The present invention is directed to the use of a compound of formula (I) as antioxidant in oil,

wherein R ¹ and R ² are independently from each other H or C _M 1 -alkyl or (CH ₂ ) _n — OH with n being an integer from 1 to 4, or R ¹ and R ² represent together a keto group,

A is CHR ³ or C(=0), and

wherein R ³ , R ⁴ and R ⁶ are independently from each other H or Ci- ₄ -alkyl, and wherein R ⁵ is H or OH or Ci- ₄ -alkyl or Ci- ₄ -alkoxy;

wherein the oil contains polyunsaturated fatty acids and/or their esters, and wherein the oil is for human consumption.

Oils containing polyunsaturated fatty acids and/or their esters are gaining more and more attention, because of their beneficial health effects in humans. Since these oils are only of limited stability, because they are oxidized very easily, there is a need to provide efficient antioxidants for their stabilization.

Detailed description of the invention

This need is fulfilled by the present invention, which is directed to the use of a compound of formula (I) as antioxidant in oil, wherein R ¹ and R ² are independently from each other H or C _M 1 -alkyl or (CH ₂ ) _n — OH with n being an integer from 1 to 4, or R ¹ and R ² represent together a keto group,

A is CHR ³ or C(=0), and

wherein R ³ , R ⁴ and R ⁶ are independently from each other H or Ci- ₄ -alkyl, and wherein R ⁵ is H or OH or Ci- ₄ -alkyl or Ci- ₄ -alkoxy;

and with the preferences for the substituents R ¹ to R ⁶ as given below;

wherein the oil contains polyunsaturated fatty acids and/or their esters, and wherein the oil is for human consumption.

Compound of formula (I)

“alkyl” and“alkoxy” in the context of the present invention encompass linear alkyl and branched alkyl, and linear alkoxy and branched alkoxy, respectively.

In a preferred embodiment of the present invention R ¹ and R ² are

independently from each other H or C _I -alkyl or (CH ₂ ) _n — OH with n being an integer from 1 to 4, A is CHR ³ , and R ³ , R ⁴ and R ⁶ are independently from each other H or Ci- ₄ -alkyl and R ⁵ is H or Ci- ₄ -alkyl or Ci- ₄ -alkoxy in the compound of formula (I).

In a further preferred embodiment of the present invention R ¹ and R ² are independently from each other H or C _I -alkyl or (CH ₂ ) _n — OH with n being an integer from 1 to 4, A is CHR ³ , and R ³ , R ⁴ and R ⁶ are independently from each other H or Ci- ₄ -alkyl and R ⁵ is H or Ci- ₄ -alkyl or Ci- ₄ -alkoxy in the compound of formula (I) with the proviso that at least one of the

substituents R ⁴ , R ⁵ and R ⁶ is not methyl.

If one of the substituents R ¹ and R ² is an C5-11 -alkyl or if one of R ¹ and R ² is a (CH ₂ ) _n — OH group with 4 C-atoms, the other substituent is preferably H.

More preferably R ¹ and R ² are independently from each other H or Ci- ₄ -alkyl or (CH ₂ ) _n — OH with n being 1 or 2, R ³ , R ⁴ and R ⁶ are independently from each other H or Ci- ₂ -alkyl, and R ⁵ is H or Ci- ₂ -alkyl or Ci- ₂ -alkoxy.

Even more preferably R ¹ and R ² are independently from each other H or C1-4- alkyl or (CH ₂ ) _n — OH with n being 1 or 2, R ³ , R ⁴ and R ⁶ are independently from each other H or Ci- ₂ -alkyl, and R ⁵ is H or Ci- ₂ -alkyl or Ci- ₂ -alkoxy with the proviso that at least one of the substituents R ⁴ , R ⁵ and R ⁶ is not methyl.

Further, more preferably R ¹ and R ² are independently from each other H or Ci- ₂ -alkyl or (CH ₂ ) _n — OH with n being 1 or 2, R ³ , R ⁴ and R ⁶ are independently from each other H or Ci- ₂ -alkyl, and R ⁵ is H or Ci- ₂ -alkyl or Ci- ₂ -alkoxy, preferably with the proviso that at least one of the substituents R ⁴ , R ⁵ and R ⁶ is not methyl.

Furthermore, more preferably R ¹ and R ² are independently from each other H or methyl or (CH ₂ )— OH, R ³ , R ⁴ and R ⁶ are independently from each other H or methyl, and R ⁵ is H or methyl or methoxy, preferably with the proviso that at least one of the substituents R ⁴ , R ⁵ and R ⁶ is not methyl.

Most preferably R ³ is H, preferably with the proviso that at least one of the substituents R ⁴ , R ⁵ and R ⁶ is not methyl, more preferably with the proviso that R ⁵ and R ⁶ are not methyl.

The compound of formula (I) is preferably selected from the group of the compounds of formulae (II) and (III), more preferably from the group of the compounds of formula (IV):

whereby A is CH ₂ or C(=0), preferably whereby A is CH ₂ ;

whereby R ^5a is H or methoxy, preferably whereby R ^5a is H;

whereby R ^1a and R ^2a are independently from each other H, CH ₂ OH or linear Ci- ₃ -alkyl or branched C _4- n-alkyl or R ^1a and R ^2a represent together a keto group (i.e. R ^1a and R ^2a are together“=0”), preferably whereby R ^1a and R ^2a are independently from each other H, methyl, CH ₂ OH or [CH ₂ -CH ₂ -CH ₂ - CH(CH ₃ )] _m CH ₃ with m being 1 or 2 or R ^1a and R ^2a represent together a keto group;

whereby R ^1b and R ^2b are independently from each other CH ₂ OH or linear C _1-3 - alkyl or branched C _4- n-alkyl, preferably whereby one of R ^1b and R ^2b is methyl and the other one of R ^1b and R ^2b is CH ₂ OH or linear Ci- ₃ -alkyl or branched C _{4- 11} -alkyl, more preferably whereby one of R ^1b and R ^2b is methyl and the other one of R ^1b and R ^2b is methyl, CH ₂ OH or [CH ₂ -CH ₂ -CH ₂ -CH(CH ₃ )] _m CI-l ₃ with m being 1 or 2;

whereby R ^1c and R ^2c are independently from each other H or linear Ci- ₃ -alkyl or branched C _4- n-alkyl, preferably whereby R ^1c and R ^2c are independently from each other H, methyl or [CH ₂ -CH ₂ -CH ₂ -CH(CH ₃ )] _m CI-l ₃ with m being 1 or 2. Preferred examples of the compound of formula (II) are the compounds of formulae (1 ), (2), (3), (4), (7), (8), (10) and (1 1 ). Preferred examples of the compound of formula (III) are the compounds of formulae (5), (6) and (9).

