WO2011076869A1

WO2011076869A1 - Isolated green plant material

Info

Publication number: WO2011076869A1
Application number: PCT/EP2010/070534
Authority: WO
Inventors: Hans-Erik ÅKERLUND
Original assignee: Aakerlund Hans-Erik
Priority date: 2009-12-22
Filing date: 2010-12-22
Publication date: 2011-06-30

Abstract

The invention relate to an isolated green plant material comprising at least 0.1 mg/g (dry weight) of zeaxanthin, a method of producing such as material as well as different products comprising said material and the use of such material and products.

Description

ISOLATED GREEN PLANT MATERIAL

FIELD OF INVENTION

BACKGROUND OF THE INVENTION

Carotenoids constitute a large group of substances with diverse biological functions. This includes light absorption in green plants and in the form of rhodopsin in bacteria and animal eye, quencher of triplet states of chlorophyll, quencher of singlet oxygen, sacrificial scavenger of peroxy radicals and coloration [Bhosale and Bernstein 2007, Eskling et al. 1997]. Carotenoids have also been suggested to be involved in modulation of membrane packing, to affect

development of different cancer forms and protection against degenerative deceases [Bhosale and Bernstein 2007, Eskling et al. 1997, Szilayi et al. 2008]. Animals can modify carotenoids but lack the ability to synthesize carotenoids and are therefore dependent on supply from food. Carotenoids are primarily produced by plants. Specific subsets of carotenoids are found in green leaves, fruits and flowers and provide red and orange color to the tissue.

A carotenoid that has attracted much interest the last decade is zeaxanthin. Zeaxanthin has together with lutein been indicated, through epidemiologic studies, to protect the human eye from age related macular degeneration (AMD)

[Whitehead et al. 2006, Carpentier et al. 2009, Krinsky et al. 2003]. Actually the yellow color of the 'yellow spot' (macula lutea) in the retina of the eye is due to the specific accumulation of zeaxanthin and its isomer lutein. The pigments are not evenly distributed [Snodderly et al. 1984, Rapp et al. 2000, Bone et al. 1988]. In the macula lutea, zeaxanthin and lutein are present at a zeaxanthin/lutein ratio of about 2 while in the peripheral retina, which is the area beyond 1 -2 mm from the fovea, the zeaxanthin/lutein ratio is only about 0.5 [Carpentier et al. 2009].

Zeaxanthin occurs in different forms, 3R3'R-, meso- and 3S3'S-xeaxanthin, where the 3R3'R- and meso-forms are dominating and present in approximately equal amounts in the central retina. Feeding experiments on rhesus monkeys showed that meso-zeaxanthin could be formed from suplemented lutein while 3R3'R- zeaxanthin only occurred in the retina when provided with the food [Johnson et al. 2005].

Age-related macular degeneration (AMD) is a degenerative eye disease that causes damage to the macula of the eye [Carpentier et al. 2009], a leading cause of blindness in senior population. Low amount of lutein and zeaxanthin in the diet, serum and retina combined with excessive exposure to blue light are thought to increase the risk of AMD. The retina is particularly susceptible to oxidative stress since it has a high consumption of oxygen, it has a high proportion of

polyunsaturated fatty acids and is exposed to visual light. In vitro, macular pigments have been shown to limit retinal oxidative damage by absorbing blue light and quenching reactive oxygen intermediates (ROI) [Beatty et al. 2000]. Although most studies has not made any distinction between the role of zeaxanthin and lutein, Gale et al. [2003] found that zeaxanthin content in plasma was significantly negatively correlated to the risk for AMD while lutein did not show such a correlation. For further discussion on relative role of zeaxanthin and lutein see Neuringer et al. [2004].

Lutein can be found in high amounts in all green plant material, particularly in dark green leaves where it take part in photosynthesis. It is an important component of the light harvesting system in higher plants and green algae

[Arvidsson et al. 1997, Cunningham and Gantt 1998]. Zeaxanthin (3R3'R-form if not otherwise stated), however, is normally found in very low amounts in green tissue. Zeaxanthin is synthesized from beta-carotene in plants, but is rapidly converted to violaxanthin under normal conditions. In the plant, violaxanthin and zeaxanthin can be interconverted in what is known as the xanthophyll cycle. When green plants are subjected to conditions where light becomes excessive to what the plant can use then violaxanthin is enzymatically converted to zeaxanthin via the intermediate antheraxanthin. It takes place on various time scales from minutes to hours [Young et al. 1997, Demmig-Adams and Adams 1996, Eskling and

Akerlund 1998, Hager 1969]. Zeaxanthin participates in a process where excessive light energy is dissipated as heat. Zeaxanthin also stabilizes membrane packing and participate in radical scavenging. In darkness or at lower light intensities zeaxanthin is again converted to violaxanthin. The balance between violaxanthin and zeaxanthin is controlled in each direction by two enzymes, violaxanthin de- epoxidase (VDE) and zeaxanthin epoxidase (ZE). Violaxanthin de-epoxidase is located in the lumen of thylakoids and the enzyme becomes active when the pH drops in the lumen due the light driven photosynthetic reaction [Szilayi et al 2008, Hager 1969, Bratt et al. 1995]. We have recently shown that thylakoids isolated at low pH (4.7) contain more than 5 -fold higher amount of zeaxanthin compared to thylakoids isolated at high pH (7) [Emek et al. 2009]

SUMMARY OF THE INVENTION

In a first aspect the invention relates to an isolated green plant material comprising at least 0.1 mg/g (dry weight) of zeaxanthin. The isolated material has been harvested from free land, green house or from in vitro cultivation in a chamber or any other cultivation forms. Thereby it is for the first time possible to provide natural plant material with the increased amount of zeaxanthin to the market.

