )-l_CP-7, (Msn 1 )-LCP-7, and LCP-9 and treatment of this mixture with a desulfurization agent provides a mixture comprising LCP-9, and desulfurized cyclopeptides (Dhb )-LCP-5 and (Dhb 1 )-LCP-7.
  • a mixture of cyclic peptides extracted from flaxseed oil comprises (Mso 1 )-LCP-5, (Msn )-LCP-5, (Mso 1 )-l_CP-7, (Msn 1 )- LCP-7, and LCP-9 and treatment of this mixture with a desulfurization agent provides a mixture comprising LCP-9, and desulfurized cyclopeptides (Abu 1 Cy 4 )-LCP-5, (Abu 1 Cy 7 )-LCP-5, (Abu Cy 6 )-LCP-7 and (Abu 1 Cy 7 )-LCP-7.
  • Another simplified mixture that is within the scope of the present application is a mixture comprising, consisting essentially of or consisting of LCP-1 , LCP-2 and LCP-3.
  • a simplified mixture of cyclic peptides comprising, consisting essentially of or consisting of LCP-N, (Mso 3 )-LCP-1 , (Mso 1 )-l_CP-2, (Mso )-LCP-3, (Mso 1 )- LCP-5 and (Abu 1 )-LCP-7.
  • the disclosure also includes a mixture or simplified mixture comprising, consisting essentially of or consisting of the cyclic peptide LCP-N, its oxidized derivative (Mso 1 )-LCP-N, and/or its subsequently oxidized derivative (Msn 1 )-LCP-N, in any ratio.
  • Another simplified mixture that is within the scope of the disclosure is a mixture comprising, consisting essentially of or consisting of any of the cyclopeptide mixtures of Table 3, in any ratio.
  • composition of claim 39 or 40 comprising the cyclopeptides as set out in Table 3 in any ratio.
  • the functional group in the cyclic peptides that is converted is a methionine and the chemical reagent is a alkylating agent that converts this form of methionine to an unstable methionine sulfonium intermediate.
  • an alkylating agent is methylbromide and its reaction product with methionine is S-methyl methionine (Smm).
  • Smm S-methyl methionine
  • the methionine sulfonium produced by reaction with an alkylating reagent can then be selectively reacted to form elimination products including alkanes, alkenes and alcohols depending on reaction conditions.
  • Linus cyclopeptides comprising methionine are treated with a nitrile containing alkylating reagent, for example, iodoacetonitrile in the presence of an amine base in an inert solvent at a temperature of about 50 °C to about 150 °C, or about 100 °C, for about 1 hour to about 48 hours to provide the corresponding "-SCH 2 CN" derivative.
  • a nitrile containing alkylating reagent for example, iodoacetonitrile in the presence of an amine base in an inert solvent at a temperature of about 50 °C to about 150 °C, or about 100 °C, for about 1 hour to about 48 hours to provide the corresponding "-SCH 2 CN" derivative.
  • novel derivatives may be treated with an oxidizing agent to provide the corresponding sulfoxide or sulfone nitrile.
  • the cyclopeptide that is converted to a nitrile derivative is LCP-7
  • alkoxycarbonyl (EOC) derivatives are prepared alongside the oxidized forms thereof.
  • a Linus cyclopeptide comprising a methionine is treated with an alkoxycarbonyl-containing alkylating reagent, for example, ethyl iodoacetate in the presence of an amine such as diisopropylamine at a temperature of about 100 °C, for about 20 hours to provide the corresponding "-SCH2COOCH2CH3" derivative.
  • Treatment of these derivatives with an oxidizing agent provides the corresponding sulfinyl or sulfonyl ethoxycarbonyl.
  • the cyclopeptide that is converted to an ethoxycarbonyl derivative is LCP-7.
  • compositions of the Application also includes the simplified mixtures of cyclopeptides obtained using the methods described herein.