Preferred examples of the compound of formula (IV) are the compounds of formulae (1 ), (2), (3), (7) and (8).

Especially preferred are the following compounds of formulae (1 ) to (1 1 ), whereby compounds of formulae (1 ) to (8) are more preferred, compounds of formulae (1 ) to (6) are even more preferred, compounds of formulae (1 ) to (4) are furthermore preferred, and the most preferred compound is the compound of formula (3):

The compound of formula (8) (chemical name: 2-(4,8-dimethylnonyl)-2- methyl-chroman-6-ol) is a novel compound. Thus, this compound is also an object of the present invention.

The compounds of the present invention are efficient as antioxidants in PUFA-containing oils for human consumption.

“PUFA(s)” means polyunsaturated fatty acid(s) such as docosahexaenoic acid (“DHA”) and/or eicosapentaenoic acid (“EPA”) and/or docosapentaenoic acid (“DPA”) and/or oleic acid and/or stearidonic acid and/or linoleic acid and/or alpha-linolenic acid (“ALA”) and/or gamma-linolenic acid and/or arachidonic acid (“ARA”) and/or the esters of all of them, whereby the term“esters” encompasses monoglycerides, diglycerides and triglycerides as well as Ci- ₆ - alkyl esters such as especially the methyl esters and the ethyl esters, whereby the triglycerides are often dominant. DHA, EPA, ALA and stearidonic acid are omega-3 fatty acids, whereas linoleic acid, gamma-linolenic acid and ARA are omega-6 fatty acids.

The term “DPA” encompasses two isomers, the omega-3 fatty acid clupanodonic acid (7Z,10Z,13Z,16Z,19Z-docosapentaenoic acid) and the omega-6 fatty acid osbond acid (4Z,7Z,10Z,13Z,16Z-docosapentaenoic acid).

In accordance with the invention, the polyunsaturated fatty acid (PUFA) is preferably DHA and/or EPA and/or DPA and/or any ester thereof, more preferably the polyunsaturated fatty acid (PUFA) is preferably DHA and/or EPA and/or any ester thereof.

Examples of PUFA-containing oils for human consumption are

- marine oil, such as preferably fish oil,

- microbial biomass containing polyunsaturated fatty acids and/or their esters (“microbial oil”), preferably containing high amounts of docosahexaenoic acid (“DHA”) and/or eicosapentaenoic acid (“EPA”) and/or docosapentaenoic acid (“DPA”) and/or their esters, and

- oil containing high amounts of PUFAs and/or their esters, preferably containing high amounts of docosahexaenoic acid (“DHA”) and/or eicosapentaenoic acid (“EPA”) and/or docosapentaenoic acid (“DPA”) and/or their esters, extracted from microbial biomass, such as fungae (“fungal oil”) or algae (“algal oil”), and

- plant oil with relatively high amounts of PUFAs and/or their esters, (“PUFA-containing plant oil”), such as e.g. canola seed oil, linseed/flaxseed oil, hempseed oil, pumpkin seed oil, evening primrose oil, borage seed oil, blackcurrent seed oil, sallow thorn/sea buckthorn oil, chia seed oil, argan oil and walnut oil.

Marine oils, microbial oils and algal oils are especially preferred.

Thus, in addition, the present invention is

(1 ) directed to the use of the compounds of formula (I) with the preferences as given above as antioxidants in marine oils, microbial oils, oils containing high amounts of PUFAs and/or their esters extracted from microbial biomass and plant oils with relatively high amounts of PUFAs and/or their esters for human consumption; as well as

(2) directed to these PUFA-containing oils for human consumption comprising such compounds of formula (I) with the preferences as given above. Further antioxidants

The compounds of formula (I) can be used in combination with one or more other antioxidants as described below.

In an embodiment of the present invention the PUFA-containing oils of the present invention comprising a compound of formula (I) additionally comprise ascorbyl palmitate.

Instead of ascorbyl palmitate other esters of ascorbic acid such as the esters of ascorbic acid with linear C _12-20 alkanols, preferably the esters of ascorbic acid with linear Ci _4-18 alkanols, may also be used, so that further embodiments of the present invention are directed to PUFA-containing oils of the present invention comprising a compound of formula (I) that additionally comprise esters of ascorbic acid with linear C _12-20 alkanols, preferably esters of ascorbic acid with linear Ci _4-18 alkanols, more preferably ascorbyl palmitate.

The PUFA-containing oils of the present invention comprising a compound of formula (I) may also comprise additionally alpha-tocopherol and/or gamma- tocopherol, whereby either an ester of ascorbic acid with a linear C _12-20 alkanol with the preferences as given above may additionally be present.

The PUFA-containing oils themselves are described in more detail below.

PUFA-containing oils

In the context of the present invention the term“PUFA-containing oil” encompasses

- marine oil, such as especially fish oil,

- microbial biomass containing polyunsaturated fatty acids (“PUFAs”), especially docosahexaenoic acid (“DHA”) and/or eicosapentaenoic acid (“EPA”) and/or docosapentaenoic acid (“DPA”) and/or their esters (“microbial oil”); - oil containing high amounts of PUFAs, especially containing high amounts of DHA and/or EPA and/or DPA and/or their esters extracted from microbial biomass as e.g., fungi (“fungal oil”) or algae (“algal oil”);

- Plant oil with high amounts of PUFAs and/or their esters (“PUFA- containing plant oil”), such as e.g. canola seed oil, linseed/flaxseed oil, hempseed oil, pumpkin seed oil, evening primrose oil, borage seed oil, blackcurrent seed oil, sallow thorn/sea buckthorn oil, chia seed oil, argan oil and walnut oil.

The term“DHA” does not only encompass the acid but also derivatives thereof such as monoglycerides, diglycerides and triglycerides as well as Ci- ₆ -alkyl esters such as the methyl and ethyl esters. The same applies for“EPA” and “DPA” and all the other PUFAs.