In a second aspect the invention relates to a method for the production of isolated green plant material comprising the steps of providing the green plant material, incubating said material in the presence of an acid or buffer at a pH of at most 7, removing said acid or buffer and obtaining a green plant material having an increased amount of zeaxanthin compared to the provided green plant material.

Said method which is simple and effective give rise to a new material that is in no need of further purification prior to being used by the mammal such as a human being due to that solely simple and acceptable acids/buffer are used which are already acceptable.

In third aspect the invention relates to products comprising said isolated and/or obtained green plant material which could be used as pharmaceutical compositions as well as food additives as defined below.

In a final aspect the invention relates to the use of the above defined material and products in different aspects relates to improving the visual performance of a mammal such as a human being or other animal, preventing cataract or age related macular degeneration as well as a food additive for animals such as domestic and wild birds.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig 1 shows the pH dependent formation of conversion of violaxanthin (filled dimonds) to (filled triangles) zeaxanthin in spinach leaves after incubation in 0.5 M acetate buffer at room temperature for 2h. Zeaxanthin and violaxanthin values are expressed as % of VAZ-pool of pigments where 100% correspond to 5 mg zeaxanthin/ 100 g leaves. Open dimonds and +-symbol reprepresent the sum of VAZ-pool of pigments normalized to lutein and fresh weight, in % of untreated material.

Fig 2 shows zeaxanthin formation from violaxanthin in spinach leaves as a function of acetate buffer concentration at pH 5 after incubation at room

temperature for 2h. Zeaxanthin and violaxanthin values are expressed as % of VAZ-pool of pigments where 100% correspond to 5 mg zeaxanthin/ 100 g leaves. Open dimonds and +-symbol reprepresent the sum of VAZ-pool of pigments normalized to luten and fresh weight, in % of untreated material.

Fig 3a and b shows time dependent formation of zeaxanthin from violaxanthin in spinach leaves at room temperature and 0.5 M of either a) acetate buffer at pH 5 or b) 0.5 M acetic acid at pH 2.5. Zeaxanthin and violaxanthin values are expressed as % of VAZ-pool of pigments where 100% correspond to 5 mg zeaxanthin/ 100 g leaves. Open dimonds and +-symbol reprepresent the sum of VAZ-pool of pigments normalized to lutein and fresh weight, in % of untreated material.

Fig 4 shows the temperature effect on zeaxanthin formation from violaxanthin in spinach leaves incubated in 0.5 M acetate buffer at pH 5.0 for 2h. Zeaxanthin and violaxanthin values are expressed as % of VAZ-pool of pigments where 100% correspond to 5 mg zeaxanthin/ 100 g leaves.

Fig 5 shows buffer dependent formation of zeaxanthin. Spinach leaves were incubated in 0.5 M of different acids or buffers at room temperature for 2h. The white, gray and black bars represent violaxanthin, antheraxanthin and zeaxanthin respectively, a) untreated leaves, b) citric acid, c) citrate buffer pH 5.0, d) lactic acid, e) lactate buffer pH 5.0, f) phosphoric acid, g) phosphate buffer pH 5.0, h) 10 mM HC1, i) acetic acid and j) acetate buffer pH 5.0. Zeaxanthin values are expressed as % of VAZ-pool of pigments where 100% correspond to 5 mg zeaxanthin/ 100 g leaves.

Fig 6 shows acid sensitivity of violaxanthin and antheraxanthin for spinach samples, a) untreated leaves, b) homogenized leaves incubated in 10 mM HC1 for 2h at room temperature, c) intact leaves incubated in acetate buffer at pH 5.0 for 2h at room temperature, d) intact leaves first incubated in acetate buffer at pH 5.0 for 2h then homogenized and incubated for 2h in 10 mM HC1. Zeaxanthin values are expressed as nmol/g of fresh weight of leaves.

Fig 7 shows the formation of zeaxanthin from violaxanthin in different green vegetables after incubation in 0.5 M acetate buffer at room temperature for 2 h. a), c), e), g) i) and k) untreated samples; b), d), f), h), j) and 1) treated samples, expressed as % of the VAZ-pool of pigments. The different species were a-b) corn salad, c-d) parsley, e-f) basil, g-h) lemon balm, i-j) sugarsnaps, k-1) peas. The white, gray and black bars represent violaxanthin, antheraxanthin and zeaxanthin respectively. The values above the bars indicate mg of zeaxanthin per 100 g of fresh weight in the treated samples.

Fig 8 shows the amount of titratable acid remaining in spinach leaves after incubation in distilled water, subsequent to treatment with 0.5 M acetate buffer for 2 h at room temperature.

DETAILED DESCRIPTION OF THE INVENTION DEFINITIONS

The term "green plant material" is intended to mean the green part of any plant material, which excludes the roots as well as the flowers. Green plant material is intended to mean that the plant cells are unbroken/intact.