  • the present application includes a composition comprising (Msn 1 )-LCP-5, (Msn )-LCP-7 and LCP-9.
  • the composition comprising (Msn 1 )-l_CP-5, (Msn 1 )-l_CP-7 and LCP-9 is obtained using the method of the present application.
  • compositions comprising (Mso 1 ,3 )-LCP-1 , (Mso 1 )-LCP-1 , (Mso 3 )-LCP-1 , LCP-1 , (Mso 1 )-LCP-2, LCP-2, (Mso 3 )-LCP-3, (Mso 3 )-LCP-3, (Mso )-LCP-3, LCP-3, (Mso )-LCP-5, LCP-5, (Mso 1 )-LCP-7, LCP-7, and LCP- 9 upon oxidation yields a simplified composition comprising (Msn 1 3 )-LCP-1 , (Msn 1 )-LCP-2, (Msn 3 )-LCP-3, (Msn )-LCP-5, (Msn 1 )-LCP-7, and LCP-9.
  • the present application includes a composition comprising LCP-9 and desulfurized cyclopeptide derivatives (Abu 1 )-LCP-5 and (Abu 1 )-LCP-7 (see Figure 2).
  • the composition comprising LCP-9, (Abu )-LCP-5, and (Abu 1 )-LCP-7 is obtained using the method of the present application.
  • the mixture of cyclic peptides extracted from flaxseed oil comprising (Mso 1 ,3 )-LCP-1 , (Mso )-LCP-2, (Mso 1 3 )-LCP-3, (Mso 1 )-LCP-5, (Mso 1 )-LCP-7, and LCP-9 is converted to (Abu 1 3 )-LCP-1 , (Abu 1 )-LCP 2, (Abu 1 ' 3 )-LCP-3, (Abu 1 )-LCP-5, (Abu 1 )-LCP-7, while LCP-9 remains unchanged.
  • the present application includes a composition comprising LCP-9 and homoserine cyclopeptide derivatives (Hse )-LCP-5 and (Hse )-LCP-7.
  • the composition comprising LCP-9, (Hse 1 )-LCP-5, and (Hse 1 )-LCP-7 is obtained using the method of the present application.
  • the mixture of cyclic peptides extracted from flaxseed oil comprising cyclopeptides (Mso 1 3 )-LCP-1 , (Mso )-LCP-2, (Mso 3 )- LCP-3, (Mso )-LCP-5, (Mso )-LCP-7, and LCP-9 is converted to (Hse 3 )- LCP-1 , (Hse 1 )-LCP-2, (Hse 3 )-LCP-3, (Hse )-LCP-5, (Hse )-LCP-7, while LCP-9 remains unchanged.
  • the present application includes a composition comprising cyclopeptide LCP-9 and 2-amino-3-butenoic acid cyclopeptide derivatives (Abe 1 )-IXP-5 and (Abe 1 )-l_CP-7.
  • the composition comprising LCP-9, 2-amino-3-butenoic acid cyclopeptides (Abe 1 )-LCP-5, anc j (Abe )-LCP-7 is obtained using the method of the present application.
  • the mixture of cyclic peptides extracted from flaxseed oil comprising cyclopeptides (Mso 1 3 )-LCP-1 , (Mso 1 )- LCP-2, (Mso 1 ' 3 )-LCP-3, (Mso 1 )-LCP-5, (Mso )-LCP-7, and LCP-9 is converted to 2-amino-3-butenoic acid cyclopeptides (Abe 1 3 )-LCP-1 , (Abe 1 )-LCP-2, (Abe 1 ,3 )-LCP-3, (Abe 1 )-LCP-5, (Abe )-LCP-7, while LCP-9 remains unchanged.