Fish oil and algal oil are commonly used by humans. Instead of fish oil and algal oil also other PUFA-containing oils may be used for human consumption, i.e.:

- microbial biomass containing PUFAs (“microbial oil”)

- oil containing high amounts of PUFAs extracted from microbial

biomass, such as especially fungal oil, and

- plant oil with high amounts of PUFAs.

The above-mentioned PUFA-containing oils may not only be used as alternative of fish oil and algal oil, but also in addition.

Details of these PUFA-containing oils for human consumption are given below.

Marine oil

Examples of suitable marine oils include, but are not limited to, Atlantic fish oil, Pacific fish oil, or Mediterranean fish oil, or any mixture or combination thereof. In more specific examples, a suitable fish oil can be, but is not limited to, pollack oil, bonito oil, pilchard oil, tilapia oil, tuna oil, sea bass oil, halibut oil, spearfish oil, barracuda oil, cod oil, menhaden oil, sardine oil, anchovy oil, capelin oil, herring oil, mackerel oil, salmonid oil, tuna oil, and shark oil, including any mixture or combination thereof.

Other marine oils suitable for use herein include, but are not limited to, squid oil, cuttle fish oil, octopus oil, krill oil, seal oil, whale oil, and the like, including any mixture or combination thereof.

For stabilizing marine oil an amount of at least one compound of formula (I) ranging from 10 to 500 ppm, preferably ranging from 30 to 300 ppm, more preferably ranging from 100 to 250 ppm, based on the total amount of the marine oil, is usually sufficient. The same applies for the other PUFA- containing oils such as microbial oil, algal oil, fungal oil and PUFA-containing plant oil.

A commercially available example of marine oil is the fish oil “MEG-3” (Bleached 30S TG Fish oil) from DSM Nutritional Products, LLC (US) whose specification and composition is shown in Tables 1 and 2 below:

Table 1

The peroxide value is defined as the amount of peroxide oxygen per 1 kilogram of oil. Traditionally this is expressed in units of milliequivalents or meq/kg. Winterization is part of the processing of fish oil, and it is performed to remove solid fat in the oil. The“cold test” is performed to check if any solid fat is present and precipitated in the oil when cooled to 0°C within a specific period of time. In this fish oil (Product Code: FG30TG), any such precipitation is checked for 3 hours at 0°C.

Table 2

“TG” = triglyceride;

“A%” =“area %” = area percentage by GC based on 24 peak analysis (meaning the 24 highest peaks have been analyzed)

Oil containing high amounts of PUFAs, especially containing high amounts of DHA and/or EPA and/or DPA and/or their esters, extracted from microbial biomass as e.g.. fungi (“fungal oil”) or algae (“algal oil”)

Algal oil

“Algal oil” is an oil containing high amounts of DHA and/or EPA and/or DPA and/or their esters extracted from algae as microbial source /biomass.

An example of algal oil is the commercially available“Algal oil containing EPA+DPA” from DSM Nutritional Products, LLC (US) whose composition is shown in the Table 3 below: Table 3

A further example of a crude oil containing high amounts of DHA and/or EPA extracted from microbial sources as e.g., algae, is the oil extracted from Algae Schizochytrium Biomass, whose specification is given in the following

Table 4.

Table 4

Microbial biomass containing polyunsaturated fatty acids (“PUFAs”). especially docosahexaenoic acid and/or eicosapentaenoic acid and/or docosapentaenoic acid (“DPA”) and/or their esters

The biomass preferably comprises cells which produce PUFAs hetero- trophically. According to the invention, the cells are preferably selected from algae, fungi, particularly yeasts, bacteria, or protists. The cells are more preferably microbial algae or fungi.

Suitable cells of oil-producing yeasts are, in particular, strains of Yarrowia, Candida, Rhodotorula, Rhodosporidium, Cryptococcus, Trichosporon and Lipomyces.

Oil produced by a microorganism or obtained from a microbial cell is referred to as“microbial oil”. Oil produced by algae and/or fungi is referred to as an algal and/or a fungal oil, respectively.

As used herein, a "microorganism" refers to organisms such as algae, bacteria, fungi, protist, yeast, and combinations thereof, e.g., unicellular organisms. A microorganism includes but is not limited to, golden algae (e.g., microorganisms of the kingdom Stramenopiles); green algae; diatoms; dinoflagellates (e.g., microorganisms of the order Dinophyceae including members of the genus Crypthecodinium such as, for example,

Crypthecodinium cohnii or C. cohnii); microalgae of the order

Thraustochytriales; yeast ( Ascomycetes or Basidiomycetes ); and fungi of the genera Mucor, Mortierella, including but not limited to Mortierella alpina and Mortierella sect, schmuckeri, and Pythium, including but not limited to Pythium insidiosum.

In one embodiment, the microorganisms of the kingdom Stramenopiles may in particular be selected from the following groups of microorganisms:

Hamatores, Proteromonads, Opalines, Developayella, Diplophrys, Labrinthulids, Thraustochytrids, Biosecids, Oomycetes,

Hypochytridiomycetes, Commotion, Reticulosphaera, Pe logo monos, Pelagococcus, Ollicola, Aureococcus, Parmales, Diatoms, Xanthophytes, Phaeophytes (brown algae), Eustigmatophytes, Raphidophytes, Synurids, Axodines (including Rhizochromulinales, Pedinellales, Dictyochales), Chrysomeri dales, Sarcinochrysidales, Hydrurales, Hibberdiales, and

Chromulinales.

In one embodiment, the microorganisms are from the genus Mortierella, genus Crypthecodinium, genus Thraustochytrium, and mixtures thereof. In a further embodiment, the microorganisms are from Crypthecodinium Cohnii. In a further embodiment, the microorganisms are from Mortierella alpina. In a still further embodiment, the microorganisms are from

Schizochytrium sp. In yet an even further embodiment, the microorganisms are selected from Crypthecodinium Cohnii, Mortierella alpina,

Schizochytrium sp., and mixtures thereof.

In a still further embodiment, the microorganisms include, but are not limited to, microorganisms belonging to the genus Mortierella, genus Conidiobolus, genus Pythium, genus Phytophthora, genus Penicillium, genus Clodosporium, genus Mucor, genus Fusarium, genus Aspergillus, genus Rhodotorula, genus Entomophthora, genus Echinosporongium, and genus Saprolegnia.