The green plant material may also be a mixture of different plants as well as different parts of the plant. It also includes beans and pods as well as different kinds of cell cultures wherein the cells and tissue parts in the culture are green parts of the plant material. Green plant material is also intended to include green algae. The green plant material may contain intact, i.e., whole or fragmented plant material as long as the main part, such as more than 80 % of the plant cells are unbroken/intact. Fragmented material may also mean powder.

The term "substantially free" or free from water" is intended to mean that the material contains less than 25% (w/w) dry weight of water or even less, such as 20, 15 , 10 or even less % (w/w). INVENTION

In a first aspect the invention relates to an isolated green plant material comprising at least 0.05 mg/g (dry weight) of zeaxanthin, such as at least 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2.0 mg/g (dry weight) of zeaxanthin or even more. Normally the plant material contains above 80 % of water and often even over 90 % water. The calculations were based upon that the plant contained 90 % water. The green plant material has been isolated and is not a cultivated plant in the soil, i.e., the green plant material is harvested plant material that could have been harvested either from the field or the green house. The material may also be in vitro culture of green plant material comprises any of the parts of the green plant material that are mentioned within the application.

The amount of zeaxanthin in said green plant material in the wet stage may be at a level of 1 mg/lOOg tissue or more and the level of zeaxantin in for example a food stuff may be 0.2 mg/lOOg tissue or more, such as at least 0.4, 0.6, 0.8, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 5.0 or 10 mg/lOOg tissue.

The green plant material has an altered ratio of zeaxanthin and violaxanthin compared to originally living plant material, such as spinach growing on free land and which has not been treated by for example the method disclosed in the application. Said green plant material comprises zeaxanthin and violaxanthin in a ratio of from at least 10:90, such as at least 15:85, 20:80, 25:75, 30:70, 35:65, 40:60, 45:55, 50:50, 55:45, 60:40, 65:35, 70:30, 75:25, 80:20, 85:15 or more.

The green plant material may be frozen, dried, substantially free from water or even totally free from water or it may be frozen after dehydration. The dehydrated tissue may be a powder, granules, flakes, sheets or chips.

In a second aspect the invention relates to a method for improving green plant material such as increasing the level of zeaxanthin in a green plant material as defined above comprising the steps of providing green plant material, incubating said vegetative plant tissue in the presence of an acid or mixtures thereof or buffers comprising said acids at a pH of up to/ at most 7, such as up to 6.5, 6.0, 5.5, 5.0, 4.5, 4.0, 3.5, 3.0, 2.5, or at most 2.0, removing said acid or buffer and obtaining a green plant material, such as a green plant material comprising at least 0.05 mg/g (dry weight) of zeaxanthin, such as at least 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2.0 mg/g (dry weight) of zeaxanthin or even more, calculated as above.

The acid may be acetic acid, citric acid, lactic acid or phosphoric acid and the buffers may comprise those acids alone or mixtures of the acids. For certain purposes the acid is acetic acid/acetate.

Additionally the method may include a step of dehydrating said tissue or tissues. Example of ways to dehydrate the tissue or tissues includes heat, air, lyophilisation, sun drying or freeze drying or combinations thereof.

The method may be performed at different temperatures depending on how fast there is an interest in driving the production of zeaxanthin in the tissue. For instance if the plant tissue is to be stored for a longer period of time, it may be desirable to incubate at a lower temperature such at about 4 °C. However, the temperature to be used is dependent on the green plant material as well as when the material is to be used the tissue or tissues could be incubated at a higher temperature such at about 37 °C. However, the temperature may be between 4 °C to 50 °C or even higher, such as 5, 6, 7, 8, 9 10, 11, 12, 13,14, 15, 16, 17, 18, 19 20, 21, 22, 23, 24, 25, 26, 27 ,28 ,29 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48 , 49 or 50 °C, or 4-37 °C or 4-21 °C.

The concentration of the acid or buffer will also influenced the speed on which zeaxanthin is produced in the green plant material and normally the concentration will be in the area of from 0.1 to 10 M, such as 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, .8.0, 8.5, 9.0, 9.5 or 10.0 M.

Additionally, the acid/buffer may also contain ascorbic acid may be at a concentration above 1 mM, such as 2, 3, 5, 10, 15 or 30 mM or other concentration optimized for the yield of zeaxanthin by said method. Ascorbic acid being a second substrate for the reaction and thereby increase the speed of zeaxanthin formation as well as being an additional antioxidant.

The method may also contain an additional step of enriching zeaxanthin from said green plant material in different ways such as extracting zeaxanthin in a lipid or an ethanol extract/fraction, such a step is well-known for a person skilled in the art. The extraction may be performed using for example acetone or ethanol or any other suitable organic solvents. The extract may for example be an ethanol extract or a lipid extract comprising zeaxanthin. The fraction/extract may also be isolated thylakoids or thylakoid membranes and/or parts of thylakoid membranes depending on the method used to obtain the lipid extract/fraction.

The method may also include a step wherein all the enzymatic reaction occurring in the plant material is interrupted, to inhibit/stop the production of more zeaxanthin or to stop the possibility of the reverse reaction wherein zeaxanthin reverts to violaxanthin. Such methods include retaining pH below 7, freezing, heating, boiling, blanching, or homogenizing the green plant material.

If the method results in that both zeaxanthin and violaxanthin is present in the obtained fraction/extract, they will be present in a ratio of from at least 10:90, such as at least 15:85, 20:80, 25:75, 30:70, 35:65, 40:60, 45:55, 50:50, 55:45, 60:40, 65:35, 70:30, 75:25, 80:20, 85:15 or more.