  • peptides extracted from flaxseed oil comprising fifteen distinct cyclopeptides (Mso 1 3 )-LCP-1 , (Mso 1 )-LCP-1 , (Mso 3 )-LCP-1 , LCP-1 , (Mso )-LCP-2, LCP-2, (Mso 3 )-LCP-3, (Mso 3 )-LCP-3, (Mso )-LCP-3, LCP-3, (Mso 1 )-LCP-5, LCP-5, (Mso 1 )-LCP-7, LCP-7, and LCP-9 is converted to LCPs (Abu 1 3 )-LCP-1 , (Abu 1 )-LCP-2, (Abu 1 3 )-LCP-3, (Abu 1 )-LCP-5 (Abu 1 )-LCP-7, and LCP-9.
  • compositions may comprise one or more carriers and/or excipients.
  • the carriers and excipients would be pharmaceutically acceptable.
  • Conventional procedures and ingredients for the selection and preparation of suitable pharmaceutical compositions are described, for example, in Remington's Pharmaceutical Sciences (2000 - 20th edition) and in The United States Pharmacopeia: The National Formulary (USP 24 NF19) published in 1999.
  • compositions of the present application are novel. Therefore, the present application includes all uses of the compositions of the present application, including, but not limited to, pharmaceutical, veterinary, diagnostic, cosmetic, in chemical synthesis, drug delivery, and nutraceutics.
  • Linus cyclopeptides is more efficient and provides sufficient quantities of cyclic peptides at lower costs than known methods. Furthermore, many of the cyclopeptide derivatives possess lower polarity and are thermally stable, making these derivatives more resistant to oxidation and available for use in high-temperature and long-term storage applications.
  • Linus cyclopeptides can potentially control parasitic infections and display antibacterial, antifungal, antimalarial, antioxidant, and antiinflammatory properties. Additionally, cyclopeptides and derivatives are known to bind to a variety of ligands and metal ions (thereby acting as cryptands), a property that can be exploited for their use in drug delivery systems, in addition to immobilization of toxic wastes leading to reduced damage to human and animal tissue. Likewise, these peptides may be combined in certain ratios to tailor the binding, transport and subsequent delivery of compounds in the blood and/or serum. Linus cyclopeptides and their derivatives may provide a synergistic effect when bound to drugs owing to their biological activities.
  • Linus cyclopeptides and their derivatives may also be useful as catalysts in asymmetric synthesis of organic compounds.
  • the antioxidant property of Linus cyclopeptides and their derivatives make them potential additives to food products to increase shelf life, in addition to the health benefits derived from them.
  • the indole-containing Linus cyclopeptides may form useful fluorescent products.
  • compositions of the application has been investigated using SPR where the binding of mixtures of cyclopeptides with Human Serum Albumin (HSA) has been shown to be modulated by the oxidation state of the peptides.
  • HSA Human Serum Albumin
  • FT-IR Fourier transform infrared
  • HPLC-DAD analysis was performed with an Agilent 1200 series HPLC system equipped with a quaternary pump, autosampler, diode array detector (wavelength range 190-300 nm), degasser, and a ZORBAX Eclipse XDB-C18 (reverse phase) column (5 ⁇ particle size silica, 4.6 ⁇ 150 mm) equipped with an online filter.
  • the mobile phase consisted of H 2 0-acetonitrile (45:55 for 6 min, to 10:90 in 18 min at a flow rate of 0.5 mL/min).
  • Preparative reverse phase chromatography was performed on BioCad Perfusion Chromatography Workstation equipped with a Prep-ODS column (10 vm particle size silica gel, 250 * 30 mm), and a UV/VIS detector operating at a wavelength of 214 nm.
  • the mobile phase consisted of H 2 0-acetonitrile (either 45:55 for 8 min, to 10:90 in 30 min or 45:55 for 30 min, to 10:90 in 1 min or 35:65 to 30:70 in 10 min, to 0: 100 in 20 min) at a flow rate of 7.0 mL/min.