In an even further embodiment, the microorganisms are from microalgae of the order Thraustochytriales, which includes, but is not limited to, the genera Thraustochytrium (species include arudimentale, aureum, benthicola, globosum, kinnei, motivum, multirudimentale, pachydermum, proliferum, roseum, striatum); the genera Schizochytrium (species include aggregatum, limnaceum, mangrovei, minutum, octosporum ); the genera Ulkenia (species include amoeboidea, kerguelensis, minuta, profunda, radiate, sailens, sarkariana, schizochytrops, visurgensis, yorkensis); the genera Aurantiacochytrium; the genera Oblongichytrium; the genera Sicyoidochytium; the genera Parientichytrium; the genera Botryochytrium; and combinations thereof. Species described within Ulkenia will be considered to be members of the genus Schizochytrium. In another embodiment, the microorganisms are from the order Thraustochytriales. In yet another embodiment, the microorganisms are from Thraustochytrium.

In still a further embodiment, the microorganisms are from Schizochytrium sp.

In certain embodiments, the oil can comprise a marine oil. Examples of suitable marine oils are the ones as given above.

The biomass according to the invention preferably comprises cells, and preferably consists essentially of such cells, of the taxon

Labyrinthulomycetes ( Labyrinthulea , net slime fungi, slime nets), in particular, those from the family of Thraustochytriaceae . The family of the Thraustochytriaceae ( Throustochytrids ) includes the genera Althomia, Aplanochytrium, Aurantiochytrium, Botryochytrium, Elnia, Japonochytrium, Oblongichytrium, Parietichytrium, Schizochytrium, Sicyoidochytrium, Thraustochytrium, and Ulkenia. The biomass particularly preferably comprises cells from the genera Aurantiochytrium, Oblongichytrium, Schizochytrium, or Thraustochytrium, more preferably from the genus Schizochytrium.

In accordance with the invention, the polyunsaturated fatty acid (PUFA) is preferably DHA and/or EPA and/or their esters as defined above.

The cells present in the biomass are preferably distinguished by the fact that they contain at least 20 weight-%, preferably at least 30 weight-%, in particular at least 35 weight-%, of PUFAs, in each case based on cell dry matter.

In a very preferred embodiment of the current invention, cells, in particular a Schizochytrium strain, is employed which produces a significant amount of EPA and DHA, simultaneously, wherein DHA is preferably produced in an amount of at least 20 weight-%, preferably in an amount of at least 30 weight-%, in particular in an amount of 30 to 50 weight-%, and EPA is produced in an amount of at least 5 weight-%, preferably in an amount of at least 10 weight-%, in particular in an amount of 10 to 20 weight-% (in relation to the total amount of lipid as contained in the cells, respectively).

Preferred species of microorganisms of the genus Schizochytrium, which produce EPA and DHA simultaneously in significant amounts, as mentioned before, are deposited under ATCC Accession No. PTA-10208, PTA-10209, PTA-10210, or PTA-10211 , PTA-10212, PTA-10213, PTA-10214, PTA-10215.

DHA and EPA producing Schizochytrium strains can be obtained by consecutive mutagenesis followed by suitable selection of mutant strains which demonstrate superior EPA and DHA production and a specific EPA:DHA ratio. Any chemical or nonchemical (e.g. ultraviolet (UV) radiation) agent capable of inducing genetic change to the yeast cell can be used as the mutagen. These agents can be used alone or in combination with one another, and the chemical agents can be used neat or with a solvent.

Methods for producing the biomass, in particular, a biomass which comprises cells containing lipids, in particular PUFAs, particularly of the order

Thraustochytriales, are described in detail in the prior art (see e.g. WO 91 /07498, WO 94/08467, WO 97/37032, WO 97/36996, WO 01 /54510). As a rule, the production takes place by cells being cultured in a fermenter in the presence of a carbon source and a nitrogen source, along with a number of additional substances like minerals that allow growth of the

microorganisms and production of the PUFAs. In this context, biomass densities of more than 100 grams per litre and production rates of more than 0.5 gram of lipid per litre per hour may be attained. The process is preferably carried out in what is known as a fed-batch process, i.e. the carbon and nitrogen sources are fed in incrementally during the

fermentation. When the desired biomass has been obtained, lipid

production may be induced by various measures, for example by limiting the nitrogen source, the carbon source or the oxygen content or combinations of these.

In a preferred embodiment of the current invention, the cells are grown until they reach a biomass density of at least 80 or 100 g/l, more preferably at least 120 or 140 g/l, in particular at least 160 or 180 g/l (calculated as dry-matter content). Such processes are for example disclosed in US

7,732,170.

Preferably, the cells are fermented in a medium with low salinity, in particular, so as to avoid corrosion. This can be achieved by using chlorine- free sodium salts as the sodium source instead of sodium chloride, such as, for example, sodium sulphate, sodium carbonate, sodium hydrogen carbonate or soda ash. Preferably, chloride is used in the fermentation in amounts of less than 3 g/l, in particular, less than 500 mg/l, especially preferably less than 100 mg/l.

PUFA-containing plant oils: Plant oils with relatively high amounts of PUFAs, especially with high amounts of DHA and/or EPA such as e.g. , canola seed oil

The plant cells may, in particular, be selected from cells of the families Brassicaceae, Elaeagnaceae and Fabaceae. The cells of the family

Brassicaceae may be selected from the genus Brassica, in particular, from oilseed rape, turnip rape and Indian mustard; the cells of the family

Elaeagnaceae may be selected from the genus Elaeagnus, in particular, from the species Oleae europaea ; the cells of the family Fabaceae may be selected from the genus Glycine, in particular, from the species Glycine max.

Examples: - Canola seed oil with a content of DHA of at least 9% by weight, of at least 12% by weight, of at least 15% by weight, or of at least 20% by weight, based on the total weight of the canola seed oil;

- Canola seed oil with a content of EPA of at least 9% by weight, of at least 12% by weight, of at least 15% by weight, or of at least 20% by weight, based on the total weight of the canola seed oil.

Examples of PUFA-containing plant oils containing high amounts of other PUFAs than EPA and/or DHA and/or DPA and/or their esters are linseed/flaxseed oil, hempseed oil, pumpkin seed oil, evening primrose oil, borage seed oil, blackcurrent seed oil, sallow thorn/sea buckthorn oil, chia seed oil, argan oil and walnut oil.