The green plant material may be any plant material that have a suitable green part which could be treated in such a way that the zeaxanthin amount in the green plant material increases to acceptable levels, such as a dicot or a monocot. Examples of green plant material includes sugar beet, kale, oil seed rape, corn, sun flower, banana, oat, wheat, barley, sorghum, rice, different kinds of vegetable such as spinach, lettuce, lemon balm, corn salad, different kinds of herbs, such as basil and parsley and peas and sugar snaps. Accordingly the green plant material may be green algae, such as sea lettuce and dead man's finger's. Examples of different kinds of lettuce include mangold, ruccola, mache, roman among other types. The green plant material may also be a mixture of different green plant tissues from different plant species as well as different varieties. The green plant material may being any part of the plant that are green, such as leaves, stems, shoots, fruits, seeds, or pods as long as they are green and contains violaxanthin. The green pant material may also be genetically modified to alter for example the amount of violaxanthin and/or zeaxanthin.

The invention also relates to a product comprising said green plant material or said extract or fraction, wherein said product is a pharmaceutical composition or a food supplement, food additive, a nutrient source, a nutrient supplement mixture or a food-stuff, for mammals such as animals or humans. The following terms food supplement, food additive, a nutrient source, a nutrient supplement mixture or a food-stuff will hereinafter be named food additive.

The invented food additive may also be mixed with other components such as antioxidants, minerals such as zinc or selen, different PUFA-s, vitamins such as E, A or C vitamins or other health improving additives/components.

The invented food additive may be included in any kind of nutritional composition, wherein for example one object is to improve visual performance. One group is mammals that are exposed to excess of blue light, which preferably should eat the developed food additive. The food additive could also be used to feed animals, such as horses, dogs, cats, cows, pigs, camels, birds, both domestic and wild, such as chicken to obtain improved eggs containing zeaxanthin.

The invented food additive may be admixed with other components such as fat, butter, margarine, oils, cream, milk, cheese, brie, flour, cereals, juices, soft drinks, flakes, spice mixes, dressings, teas either prior to being added to a food product or during the addition to the food product.

Said food additive may be solid, semisolid or in a liquid form. Further it may be dried, freeze dried, sun dried, spray dried or lyophilized. The invented food additive may be used in any kind of food product as well as being used alone. Examples of food products are fat, butter, margarine, oils, cream, cereals, milk, cheese, brie, flour, juices, soft drinks, teas. Other examples are yoghurt, ice cream, cakes, bread and dressing.

The invented green plant material or the extract/fraction obtained by the invented method may also be used as a pharmaceutical composition. The pharmaceutical composition comprises additionally a pharmaceutically acceptable buffer, excipient, carrier or diluent. The composition may be used in all cases wherein there is a need of improving visual performance or to prevent the development of for example cataract or age-related macular degeneration (AMD) or other diseases or disorders in which there is a need of increasing the amount of zeaxanthin present in the mammal.

"Pharmaceutically acceptable" means a non-toxic material that does not decrease the effectiveness of the biological activity of the active ingredients, i.e., zeaxanthin. Such pharmaceutically acceptable buffers, carriers or excipients are well-known in the art (see Remington's Pharmaceutical Sciences, 18th edition, A. R Gennaro, Ed., Mack Publishing Company (1990) and handbook of

Pharmaceutical Excipients, 3rd edition, A. Kibbe, Ed., Pharmaceutical Press (2000).

The term "buffer" is intended to mean an aqueous solution containing an acid -base mixture with the purpose of stabilising pH. Examples of buffers are Trizma, Bicine, Tricine, MOPS, MOPSO, MOBS, Tris, Hepes, HEPBS, MES, phosphate, carbonate, acetate, citrate, glycolate, lactate, borate, ACES, ADA, tartrate, AMP, AMPD, AMPSO, BES, CABS, cacodylate, CHES, DIPSO, EPPS, ethanolamine, glycine, HEPPSO, imidazole, imidazolelactic acid, PIPES, SSC, SSPE, POPSO, TAPS, TABS, TAPSO and TES.

The term "diluent" is intended to mean an aqueous or non-aqueous solution with the purpose of diluting zeaxanthin in any form in the pharmaceutical preparation. The diluent may be one or more of saline, water, polyethylene glycol, propylene glycol, ethanol or oils (such as safflower oil, corn oil, ethyl-oleate, peanut oil, cottonseed oil or sesame oil).

The excipient may be one or more of carbohydrates, polymers, lipids and minerals. Examples of carbohydrates include lactose, sucrose, mannitol, and cyclodextrines, which are added to the composition, e.g., for facilitating

lyophilisation. Examples of polymers are starch, cellulose ethers, cellulose carboxymethylcellulose, hydroxypropylmethyl cellulose, hydroxyethyl cellulose, ethylhydroxyethyl cellulose, alginates, carageenans, hyaluronic acid and derivatives thereof, polyacrylic acid, polysulphonate,

polyethylenglycol/polyethylene oxide, polyethyleneoxide/polypropylene oxide copolymers, polyvinylalcohol/polyvinylacetate of different degree of hydrolysis, and polyvinylpyrrolidone, all of different molecular weight, which are added to the composition, e.g., for viscosity control, for achieving bioadhesion, or for protecting the lipid from chemical and proteolytic degradation. Examples of lipids are fatty acids, phospholipids, mono-, di-, and triglycerides, ceramides,

sphingolipids and glycolipids, all of different acyl chain length and saturation, egg lecithin, soy lecithin, hydrogenated egg and soy lecithin, which are added to the composition for reasons similar to those for polymers. Examples of minerals are talc, magnesium oxide, zinc oxide and titanium oxide, which are added to the composition to obtain benefits such as reduction of liquid accumulation or advantageous pigment properties.