  • the mobile phase consisted of a linear gradient of solvent A, H 2 0 containing 0.1 % formic acid, and solvent B, acetonitrile containing 0.1 % formic acid; A:B (70:30 to 40:60 in 40 min, then to 0: 100 in 10 min) at a flow rate of 0.25 mL/min.
  • Example 1 Fresh flax oil extract contains at least thirteen unique cyclopeptides (Mso 1 3 )-LCP-1 , (Mso 1 )-LCP-1 , LCP-1 , (Mso 1 )-LCP-2, LCP-2, (Mso 1 3 )-LCP-3, (Mso 3 )-LCP-3, LCP-3, (Mso 1 )-IXP-5, LCP-5, (Mso 1 )-LCP-7, LCP-7, and LCP-9 ( Figure 3a).
  • the tryptophan (Trp) containing LCPs such as (Mso 1 3 )-LCP-1 , (Mso 1 )-LCP-1 , LCP-1 , (Mso 1 )-LCP-2, LCP-2, (Mso 1 3 )-LCP-3, (Mso 3 )-LCP-3, and LCP-3 are detected in trace amounts, or are completely lost. Controlled treatments are required to produce a high yield of simplified peptides.
  • Example 1a Isolation of LCPs from flaxseed oil
  • Trysil 40 kg was added to 1860 kg of flax oil and the oil was passed through an industrial plate and frame filter. After filtration, residual oil in the filter was reduced by passing pressurized nitrogen through the filter body. Eighty kg of oil-soaked Trysil cake was stored in plastic pails at 4 °C and processed in the laboratory in 0.5-2 kg lots to obtain crude peptide fractions. Oil-soaked Trysil cake (500 g), was extracted with hexane (1 L) in a beaker for 30 minutes with periodic stirring, followed by filtration through a sintered glass funnel. The residue was re-extracted under similar conditions with hexane (1 L x 2).
  • the recovered silica was sequentially extracted with 50% (v/v) ethyl acetate in hexane (1 L * 3) and 10% methanol (MeOH) in dichloromethane (CH 2 CI 2 ) (1 L 3). An aliquot of each filtrate was concentrated using a rotary evaporator and was dissolved in MeOH followed by HPLC-DAD analysis.
  • the chromatography conditions were: column flow: 0.50 mL/min; Solvent A: 45% d.d. H 2 0; Solvent B: 55% MeCN.
  • the solvent gradient was as follows:
  • a portion of the cyclopeptide residue from example 1 a was dissolved in EtOAc (500 mL), transferred to a separatory funnel, and was extracted with a saturated solution of sodium bicarbonate (aq. NaHCC>3, 500 mL). After phase separation, the middle phase was extracted with EtOAc (200 mL). The organic layers from the first and second extraction were combined and were extracted with saturated aq. NaHC0 3 (700 mL). The organic layer was then extracted with brine (saturated aq. NaCI) to remove excess water.
  • a portion of the crude cyclopeptide extract from example 1 b was further separated by silica gel chromatography.
  • the solvent systems used included, in the following order: 80% EtOAc in hexane, 100% EtOAc, 2.5% MeOH in CH 2 CI 2 , 5% MeOH in CH 2 CI 2 , 7.5% MeOH in CH 2 CI 2 , and 10% MeOH in CH 2 CI 2 .
  • Fractions containing predominantly cyclopeptides (Mso 1 )- LCP-5 and (Mso 1 )-l_CP-7 were retained separately from those containing predominantly cyclopeptides (Msn )-LCP-5, (Msn )-LCP-7, and LCP-9.
  • Example 1 e Isolation of individual cyclopeptides from crude extract
  • a portion of the crude cyclopeptide extract from example 1 c was further separated by preparative chromatography on a BioCad Perfusion Chromatography Workstation equipped with a UV/Vis detector (operating at a wavelength of 214 nm), and a Hypersil octadecylsilane (ODS) column (10 ⁇ particle size silica, 250 x 30 mm).