Preferred embodiments of the present invention

The compounds of formulae (4), (6), (8) and (9), preferably the compounds of formulae (4), (8) and (9), more preferably the compounds of formulae (4) and (8), are especially suitable for stabilizing marine oil, microbial oil and algal oil.

The invention is now further illustrated in the following non-limiting examples.

Examples

Examples 1 -10: Syntheses of compounds of formulae (1 ) to (4) and (6) to illi

Compound of formula (5) (PMC = Pentamethylchromanol, CAS 950-99-2) is commercial and is e.g. purchased from Aldrich (catalog# 430676). Example 1 : Synthesis of compound of formula (1 ) (6-chromanol) (see Fig.

2)

First step a): HC(OEt) ₃ , 70% HCIO _{4 -} 1 ) room temperature, 1 hour; 2) 100°C,

5 minutes in water, then room temperature (yield: 95%);

Second step b): H ₂ , Pd/C, 40° C, 8 hours, 5 bar (yield: 56%).

The procedure is described by J. C. Jaen, L. D. Wise, T. G. Heffner, T. A. Pugsley, L. T. Meltzer:“Dopamine autoreceptor agonists as potential antipsychotics. 2. (Aminoalkoxy)-4A7-1 -benzopyran-4-ones.” in J. Med.

Chem. 1991 , 34, 248-256. It is followed for the first step a), which furnishes the intermediate in high yield and good selectivity. The crude intermediate enone is then hydrogenated using Pd/C in a second step b), furnishing ch roman -6-0I in 53% yield over two steps.

Example 2: Synthesis of compound of formula (2) (2-methyl-6-chromanol) (see Fig. 3)

First step a): 1 ) KOtBu, tetrahydrofuran; 2) CuCl ₂ ; 3) NaOH; 24 hours at room temperature; yield: 46%.

Second step b): H ₂ , Pd/C; EtOH; 70° C, 7 days, 10 bar, yield: 20%.

Starting from 2-acetyl- 1 ,4-phenylene diacetate a base-mediated

intramolecular aldol condensation furnishes the enone intermediate as described before (A. O. Termath, Stereoselektive Totalsynthese von o- Tocopherol durch Cu-katalysierte asymmetrische 1 ,4-Addition an ein Chromenon, Dissertation, Universitat zu Koln, ISBN 978-3-8439-1407-9, Verlag Dr. Hut, MLinchen, 2013; Chapter 6.2.2, page 196-197.). The subsequent hydrogenation, using similar conditions as for compound of formula (1 ) above, furnishes the product in 20% yield.

Example 3: Synthesis of compound of formula (3) (2.2-dimethyl-6- chromanol) (see Fig. 4)

Conditions: Formic acid (80%), 110°C, 4 h; yield 37%. Compound of formula (3) is prepared following the literature procedure: . Wang, X. She, X. Ren, J. Ma, X. Pan.“The First Asymmetric Total Synthesis of Several 3,4-Dihydroxy-2,2-Dimethyl-Chroman Derivatives.”, Tetrahedron: Asymmetry 2004, 15, 29-34.

Example 4: Synthesis of compound of formula (4) (7-methoxy-2.2- dimethyl-6-chromanol) (see Fig. 5)

Compound of formula (4) (= Lipochroman-6®, CAS 83923-51 -7) is prepared according to literature procedures (see Fig. 5 for the reaction scheme).

First Step a): MeSQ ₃ H (solvent), P ₂ Os (50 mol-%, based on 2-methoxy-1 ,4- hydroquinone), yield: 91%. (F. Camps, J. Coll, A. Messeguer, M. A. Pericas,

S. Ricart, W. S. Bowers, D. M. Soderlund, Synthesis 1980, 725-727:“An Improved Procedure for the Preparation of 2,2-Dimethyl-4-chromanones.”)

Second Step b): LiAIFU, Et ₂ 0, yield: 87%.

(P. Anastasis, P. E. Brown, J. Chem. Soc., Perkin Trans. 1 1982, 2013-2018: “Analogues of antijuvenile hormones”.)

Example 5: Synthesis of compound of formula (6) (2-hydroxymethyl- 2.5.7.8-tetramethyl-6-chromanol)

Compound of formula (6) (Trolol, CAS 79907-49-6) is prepared according to the following literature procedure: J.-W. Huang, C.-W. Shiau, J. Yang, D.-S. Wang, H.-C. Chiu, C.-Y. Chen, C.-S. Chen. Development of Small-Molecule Cyclin D1 -Ablative Agents. J. Med. Chem. 2006, 49, 4684-4689.

Example 6: Synthesis of compound of formula (7) (2-(4-methylpentyl)-2- methyl-chroman-6-ol) (see Fig. 6)

A 1500 ml. 4-necked flask with magnetic stirrer, oil bath, thermometer and argon supply was charged with hydroquinone (95.0 g, 864 mmol, 4.0 mol equiv.), 7-dimethyloct-1 -en-3-ol (34.0 g, 216 mmol, 1.0 mol equiv.) and dissolved in ethylene carbonate (EC, 400 ml.) and heptane (300 ml_), forming a 2-phase system. Then, p-toluenesulfonic acid monohydrate (0.38 g, 2.16 mmol, 1 mol%) was added and the mixture was heated to reflux. After 1 h, deionized water (500 ml.) was added to the reaction mixture and the hot reaction phases were separated. The lower EC phase was extracted twice with a total of heptane (600 ml_). The combined heptane phases were dried over Na ₂ S0 ₄ and concentrated in vacuo (40 °C/50-20 mbar). The residue was purified by column chromatography (eluent: heptane/EtOAc 95:5 to 85: 15, w/w). The combined pure fractions were concentrated in vacuo (40° C/200- 10 mbar) and dried under high vacuum at 45 ° C, furnishing 2-(4- methylpentyl)-2-methyl-chroman-6-ol as colorless oil (31 .7 g, 97% purity by qNMR, 124 mmol, 58% yield).

HRMS: Calcd. for CI ₆ H ₂ 40 ₂ (M ⁺ ) 248.1776, found 248.1800.

¹ H NMR (300 MHz, CHLOROFORM-d) d 0.88 (d, J = 6.6 Hz, 6 H), 1 .12-1 .23 (m, 2 H), 1 .27 (s, 3 H), 1 .33-1 .46 (m, 2 H), 1 .46-1 .62 (m, 3 H), 1 .64-1 .89 (m, 2 H), 2.71 (t, J = 6.9 Hz, 2 H), 4.51 (s, 1 H, OH), 6.54-6.63 (m, 2 H), 6.66 (d, J = 8.9 Hz, 1 H) ppm.