The pharmaceutical compositions may be subjected to conventional pharmaceutical operations such as sterilisation and/or may contain conventional adjuvants such as preservatives, stabilisers, wetting agents, emulsifiers, buffers, fillers, etc., e.g., as disclosed elsewhere herein.

The pharmaceutical compositions will be administered to a patient in a pharmaceutically effective dose. By "pharmaceutically effective dose" is meant a dose that is sufficient to produce the desired effects in relation to the condition for which it is administered. The exact dose is dependent on the, activity of the compound, manner of administration, nature and severity of the disorder, age and body weight of the patient different doses may be needed. The administration of the dose can be carried out both by single administration in the form of an individual dose unit or else several smaller dose units and also by multiple administration of subdivided doses at specific intervals.

The present invention concerns both humans and other mammal such as horses, dogs, cats, cows, pigs, camels, birds, among others. Thus the methods are applicable to both human therapy and veterinary applications. The objects, suitable for such a treatment may be identified by well-established hallmarks. The treatment will in most cases be a pretreatment.

The following examples are included to demonstrate preferred

embodiments of the invention. It should be appreciated by those skilled in the art that the disclosed techniques that follows represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, a person skilled in the art, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and the scope of the invention.

EXAMPLES

In this work whole leaves or tissues of commercially harvested plant material were used. Conditions were established to convert violaxanthin into zeaxanthin and thus improve the nutritional value of the vegetable. It was found that incubation of vegetables at moderately low pH in the presence of a membrane permeable weak acid, acetic acid, could efficiently increase zeaxanthin content by a factor of up to 30. The method is suited both for food industry and for the common household, and would help the consumer to obtain increased amount of zeaxanthin.

Spinach (baby spinach, Spinacia oleracea), basil (Ocimum basilicum), parsley {Petroselinum crispum), corn salad (Valerianella locusta), lemon balm {Melissa officinalis), peas and sugarsnaps (Pisum sativum) were bought in a local market. Spinach, parsley, corn salad and peas were kept at 4°C until use, within one week from purchase. Basil and lemon balm were bought as plants and were kept at room temperature until use, within two days from purchase. To extract pigments for HPLC analysis, leaf material from six individual spinach leaves (50-100 mg fresh weight) were ground under liquid nitrogen to a fine powder, together with a small amount of CaCO₃(s) (MERCK p. a.). Then 0.2 ml water and 0.8 ml ice-cold acetone (Scharlau, analytical grade) were added and the samples were incubated on ice in darkness for at least 15 min. After extraction, the samples were centrifuged twice for four minutes in a table-top eppendorf centrifuge at maximum speed. To 0.8 ml of the supernatant, 160 μΐ water was added and the pigments were separated using HPLC (see below). Sample preparation for other species was done in a similar manner, using approximately 50 mg of leafy vegetables and 150-300 mg of pea pods.

Spinach leaves were incubated at room temperature (21 ± 2 °C), at 4 ± 3 °C or at 37 ± 0.5 °C for a time, in buffer solution and at pH specified in each experiment. The remaining species were all incubated in 0.5 M acetate buffer, pH 5.0, for 2h at room temperature. Untreated controls were analyzed in all experiments.

For preparation of buffers, acetic acid (Acros organics, 99.8%), sodium acetate (Sigma Ultra 99%), ortho-phosphoric acid (85% MERCK), sodium dihydrogen phosphate (MERCK p. a.), L(+)-lactic acid (90% Acros organics), citric acid (MERCK p.a.), tri-sodium citrate (MERCK p.a.), sodium hydroxide (ICN

Biomedicals Inc p.a.), and hydrochloric acid (37%, MERCK p.a.) were used. All solutions were prepared in deionized (Milli-Q) water.

Samples incubated in 10 mM HCl were prepared in the following manner: Spinach leaves were either used directly (violaxanthin samples) or were incubated in 0.5 M acetate buffer, pH 5.0, at room temperature for two hours (zeaxanthin samples). Approximately 60 mg of spinach, taken from six individual leaves, were ground under liquid nitrogen. The fine powder was then incubated with 10 mM HCl, at 37 °C and in darkness, for two hours with occasional stirring. The samples were centrifuged in a table-top eppendorf centrifuge at maximum speed for four minutes and the supernatant was removed. The pellet was resuspended in 200 μΐ water and a small amount of CaCO (s) was added. The xanthophylls were extracted with 0.8 ml acetone, as described above. The pigments extracted as described above were analyzed by reversed-phase HPLC (Waters 600E), using conditions essentially as described by Thayer and Bjorkman [1990]. Samples were applied to a Zorbax ODS 4.6 x 250 mm non-end capped cartridge column and the xanthophylls were separated using a

methanol/acetonitrile mixture (15%/85% v/v) for 6 min. Other pigments were eluted using a methanol/ethylacetate mixture (50%/50% v/v) for 6 min. The solvents used were all of HPLC grade and the flow rate was 1 ml/min. Pigments were detected at 445 nm and were quantified by peak area integration using data acquisition software (Waters Millennium) and the mM extinction coefficients 120 (Neoxanthin), 140 (Violaxanthin), 130 ( Anther axanthin), 126 (Lutein and

Zeaxanthin). Each datapoint represent the average of two separate experiments.