  • the chromatography conditions were: Column flow: 7.0 mL/min; Solvent A: 45% d.d. H 2 0; Solvent B: 55% of MeCN.
  • the elution gradient was as follows:
  • Flaxseed oil was loaded into a column packed with silica gel (silica to oil 1 : 10 w/v) and was sequentially eluted with hexane (1 L x 2), 50% (v/v) EtOAc in hexane (1 L), 100% EtOAc (2 L), and 10% MeOH in CH 2 CI 2 (2 L). An aliquot of each fraction was concentrated using a rotary evaporator and was taken in MeOH for HPLC-DAD analysis (HPLC parameters and chromatographic conditions were identical to those in example 1 a).
  • the following example discloses a different method for isolating hydrophobic peptides from flax oil.
  • the crude cyclopeptide extract of Trysil cake contains cyclopeptides (Mso 1 )-LCP-5, (Msn )-IXP-5, (Mso 1 )-LCP-7, (Msn )-LCP-7, and LCP-9, of which cyclopeptides (Mso 1 )-LCP-5 and (Msn 1 )- LCP-7 co-elute under the present chromatographic conditions.
  • Example 3a Oxidation of crude methionine-containing cyclopeptide extract
  • Crude cyclopeptide extract (1 g) previously obtained from similar methods as those described in example 1 c, was dissolved in CH2CI2 (20 mL). The mixture was treated with m-CPBA (90 mg, 3 equivalents) at room temperature with constant stirring. After 1 h, the reaction mixture was sequentially washed with a solution of 10% sodium thiosulphate (Na 2 S 2 0 3 ) (5 ml_), saturated aq. NaHC0 3 and d.d. H 2 0 to yield an oxidized crude cyclopeptide extract (960 mg).
  • the oxidized crude cyclopeptide extract comprised Linus cyclopeptides (Msn 1 )- LCP-5, (Msn 1 )-LCP-7, and LC p-9 ( Figure 5) .
  • Example 3b Isolation of individual cyclopeptides from oxidized crude extract
  • Example 5b Desulfurization of cyclopeptide (Mso 1 )-LCP-5 to (Abu 1 )-LCP-5 [00136] Cyclopeptide (Mso 1 )-LCP-5 (200 mg) was dissolved in EtOH (50 mL). To this solution, a slurry of Raney Nickel (2 g) in EtOH was added. The reaction mixture was left to stand at room temperature and was monitored by HPLC-DAD. After 48 h, the mixture was filtered through a celite column and was concentrated by rotary evaporation to yield the product.
  • Example 5c Desulfurization of cyclopeptide (Mso 1 3 )-LCP-1 to (Abu 1 3 )-LCP-1 [00137] Cyclopeptide (Mso 1 3 )-LCP-1 (50 mg) isolated from example 2 was dissolved in EtOH (15 ml_). To this solution, a slurry of Raney Nickel (0.5 g) in EtOH was added. The reaction mixture was left to stand at room temperature and was monitored by HPCL-DAD. After 48 h, the mixture was filtered through a celite column and was concentrated by rotary evaporation to yield the product. The HPLC-DAD and MS analyses indicated that the desulfurization of sulfoxides in (Mso 1 3 )-LCP-1 to (Abu 3 )-LCP-1 is completed and no further purification is required (Figure 7c).
  • Example 5d Desulfurization of cyclopeptide (Msn 1 )-LCP-5 to (Abu 1 Cy 4 )-LCP- 5
  • Example 6a Generation of olefin from cyclopeptide (Mso 1 )-LCP-7
  • Neat cyclopeptide (Mso 1 )-LCP-7 was heated (240 °C) for 16 h in Kugelrohr apparatus under vacuum. The reaction was monitored by HPLC- DAD. The reaction mixture was subjected to HPLC-MS analyses which indicated a quasimolecular ion at m/z 1010.6057 [M+H] + corresponding to a molecular formula of CssHeoNgOg ( Figure 8a). The starting material was not detected by HPLC analyses, suggesting complete conversion of cyclopeptide (Mso 1 )-LCP-7 to olefin (Dhb )-LCP-7.