¹³ C NMR (75 MHz, CHLOROFORM-d) d 21 .4 (1 C), 22.4 (1 C), 22.6 (2 C), 24.1 (1 C), 27.9 (1 C), 30.9 (1 C), 39.4 (1 C), 39.7 (1 C), 75.9 (1 C), 1 14.4 (1 C), 1 15.4 (1 C), 1 17.8 (1 C), 122.0 (1 C), 147.9 (1 C), 148.4 (1 C) ppm.

Example 7: Synthesis of compound of formula (8) (2-(4.8-dimethylnonyl)- 2-methyl-chroman-6-ol) (see Fig. 7)

A 200 mL 4-necked flask equipped with magnetic stirrer, oil bath, thermometer and argon supply was charged with 1 ,4-hydroquinone (12 g,

109 mmol, 4.0 mol equiv.), 3,7, 1 1 -trimethyldodec-1 -en-3-ol (6.39 g, 27.2 mmol, 1 .0 mol equiv.) and dissolved in ethylene carbonate (EC, 50 mL) and heptane (50 mL) forming a 2-phase system. Then, p-toluenesulfonic acid monohydrate (0.10 g 0.54 mmol, 2 mol%) was added and the mixture heated to reflux. After 90 min, the reaction mixture was cooled to 80° C and the phases were separated. The lower EC phase was extracted with heptane (25 mL). The combined organic phases were dried over sodium sulfate and concentrated in vacuo (40° C/50-20 mbar). The residue was purified by column chromatography (eluent heptane/EtOAc 95:5 to 85: 15 w/w). The combined pure fractions were concentrated in vacuo (40° C/200-10 mbar) and dried under high vacuum at 40°C, furnishing 2-(4,8-dimethylnonyl)-2- methyl-chroman-6-ol as light beige oil (4.95 g, 98.4% purity by qNMR, 15.3 mmol, 56% yield).

HRMS: Calcd. for C ₂₁ H ₃₄ O ₂ (M ⁺ ) 318.2559, found 318.2370.

¹ H NMR (300 MHz, CHLOROFORM-d) d 0.86 (d, J = ~ 6 Hz, 3 H), superimposed by 0.88 (d, J = 6.6 Hz, 6 H), 1 .04-1 .46 (m, 1 1 H), superimposed by 1.27 (s, 3 H), 1.46-1 .67 (m, 3 H), 1 .70-1 .87 (m, 2 H), 2.71 (t, J = 6.8 Hz, 2 H), 4.54 (br s, 1 H, OH), 6.53-6.62 (m, 2 H), 6.66 (d, J = 8.5 Hz, 1 H) ppm.

¹³ C NMR (75 MHz, CHLOROFORM-d) d 19.6, 21 .1 , 22.3, 22.6, 22.7, 24.1 ,

24.8, 28.0, 30.8, 30.9, 32.7, 37.3, 37.5, 39.3, 39.78, 39.82, 76.0, 114.4, 1 15.4, 1 17.8, 122.0, 147.9, 148.4 ppm.

Example 8: Synthesis of compound of formula (9) (2.5.7.8-tetramethyl)- 2-(4-methylpentyl)-chroman-6-ol) (see Fig. 8)

A 1 .5 L 4-necked flask equipped with mechanical stirrer, oil bath, thermometer and argon supply was charged with 2,3,5-trimethyl-1 ,4- hydroquinone (134 g, 853 mmol, 4.0 mol equiv.), 3,7,1 1 -trimethyldodec-1 - en-3-ol (34 g, 213 mmol, 1 .0 mol equiv.) and dissolved in ethylene carbonate (EC, 300 mL) and heptane (300 mL) forming a 2-phase system. Then, p-toluenesulfonic acid monohydrate (0.37 g 0.54 mmol, 1 mol%) was added and the mixture heated to reflux. After 90 min, the reaction mixture was cooled to 60 °C and the phases were separated. The lower EC phase was extracted with heptane (2x 250 mL). The combined heptane phases were extracted with water (500 mL), dried over magnesium sulfate and concentrated in vacuo (40° C/50-20 mbar). The residue was purified by column chromatography (eluent heptane/EtOAc 95:5 to 80:20 w/w). The combined pure fractions were concentrated in vacuo (40° C/200-10 mbar) and dried under high vacuum at 40°C, furnishing 2,5,7,8-tetramethyl)-2-(4- methylpentyl)-chroman-6-ol as off-white solid (26.8 g, 98.9% purity by qNMR, 91 mmol, 43% yield). Spectral data are in agreement with literature data: L. Rotolo, E. C.

Gaudino, D. Carnaroglio, A. Barge, S. Tagliapietra, G. Cravotto, RSC Adv. 2016, 6, 63515-63518.

Example 9: Synthesis of compound of formula (1 1 ) (6-hydroxychroman-2- one) (see Fig. 9)

A 100 ml. 3-necked round-bottom flask equipped with magnetic stirrer and septa was charged with 6-hydroxycoumarin (1 .38 g, 8.17 mmol), THF (30 ml_). Pd/C (10% Pd on C, 208 mg, 0.20 mmol, 2.4 mol%) was then added to this suspension. The flask was then inertized with argon (3x evacuation & flush with argon) and finally a hydrogen balloon was attached. The reaction was stirred at room temperature for 18 h and monitored by HPLC (full conversion after 18 h). The catalyst was filtered off and washed with THF (3x 5 ml_). The filtrate was concentrated in vacuo (60 °C/2 mbar), furnishing 1 .41 g of crude product. Thus, the residue was suspended in Et ₂ 0 (6 g) and stirred for 18 h at room temperature. The solid was isolated by filtration, washed with Et ₂ 0 (2x 3 ml.) and dried at 60°C/20 mbar for 5 h. The product was obtained as colorless solid (1 .25 g, 97.1% by qNMR, 91% yield). mp 161 -162 °C

¹ H NMR (300 MHz, DMSO-cfe) d 2.66-2.75 (m, 2 H), 2.82-2.94 (m, 2 H), 6.61 - 6.67 (m, 2 H), 6.82-6.89 (m, 1 H), 9.33 (s, 1 H) ppm.