The remaining acid in leaves were determined by pH stat titration (TIM854 Radiometer Analytical SAS, Cedex, France) using 15 ml buffer containing 2 mM Tris-maleate, pH 7. As titrant 0.1 M NaOH was used.

Results

From earlier studies on isolated thylakoids or purified violaxanthin de-epoxidase the parameters expected to influence conversion of violaxanthin to zeaxanthin would be pH, time of reaction, and temperature. To determine the influence of pH on conversion of violaxanthin to zeaxanthin, spinach leaves were incubated at different pH, with all other parameters kept constant. Acetate-acetic acid was used as a membrane permeable buffer since intact, living leaves were expected to resist internal pH changes imposed by changes in the surrounding medium.

As shown in Fig 1 (filled dimonds) incubation of leaves at pH 6.5 and above gave less than 5% of the xanthophyll cycle pigments (VAZ) in the zeaxanthin form. This amount corresponds to 2.5 μg zeaxanthin/g leaves and typical for untreated and dark-adapted leaves. At lower pH a gradual decrease in violaxanthin and increase in zeaxanthin was seen, with a local maximum at pH 4, where 65-70% of VAZ in the zeaxanthin form. This corresponds to 33-36 μg/g leaf. A slightly more acidic pH was required than observed for the pure enzyme and the purified thylakoids [Bratt et al. 1995], probably due to the restricted accessibility of the buffer in the intact system used here, or to limitations in the availability of the endogenous second substrate ascorbic acid [Bratt et al. 1995].

An unexpected finding was that acetic acid (without acetate), which gave a pH of 2.5 in the medium surrounding the leaves, caused a high production of zeaxanthin. This result was in contrast to the results obtained for the purified systems, where no conversion could be seen at such a low pH [Bratt et al. 1995]. It is of note that the sum of xanthophyll cycle pigments (violaxanthin, antheraxanthin and zeaxanthin) remained close to 100% of the control value when related either to lutein (Fig 1, dimonds) or wet weight (Fig 1, plus). The high degree of scatter between the values related when related to wet weight is probably due to the variable efficiancy in removal of excess medium from the leafs afer incubation. However, the retention of the sum of xanthophyll cycle pigments indicate that the pH within the thylakoids in the leaf did not drop to the low values of the external medium. The reason may again be due to accessibility restrictions. Anyhow, the results show that a 10 - 20 fold increase in zeaxanthin could be obtained in intact leaves in the pH range of 2.5 - 5.5.

Since the internal pH change in the leaves was expected to be due to the entrance of acetic acid through the membrane, not only the pH but also the concentration of acetic acid/acetate buffer was expected to be important. Indeed changing the buffer concentration, with all other parameters kept constant, showed that conversion of violaxanthin to zeaxanthin was more efficient at higher buffer concentration (Fig 2). Starting at the control value of around 3% zeaxanthin of the VAZ-pool to a limiting value of close to 70 % at 2 M acetate buffer, with a half- maximal value reached at 0.25 M acetate buffer. No threshold was seen as function of acetate buffer concentration but rather a gradual increase of

zeaxanthin, which means that any concentration within the range tested caused increase in the amount of zeaxanthin. No visual change of texture of the leaves was seen up to 0.5 M acetate buffer. At increasing acetate concentrations the leaves lost turgor, seen in Fig 2 (plus) as an increase in VAZ in relation to wet weight, and became soft. .

Another important parameter is the time of incubation. With either 0.5 M acetate buffer at pH 5 (Fig 3a) or 0.5 M acetic acid at pH 2.5 (Fig 3b) a gradual increase in zeaxanthin was found up to a value of around 80% of the VAZ-pool. A faster kinetic was seen at pH 2.5, probably because the membrane permeable form

(acetic acid) was dominating at this pH, while at pH 5 it constituted less than half of the acetate/acetic acid couple in the buffer (pKa = 4.7 for acetic acid). Still, the half maximal conversion required 40-70 min of incubation, which is

approximately 3-8 fold slower than for the purified system [Arvidsson et al. 1997]. Also here, no threshold was seen which means that the level of zeaxanthin could be increased using any time of incubation, although to a different degree. At longer incubation times a gradual loss of turgor was observed and at the last time point (5h) even conversion of chlorophyll to feofytin was observed, especially at pH 2.5 and for thin leaves (not shown).

Temperature had a dramatic effect on the degree of conversion of violaxanthin to zeaxanthin in intact leaves (Fig 4). At 4°C the zeaxanthin level increased to about 7% of the VAZ-pool, which was just a doubling compared to the untreated material. Increasing the temperature to room temperature or up to 37°C caused a drastic increase in conversion of violaxanthin to zeaxanthin. These results are in accordance with earlier results from studies on the purified system [3, Arvidsson et al. 1997, 20]. It was earlier observed that temperature had limited effect on the kinetics of the reaction. However, the maximum degree of conversion was strongly dependent on temperature. The reason for this was suggested to be due to changes in organization and packing of the lipid matrix of the thylakoid membrane at different temperatures. From the results presented in Fig 4 and from the earlier published results it is obvious that an efficient conversion of violaxanthin in leaves requires room temperature or above. The upper limit has not been determined but is expected to be around 40-50 °C where several cellular processes are affected. At 4°C and room temperature the leaves retained a fresh appearance while at 37°C leaves became soft and some conversion of chlorophyll to pheophytin was observed (not shown).