  • Example 6b Generation of olefin from cyclopeptide (Mso 1 )-LCP-5
  • Neat cyclopeptide (Mso 1 )-LCP-5 was heated to 240 °C for 16 h in Kugelrohr apparatus under vacuum. The reaction was monitored by HPLC- DAD. The reaction mixture was subjected to HPLC-MS analyses which indicated a quasimolecular ion at m/z 913.5390 [M+H] + corresponding to a molecular formula of C50H73 8O8 ( Figure 8b). HPLC-DAD analysis showed full conversion of (Mso 1 )-LCP-5 to 0
  • Example 6c Thermal stability of cyclopeptide (Msn 1 )-LCP-7
  • Neat cyclopeptide (Msn 1 )-LCP-7 was heated to (240 °C) for 16 h in Kugelrohr apparatus under vacuum.
  • the HPLC analyses of the reaction mixture showed the presence of cyclopeptide (Msn 1 )-l_CP-7 and no other detectable products of reaction.
  • Example 6d Thermal stability of cyclopeptide (Msn 1 )-LCP-5
  • Neat cyclopeptide (Msn )-LCP-5 was heated to (270 °C) for 4 h in Kugelrohr apparatus under vacuum.
  • the HPLC-DAD analyses of the reaction mixture showed the presence of cyclopeptide (Msn 1 )-LCP-5 and no other detectable products of reaction.
  • Example 7a Generation of nitrite (MetCN 1 )-LCP-7 from cyclopeptide LCP-7
  • Cyclopeptide LCP-7 50 mg was dissolved in N- methylpyrrolildinone (2.5 mL) at room temperature.
  • the reaction mixture was extracted with EtOAc and the combined organic extract was washed with water and was subsequently concentrated under high vacuum to yield a crude mixture.
  • HPLC analyses indicated full conversion of cyclopeptide LCP-7 to (MetCN )-LCP-7.
  • Example 7b Oxidation of (MetCN 1 )-LCP-7 to (MsoCN 1 )-LCP-7
  • Example 7c Oxidation of (MetCN 1 )-LCP-7 to (MsnCN 1 )-LCP-7
  • (MetCN )-LCP-7 (Example 7a, 9.4 mg) was dissolved in dichloromethane (1 mL) to which m-CPBA (6.4 mg) was added. The reaction mixture was stirred at room temperature for 1 h, and was sequentially washed with a solution of 10% Na 2 S 2 0 3 (5 mL), saturated aq. NaHC0 3 , and d.d. H 2 0 to yield sulfonyl nitrile (CMsn 1 )-LCP-7 (10 mg).
  • CMsn 1 sulfonyl nitrile
  • Example 8a Generation of ethoxycarbonyl (MetEOC 1 )-LCP-7 from cyclopeptide LCP-7
  • Cyclopeptide LCP-7 (300 mg) was dissolved in N- methylpyrrolildinone (10 ml_) at room temperature. Diisopropylethylamine (0.123 ml_) and ethyl iodoacetate (0.234 ml_) were sequentially added to the solution and the resulting mixture was heated to 100 °C for 20 h. The reaction mixture was extracted with EtOAc and the combined organic extract was washed with water and was subsequently concentrated under high vacuum to yield a crude mixture. HPLC analyses indicated full conversion of cyclopeptide LCP-7 to (MetEOC 1 )-LCP-7.
  • Example 8b Oxidation of (MetEOC 1 )-LCP-7 to (MsnEOC 1 )-LCP-7
  • Example 9 Asymmetric catalytic epoxidation of chalcone with cyclopeptides
  • HSA Human serum albumin
  • Running buffer for all experiments was 1 x phosphate- buffered saline (PBS; 10 mM phosphate buffer, 2.07 mM KCI, 137 mM NaCI, pH 7.4) containing 5% DMSO.