Example 10: Synthesis of compound of formula (12) (6-hydroxy-7- methoxy-2.2-dimethyl-chroman-4-one) (see Fig. 10)

Compound of formula (12) is an intermediate in the synthesis of compound of formula (4) as described in example 4.

Conditions: MeS0 ₃ H (solvent), P ₂ Os (50 mol-%, based on 2-methoxy-1 ,4- hydroquinone), yield: 91%. (F. Camps, J. Coll, A. Messeguer, M. A. Pericas,

S. Ricart, W. S. Bowers, D. M. Soderlund, Synthesis 1980, 725-727:“An Improved Procedure for the Preparation of 2,2-Dimethyl-4-chromanones.”) Example 1 1 : Antioxidant activities of compounds of formulae (4). (6). (8) and (9) in fish oil

The compounds of formulae (4), (6), (8) and (9) have been tested. The blank oil, i.e. oil without any antioxidant, and oil containing“MNT” have been used as benchmark. Any compound better in antioxidant activity than the blank oil indicates that it has antioxidant activity. The comparison with MNT gives an indication about the amount of the antioxidant effect, relative to the activity of MNT.

“MNT” are mixed natural tocopherols commercially available as e.g., “Tocomix 70 IP” from AOM (Buenos Aires, Argentina). Tocomix 70 IP comprises d-alpha-tocopherol, d-beta-tocopherol, d-gamma-tocopherol and d-delta-tocopherol, whereby the total amount of tocopherols is at least 70.0 weight-% and the amount of non-alpha tocopherols is at least 56.0 weight-%.

The compounds of formulae (4), (6), (8) and (9) were evaluated primarily for their oxidative stability by the Oil Stability Index (OSI) measurements. Two different levels of these antioxidants (0.5 and 2 mg/g) were used in 5 g of natural fish oil (Product code: FG30TG) and used in the Oxidative

Stability Instrument at 80°C with the air flow rate of 40 psi. The solubility of different amounts and types of antioxidants used in OSI was checked before and after the application.

For the determination of Oil Stability Indices of crude algal oil (Lot#

VY00010309), only oil soluble compounds were used since the compounds which were not soluble in fish oil clearly showed relatively low stability indices. Crude algal oil contained about 1.6 mg/g of mixed natural tocopherols (MNT) prior to use in these experiments whereas fish oil did not contain any antioxidants.

The compounds of formulae (6) and (9) (see Table 6 below), compound of formula (4) (see Table 7 below) and compound of formula (8) (see Table 8 below) were used at different times in the Oxidative Stability instruments under similar operational conditions used for fish oil evaluations. MNT and the oil without any antioxidants (“Blank”) were always used to compare. Also, the synergistic effect of only oil soluble compounds with ascorbyl palmitate was determined using the OSI values. The polymers generated at the end of the experiment were determined by LC (LC = liquid

chromatography).

The solubility of the compounds used in the oxidative stability study are shown in Table 5.

Table 5: Solubility of compounds of formulae (4), (6), (8) and (9) in fish oil

Oil Stability Index for these compounds at 500 and 2000 ppm levels, in comparison with the same amounts of MNT, are shown in Tables 6-8.

Preliminary OSI results indicate that compound of formula (6) is comparable to the effect of MNT for the same level used (see Table 6). Table 6: Oxidative stability of FG30TG fish oil with compounds of formulae (6) and (9) (SD = standard deviation)

Table 7: Oxidative stability of FG30TG fish oil with compound of formula (4) (SD = standard deviation)

Table 8: Oxidative stability of FG30TG fish oil with compound of formula (8) (SD = standard deviation)

The Protection Factors of the corresponding antioxidant compounds in fish oil are shown as a percentage in Tables 9-1 1.

The Protection Factors (PF) for each compound in oil were calculated in percentage as: 100% x (OSI of the sample with compound - OSI of the sample without compound)

PF (%) =

OSI of the sample without compound

Table 9: Protection Factors of compounds of formulae (6) and (9) in FG30TG

fish oil

Table 10: Protection Factors of compound of formula (4) in FG30TG fish oil

Table 1 1 : Protection Factors of compound of formula (8) in FG30TG fish oil

Improvement of the oxidative stability of oil soluble compounds of formulae

6 and 9 when combined with AP is shown in Table 12, whereas Table 13

shows the same synergistic effect of compound of formula (8) with AP.

Table 12: Improvement of the effect of the compounds of formulae (6) and

(9) in FG30TG fish oil using AP (SD = standard deviation)

Table 13: Improvement of the effect of compound of formula (8) in FG30TG fish oil using a synergistic compound (AP) (SD = standard deviation)

Improvements of the Protection Factors of these oil soluble compounds with AP in fish oil are shown in Tables 14 and 15.

Table 14: Improvement of the Protection Factors of compounds of formulae (6) and (9) with AP in FG30TG fish oil

Table 15: Improvement of the Protection Factor of compound of formula (8) with AP in FG30TG fish oil

Polymers generated at the end of the stabilization experiment of fish oil with compounds of formulae 6 and 9 and AP are shown in Table 16. Table 16: Reduction of polymers in FG30TG oil with a compound (AP) synergistic to compounds of formulae (6) and (9) (SD = standard deviation)

Tables 17, 18 and 19 show the PV (peroxide value), p- AV (p-anisidine value) and CD (Conjugated dienoic acid %) of the fish oil samples stabilized with compounds of formulae (6), (8) and (9), respectively.

Table 17: Variation of PV with compounds of formulae (6), (8) and (9) in FG30TG

Table 18: Variation of p- AV (p-anisidine value) with compounds of formulae (6), (8) and (9) in FG30TG

Table 19: Variation of CD (conjugated dienoic acid in %) with compounds of formulae (6), (8) and (9) in FG30TG

Results:

To evaluate the efficacy of the compounds of formulae (4), (6), (8) and (9) in algal oils, only oil soluble compounds were used. Some of these compounds showed very similar pattern of OSI in fish oil. The compounds of formulae (6) and (9) had clearly lower OSI values than those of MNT in algal oil and both compounds seem to have possible prooxidant effect at higher levels (2 mg/g). Although some of these novel compounds did not have better oxidative stabilities than the commonly used MNT, their antioxidant effect can be improved to a considerable extent by combining with ascorbyl palmitate (“AP”) (Tables 12-13). Protection Factors of all these

compounds, including MNT, in fish oil could be improved by the addition of AP (Tables 14-1 5) indicating the possibility of combining AP to all these novel compounds to improve the oxidative stability of matrices containing high amounts of unsaturated fatty acids such as fish oil.