Other buffers also supported conversion of violaxanthin to zeaxanthin in intact leaves (Fig 5). At pH 5 (Fig 5c,e,g,j) the order of efficiency was acetate> lactate>citrate=phosphate. In the unbuffered systems (Fig 5b,d,f,h,i) the order of efficiency was acetic acid> phosphoric acid>citric acid>lactic acid. Although the conversion of violaxanthin to zeaxanthin was more efficient with the pure acids destruction of pigments, primarilly chlorphyll, was seen in the pigment analysis . Thus citric acid and lactic acid gave some pheophytin formation from chlorophyll while phosphoric acid caused massive pheophytin formation and the leaves became brown (not shown). Marginal changes in appearance and no pheophytin production were seen at pH 5 for the acids tested.

An interesting question was if HC1 could be used for conversion, as ingested food will reach the stomach where the gastric juice has a pH close to 2. This

corresponds to an HC1 concentration of 10 mM. To mimic the situation of eating vegetables, leaves in the violaxanthin form or zeaxanthin form were grinded and incubated in 10 mM HC1 followed by pigment analysis (Fig 6). Strikingly, violaxanthin (and antheraxanthin) almost completely disappeared while the zeaxanthin content was unchanged. The explanation for the disappearance of violaxanthin and antheraxanthin most certainly is that epoxides are known to be sensitive to low pH [Nagy et al. 2009, Barua and Olson 2001]. In whole leaf experiments violaxanthin was only converted to antheraxanthin and zeaxanthin and no destruction of any of the xanthophyll cycle pigments was observed. The ratio of xanthophyll cycle pigments to lutein and in relation to sample weight stayed within 10% and 25% respectively (not shown) This indicates that the pH inside the intact leaf did not drop to such a low value. The results indicate that zeaxanthin may not be formed in the stomach despite the low pH. However, zeaxanthin, lacking epoxide groups, are more stable at low pH and could stand the gastric juice. Thus, treatment of vegetables to convert of violaxanthin to

zeaxanthin also means that the total amount of carotenoids that can pass the stomach would be higher.

The post harvest method to increase zeaxanthin content was not limited to spinach, but was efficient in all green vegetables tested (Fig 7). Under the conditions tested, corn salad gave the highest degree of change, 22-fold. Even for peas, that showed the smallest change, the increase was 3-fold. However, note that the absolute amount of zeaxanthin that can be formed is dependent of the amount of VAZ-pool of pigments to start with. Thus the leafy sourses gave far more zeaxanthin than peas and sugar snaps. Note also that the optimal conditions for each species was not established, which means that even higher values should be possible to obtain.

Although acetic acid is commonly used in the household, it may not be desired to have it present in all products and not at such high concentration as used in some of the experiments. The acetic acid outside the leaves is easily washed off with water, which would be sufficient for most applications. However, the acetic acid taken up by the leaves may also be of interest to remove. Spinach leaves were therefore loaded for 2 h with 0.5 M acetic and then transferred the leaves to distilled water. The remaining acid in the leaves was determined by pH stat titration. The amount exceeding the value obtained for untreated leaves was assumed to correspond to the amount of acetic acid remaining in the leaves. As seen in Fig 8 a gradual loss of acetic acid with a half time of approximately 45 min was found. Thus the excess of acetic acid could easily be removed to a large extent.

Discussion

The results clearly show that post harvest treatment could be used to improve the zeaxanthin content of green vegetables. A wide range of conditions supported the conversion of violaxanthin to zeaxanthin in intact tissue, using acetic acid/acetate as a buffer. In this work one parameter was studied at the time. However, as the parameters are dependent on each other, many different conditions are in principle expected to give the maximal formation of zeaxanthin. The limit of formation was strongly dependent on temperature (Fig 4), in agreement with earlier studies on isolated thylakoids [Arvidsson et al. 1997, Szilagyi et al. 2007]. Note also that the amount of xanthophyll cycle pigments in green plant tissue is strongly dependent on age and growth conditions, and is highest in young tissue and is dramatically increased, within a few days, upon light stress [Demmig-Adams and Adams 1996, Eskling M and Akerlund H-E 1998].

Acetic acid/acetate was chosen as the buffer for the conversion of violaxanthin to zeaxanthin in leaves as it is a membrane permeable buffer and also that it is commonly used in household to cure and preserve food and thus compatible with food production. Acetic acid/acetate was also found to be the most efficient buffer but other acids like citric acid, lactic acid and phosphoric acid supported conversion to some degree, with a preference for the more membrane permeable buffers (Fig 5). Acetate is the buffer component with the lowest molecular weight and thus even more efficient than the others on a weight basis. The concentration of acetate buffer used in this study was in most cases 0.5 M, corresponding to only 3 %, which can be compared to table vinegar that has 4-6 %. In addition the acetate was easy to remove from leaves after treatment. Although different treatments could be used to obtain maximal conversion of violaxanthin to zeaxanthin, extreme conditions also gave destruction of chlorophyll and changes in texture of the plant material, and should be avoided when a fresh appearance of the product is required. With acetic acid/acetate buffer at pH 5 there was no problem of finding good zeaxanthin formation without visible changes in the appearance of the vegetables.