  • Running buffers were filtered and degassed daily before use.
  • Analyte solutions containing the Linus cyclopeptides were prepared by diluting a stock solution (10 mM in DMSO) into 1.05 X PBS, giving a final 1 X PBS, pH 7.4 buffer containing a 5% DMSO. Fresh solutions were prepared preferably just before use, but could be stored at 4 °C for 3 days.
  • HSA was covalently immobilized onto a CM5 sensor surface at 25 °C using standard amine-coupling chemistry. Briefly, CM5 sensor chips were preconditioned prior to HSA immobilization by priming the instrument 3 times with HBS-EP buffer, followed by three successive 1 min injections of 50 mM NaOH at 50 pL/min. Flow cell 1 (Fc1 ) and flow cell 2 (Fc2) were activated by a 7 min injection of a 1 : 1 mixture of 0.1 M NHS and 0.4 M EDC using a flow rate of 5 pL/min. Immediately after activation, HSA ( 30 pg/mL, 10 mM acetate buffer pH 5.3) was immobilized onto Fc2.
  • Example 12 Ratio of peptide levels in Flax lines
  • Figure 16 shows the HPLC chromatogram of the oxidized extract of flax line CDC Bethune
  • Figure 17 shows the ' HPLC chromatogram of flax lines Hollandia and Z1 1637 in comparison to CDC Bethune, showing an additional peak at 3.1 minutes.
  • Figure 18 further shows the peak at 3.1 from the Hollandia and Z1 1637 (Figure 18a) flax lines from different years, showing the peak is generally constant in these lines.
  • Figure 18b shows the same peak 3.1 from the Saskatoon and Morden flax lines from different years.
  • Figure 19 shows the structure of LCP-N identified as the peak at 3.1 minutes as described above.
  • the mass spectrograph and chromatogram of LCP-N are shown in Figure 20.
  • the structure of the oxidized form of LCP-N being (Mso 1 )-LCP-N is subsequently shown in Figure 21 , and the mass spectrograph and chromatogram of (Msn 1 )-l_CP-N a re shown in Figure 22.
  • Figure 25 shows the amino acid sequence of (Mso )- LCP-5 (MLVFPLFI), LCP-7 (MLIPPFFVI) and LCP-9 (ILVPPFFLI), while Figure 26, showing the sequence homology of 1-Met-CLN (MLIPPFFLI).
  • Table 1 LCP proposed new naming system, genes and CAS Number.
  • CL Cyclolinopeptide
  • LCP Linus cyclopeptide
  • Mso methionine sulfoxide
  • Msn methionine sulfone.
  • Ta ble 3 Peptide levels of F ax lines in mAu
  • CN refers to Canadian National accession number at Plant Gene
  • TMP refers to a temporary numbering system that were acquired but not 5 added to the PGRC collection.
  • G is (Mso 3 )-LCP-1
  • E is (Mso 1 )-LCP-5
  • v) ACE is the sum total of "peak areas" of the three groups of peptides (A+C+E) in the extract.
  • DFG is the sum total of "peak areas" of the three groups of
  • SDG secoisolariciresinol diglucoside
  • Prasad, K Reduction of serum cholesterol and hypercholesterolemic atherosclerosis in rabbits by secoisolariciresinol diglucoside isolated from flaxseed. Circulation, 1999, 99, 1355-1362. 1 1 . Prasad, K. Flaxseed and cardiovascular health. Journal of Cardiovascular Pharmacology, 2009, 54, 369-377.
  • Polonica 2001 48, 1 125-1 129.
  • Cyclolinopeptides B-E new cyclic peptides from Linum usitatissimum. Tetrahedron 1999, 55, 967-976.