A combination of complex, polymeric compounds generated at the end of the oxidation cascade of unsaturated fatty acids indicate the levels of overall oxidation of the matrix. The generation of such polymers in fish oil containing these novel antioxidant compounds could be reduced

considerably when AP was added as a synergistic compound (Table 16).

For the storage stability study oil soluble compounds were used in fish oil at 2 mg/g level only. Compared to the same level of MNT, all compounds showed much higher PVs than those of MNT (Table 17). There was no considerable variation in p-AV and CD (Tables 18-19) during the storage.

All compounds showed antioxidant properties in fish oil at different levels.

The oxidative stability of fish oil with compound of formula (9) is

comparable to the antioxidative effect of MNT.

Compound of formula (4) which is not soluble in fish oil clearly had lower OSI values than those of MNT indicating poorer antioxidant activity than MNT.

Example 12: Antioxidant activities of compounds of formulae (3) and (7) in fish oil

The compounds of formulae (3) and (7) have been tested. The blank oil, i.e. oil without any antioxidant, and oil containing“MNT” (as used also in example 12) have been used here as benchmark. Any compound better in

antioxidant activity than the blank oil indicates that it has antioxidant

activity. The comparison with MNT gives an indication about the amount of

the antioxidant effect, relative to the activity of MNT.

The compounds of formulae (3) and (7) were used in both fish and algal oils

to see their antioxidant effect in these oils. Antioxidant effect was

determined using mainly the Oil Stability Index (OSI).

A storage stability study was performed to compare the variation of primary

oxidation products, the hydroperoxides, generated during oxidation,

measured in terms of peroxide value (PV) and the secondary oxidation

products which were measured and determined as anisidine reactive

substances or p-anisidine value (p-AV) of oil samples containing these

compounds.

Oxidative stability

Two concentration levels were used: 0.5 mg/g (low level) and 2 mg/g (high

level), respectively, have been added to 5 g of oil and used in the Oxidative

Stability Instrument operated at 80°C with the continuous air flow rate at

-40 psi. All samples were run in duplicate. The Protection Factors (PF) for

each compound, as percentage, in oil were calculated as, 100% x (OSI of the sample with compound - OSI of the sample without compound)

OSI of the sample without compound

Storage stability

The two different concentrations of compounds of formulae (3) and (7) were

used for the storage stability study. Each compound and MNT were added,

each individually, to 40 g of fish oil samples in 60 ml amber bottles at 0.5

mg/g and 2 mg/g levels, thoroughly mixed and stored at ambient

temperature for 19 days. All sample bottles were stored open to air, away

from light. Compounds of formulae (3) and (7) were not readily soluble in oil, so they had to be thoroughly mixed with the oil around 40°C to dissolve completely. Peroxide values (PV) and p-anisidine values (p-AV) were determined at different times for 19 days. Results

OSI values of the fish oil samples containing the compounds of formulae (3) and (7), in comparison to the same levels of MNT, are shown in Table 20.

Table 20: Oxidative Stability Indices (OSI) of FG30TG fish oil with compounds of formulae (3) and (7) (SD = standard deviation)

Based on the Oil Stability Index data, compounds of formulae (3) and (7) have antioxidant properties since all fish oil samples containing these compounds, at both low and high levels, showed higher OSI values than those of the oil without any antioxidant (Blank- FG30TG).

Compound of formula (3) showed slightly higher Oil Stability Indices than those of MNT indicating that compound of formula (3) possesses relatively higher antioxidant properties than MNT at the specified concentration levels. Compound of formula (10) showed comparable antioxidant activity to MNT.

Peroxide values of fish oil samples at low (0.5 mg/g) and high levels (2 mg/g) are shown in Tables 21 and 22, respectively, whereas the p-AV of the same samples at low (0.5 mg/g) and high levels (2 mg/g) are shown in Tables 23 and 24, respectively. Table 21 : Peroxide values (PV, meq/kg) of compounds of formulae (3) and (7) during storage at 25°C (0.5 mg/g level)

Table 22: Peroxide values (PV, meq/kg) of compounds of formulae (3) and (7) during storage of compounds of formulae (3) and (7) at 25°C (2 mg/g level)

Table 23: p-Anisidine value (p-AV) of compounds of formulae (3) and (7) during storage at 25°C (0.5 mg/g level)

Table 24: p-Anisidine value (p-AV) of compounds of formulae (3) and (7) during storage at 25°C (2 mg/g level)

Primary oxidation products (hydroperoxides) generated in the fish oil samples containing compound of formula (3) or compound of formula (7) or MNT, determined as peroxide values (PV), did not show a considerable difference among them at both concentration levels used. Also, there was no considerable difference in the variation of p-anisidine values (p-AV) in all these samples except the sample which did not contain any antioxidants. The sample which did not contain any antioxidant had relatively higher p-AV values than all other samples showing that the compounds of formulae (3) and (7) possess antioxidant properties.

The Oil Stability Indices (OSI) of crude algal oil with levels of 0.5 mg/g and 2 mg/g of compounds of formulae (3) or (7) are shown in Table 25. Table 25: Oxidative stability of crude algal oil with compounds of formulae (3) or (7) (SD = standard deviation)

The compounds of formulae (3) and (7) improved the oxidative stability of crude algal oil when compared with an oil sample which did not contain any of these compounds (Table 25) showing the antioxidant effect of these compounds in algal oil as well.

Tables 26 and 27 show the Protection Factors of the compounds of formulae (3) and (7) in fish oil and crude algal oil, respectively. Table 26: Protection Factors of compounds of formulae (3) and (7) in fish oil (80°C)

Table 27: Protection Factors of compounds of formulae (3) and (7) in crude algal oil (80°C)

Results

The compounds of formulae (3) and (7) have antioxidant properties that are comparable in activity with MNT in fish oil. Compound of formula (3) has slightly higher Oil Stability Indices compared to MNT.

Application of compound of formula (3) also resulted in the lowest level of secondary oxidation products (p-AV) at levels at 0.5 mg/g concentration. There was no considerable difference in PV between compounds of formulae (3), (7) and MNT during storage.