Violaxanthin is a carotenoid that contains two epoxide groups, which are very sensitive to low pH (Fig 6 and [Nagy et al. 2009, Barua and Olson 2001]). Thus, violaxanthin (as well as neoxanthin and antheraxanthin) are expected to be destroyed in the stomach. However, zeaxanthin that lack epoxide groups are much more stable and can survive acidic conditions. By converting violaxanthin to zeaxanthin the total amount of available carotenoids will increase by 10-20%, and thus add to the nutritional value.

The method described here should be easy to perform in the common household and is also suitable for industrial scale. It is of note that in the natural situation plants convert violaxanthin to zeaxanthin under conditions of light stress [Eskling et al. 2007, Demmig-Adams and Adams 1996], through the process of

photosynthetically driven acidification of the thylakoid lumen, the compartment harboring the violaxanthin de-epoxidase. Thus, leaves picked directly from the plant on a day with full sunlight usually contain increased levels of zeaxanthin [Eskling et al. 2007]. However, in low light or darkness, zeaxanthin, is reconverted to violaxanthin. This is typically the condition after harvest of vegetables. In principle, strong light could be used in an industrial post harvest process to convert violaxanthin to zeaxanthin in vegetables, but the time for illumination, intensity of light required and to get all parts of the vegetable illuminated makes such an approach less attractive.

The daily requirement of zeaxanthin is not known but ordinary consumption of zeaxanthin is typically in the range of 0.2 - 0.5 mg/day [ app et al. 2000]. Just for comparison, assume that 0.5 mg zeaxanthin/day would be a desired amount. To obtain that amount from untreated spinach, approximately 300 gram of spinach is required each day. However, after the treatment outlined in this paper the same amount of zeaxanthin could be obtained with just 12 g of leaves, an amount that more people find acceptable.

Supplementary diet studies have normally been performed with up to 10 mg/day of zeaxanthin or a combination of zeaxanthin and lutein, without any negative effects. Rats given 1 g zeaxanthin/kg bw per day, dogs 442 mg/kg bw per day and monkeys 10 mg/kg bw showed no negative effects in relation to zeaxanthin [Joint FAO/WHO Expert Committee on Food Additives 2004]. An ADI (acceptable daily intake) value of up to 2 mg/kg bw has been recommended by WHO [Joint FAO/WHO Expert Committee on Food Additives (2004)]. This would correspond to 140 mg of zeaxanthin/day for a person of 70 kg. Thus, there is no risk to come close of that value by eating common vegetables treated to increase zeaxanthin content in the way presented in this work.

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Claims

1. An isolated green plant material comprising at least 0.1 mg/g (dry weight) of zeaxanthin.

2. The isolated green plant material according to claim 1 , comprising at least 0.2mg/g (dry weight) of zeaxanthin.

3. The green plant material according to any of claims 1-2, comprising at least 1.0 mg/g (dry weight) of zeaxanthin.

4. The isolated green plant material according to any of preceding claims, wherein said tissue or tissues is dried or substantially free from water.

5. The isolated green plant material according to any of preceding claims, wherein said tissue comprises zeaxanthin and violaxanthin in a ratio of at least 10:90.

6. The isolated green plant material according to any of preceding claims, wherein said material comprises zeaxanthin and violaxanthin in a ratio of at least 25:75.

7. The isolated green plant material according to any of preceding claims, wherein said material comprises zeaxanthin and violaxanthin in a ratio of at least 50:50.

8. The isolated green plant material according to any of preceding claims, wherein said tissue is leaf plant material.

9. A method for the production of isolated green plant material comprising the steps of a) providing the green plant material,

b) incubating said material in the presence of an acid or buffer at a pH of at most 7

c) removing said acid or buffer and

d) obtaining a green plant material having an increased amount of

zeaxanthin compared to the provided green plant material.

10. The method according to claim 9, wherein said improved green plant

material comprises at least 0.1 mg/g (dry weight) of zeaxanthin.

11. The method according to claims 9-10, wherein said acid is acetic acid, citric acid, lactic acid or phosphoric acid.

12. The method according to any of claims 9-11, wherein said buffer is acetic acid/acetate.

13. The method according to any of claims 9-12, wherein said buffer comprises ascorbic acid.

14. The method according to any of claims 9-13, wherein said method

comprises a step of dehydrating said isolated green plant material, such as by heat, air, lyophilisation, spray drying, sun drying or freeze drying.

15. The method according to any of claims 9-14, wherein said method is

performed in a temperature of from 4 °C to 50 °C or more.

16. The method according to any of claims 9-15, wherein said method further comprises a step of purifying said zeaxanthin, such as by an organic solvent such as by acetone or ethanol and obtaining a lipid or ethanol

extract/fraction or isolated thylakoids or fragments of isolated thylakoids.

17. The method according to any of claims 9-16, comprising an additional step wherein all the enzymatic reaction occurring in the plant material is interrupted, to inhibit/stop the production of more zeaxanthin or to stop the possibility of the reverse reaction wherein zeaxanthin reverts to

violaxanthin.

18. An isolated green plant material obtainable by the method according to any of claims 9-17.

19. A product comprising said isolated green plant material according to any of claims 1-8 or claim 18.

20. The product according to claim 19, wherein said product is a

pharmaceutical product or a food additive.