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Abstract

La présente invention concerne des procédés d'isolement de peptides cycliques provenant d'une graine de lin par la simplification des extraits peptidiques pour augmenter l'efficacité de chromatographie pour la récupération du ou des peptides spécifiques. L'invention concerne également de nouveaux mélanges simplifiés de cyclopeptides provenant d'une graine de lin et leurs utilisations.
PCT/CA2012/001165 2011-12-19 2012-12-19 Mélanges de peptides cycliques provenant d'une graine de lin et leurs utilisations WO2013091070A1 (fr)

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KR101763475B1 (ko) 2015-09-04 2017-08-01 성균관대학교산학협력단 고리형 펩티드 혼합물을 포함하는 염증질환 치료용 조성물
FR3077202A1 (fr) * 2018-02-01 2019-08-02 Sederma Utilisation de cyclopeptides en cosmetique
CN111411004A (zh) * 2020-03-31 2020-07-14 广东石油化工学院 一种利用变压吸附原理高效提纯沼气的方法和装置
CN112521451A (zh) * 2020-11-11 2021-03-19 北京大学深圳研究生院 一种稳定多肽及其制备方法
CN113105523A (zh) * 2021-03-19 2021-07-13 暨南大学 一种低环肽a含量的亚麻籽环肽混合物的制备方法
CN113684099A (zh) * 2021-07-23 2021-11-23 南昌大学 一种从废白土中提取亚麻籽油和环肽的方法
CN114831171A (zh) * 2022-05-17 2022-08-02 天津科技大学 适用于鲜果贮藏的高氧应激保鲜方法和应用
CN114920797A (zh) * 2022-06-30 2022-08-19 暨南大学 一种从亚麻籽粕饼制备高纯度亚麻籽环肽的方法

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WO2009079792A1 (fr) * 2007-12-21 2009-07-02 University Of Saskatchewan Récupérations de peptides hydrophobes à partir d'huiles

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MORITA, H. ET AL.: "Cyclolinopeptides B - E, new cyclic peptides from Linum usitatissimum", TETRAHEDRON, vol. 55, no. 4, 1999, pages 967 - 976, XP004151321 *

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101763475B1 (ko) 2015-09-04 2017-08-01 성균관대학교산학협력단 고리형 펩티드 혼합물을 포함하는 염증질환 치료용 조성물
JP2021513520A (ja) * 2018-02-01 2021-05-27 セダーマ 化粧品における環状ペプチドの使用
FR3077202A1 (fr) * 2018-02-01 2019-08-02 Sederma Utilisation de cyclopeptides en cosmetique
WO2019149450A1 (fr) 2018-02-01 2019-08-08 Sederma Utilisation de peptides cycliques dans un produit cosmétique
CN111670027A (zh) * 2018-02-01 2020-09-15 赛德玛公司 环肽在美容中的用途
JP7285848B2 (ja) 2018-02-01 2023-06-02 セダーマ 化粧品における環状ペプチドの使用
CN111411004A (zh) * 2020-03-31 2020-07-14 广东石油化工学院 一种利用变压吸附原理高效提纯沼气的方法和装置
CN112521451A (zh) * 2020-11-11 2021-03-19 北京大学深圳研究生院 一种稳定多肽及其制备方法
CN113105523A (zh) * 2021-03-19 2021-07-13 暨南大学 一种低环肽a含量的亚麻籽环肽混合物的制备方法
CN113105523B (zh) * 2021-03-19 2023-10-10 暨南大学 一种低环肽a含量的亚麻籽环肽混合物的制备方法
CN113684099A (zh) * 2021-07-23 2021-11-23 南昌大学 一种从废白土中提取亚麻籽油和环肽的方法
CN114831171A (zh) * 2022-05-17 2022-08-02 天津科技大学 适用于鲜果贮藏的高氧应激保鲜方法和应用
CN114920797A (zh) * 2022-06-30 2022-08-19 暨南大学 一种从亚麻籽粕饼制备高纯度亚麻籽环肽的方法

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