EP1451170A1

EP1451170A1 - Zeolites as matrices

Info

Publication number: EP1451170A1
Application number: EP02783918A
Authority: EP
Inventors: Hakan Eriksson; Annika Olsson
Original assignee: Biosilvio Munkfors AB
Current assignee: Eriksson Hakan
Priority date: 2001-11-07
Filing date: 2002-11-06
Publication date: 2004-09-01
Also published as: WO2003040123A1; SE0103693L; SE525503C2; SE0103693D0

Abstract

A method and a kit for the extraction and purification of anthocyans from plant materials, comprising the steps of providing plant material(s) comprising anthocyans, providing one or more hydrophobic dealuminised zeolite(s), adding the plant material(s) to one or more hydrophobic dealuminised zeolite(s), allowing adsorption of the anthocyans to one or more hydrophobic zeolite(s), eluting the anthocyans from the hydrophobic zeolite(s), and obtaining the anthocyans which have been extracted and purified form the plant material(s). A method for separating the compounds anthocyanidines and anthocyanins. The use of extracted product(s) preferably for direct or indirect use for human consumption, in food or pharmaceutical production.

Description

ZEOLITES AS MATRICES

FIELD OF INVENTION

The invention relates to a new improved method to enable extraction and purification of anthocyans from plant material(s) using hydrophobic or

"dealuminised" zeolites. Anthocyans are known to be antioxidants and are meant to be used, directly or indirectly, for human consumption, or in the production of food or pharmaceuticals.

BACKGROUND OF THE INVENTION

Antioxidants are thought to reduce risk for cardiovascular diseases and various forms of cancer. A large and significant group of antioxidants are anthocyans found in many different plants. These molecules or pigments confer colour to flowers and berries in red and blue hues at low pH values; at higher pH values the molecules are rearranged to another form and the colour disappears. The pigments are relatively unstable and easily oxidised. Anthocyans are thought to have a positive effect on sight, strengthen capillary circulation and reduce capillary permeability (EP 90308197.4, US 5,320,841). They have also been shown to possess anti-inflammatory properties and encourage the healing process. In Europe, anthocyanins are used as natural colourants under classification E163 (see for example, Goiffon, J-P. et al. Analytica Chimica Acta, 382 (1999)).

Anthocyans comprise anthocyanins and anthocyanidines. A number of anthocyanidines form glycosides with different carbohydrates resulting in a large number of different anthocyanins. Anthocyanidines are unstable and they are therefore only found in low concentrations.

Isolation of such compounds has proven to be a difficult task. Earlier attempts using talc have not functioned satisfactorily since talc has little capacity for adsorption and would require large amounts to be effective. Others have tried using anion exchangers, but in this case, adsorption lacks selectivity and subsequent elution must be performed at a pH which may be harmful to the quality of the resultant desired product. Moreover, anion exchangers are expensive and it is difficult to regenerate the ion exchange matrix if it becomes contaminated with microorganisms or became clogged due to small particles in the sampling fluid.

US 4,260,388 describes a method to purify aqueous solutions of anthocyans using a polystyrene coated metal oxide as the adsorbent. Besides being a more expensive matrix, the need for a polymer coating process implies the need for toxic organic solvents, for example, methylene chloride and benzene, as well as a risk for the potential presence of toxic monomers within the coating formulation. Thus, there is a need for an improved method to extract and purify anthocyans possessing no or very minimal risks for toxicity.

Zeolites are hydrated aluminiumsilicate minerals containing alkali and alkaline earth metals, such as Na and Ca, as well as K, Ba and Sr. They are noted for their lability toward ion exchange; for ex., Na and K atoms easily exchanged with Ca and Mg, as well as for their lability toward reversible dehydration. Zeolites have a framework structure that encloses interconnected cavities occupied by large metal cations (possibly charged ions) and water molecules. On dehydration, some zeolites form network-like structures with regular diffusion channels. This network structure in zeolites has, in the last years, been heavily exploited in uses including the fractionation of hydrocarbons, such as in petroleum refining, drying of gases and liquids and pollution control by selective molecular adsorption.

The zeolites mentioned above have been classified as "hydrophobic" or "dealuminised" zeolites. The degree of hydrophobicity is dictated by the Si/Al ratio. Zeolites with a high Si/Al ratio carry less framework charge and are commonly referred to as "hydrophobic" or "dealuminised"; the opposite is true for high alumina content zeolites which are labelled "hydrophilic". Some examples of hydrophobic zeolites include silicalite, mordenite and zeolite Y. One of the differences which exist between these zeolites lie in the size and availablility of the pores present in the zeolite crystals. For example, silicalite and zeolite Y have three- dimensional pore systems which are easily accessible, while the pore system in mordenite is two-dimensional and is therefore less easily accessible. With respect to pore size, both zeolite Y and mordenite belong to the largest known groups of zeolites with pore size respectively about 7 and 7.5 A; silicalite has on the other hand, a pore diameter of about 5.5 A (see D.W.Breck, Zeolite Molecular Sieves, Wiley, New York, 1974.).

The use of hydrophobic zeolites for adsorption of specific organic compounds in water has been described previously. US 5,108,617 describes the adsorption of detergents using hydrophobic zeolites, while WO 9965826 describes a method for purifying water using hydrophobic zeolites. EP 0956155 describes a method for removing preservatives from polypeptide solutions using hydrophobic zeolites. There is, however, a size limit to selective molecular adsorption by hydrophobic dealuminised zeolites, and in many cases, hydrophobic zeolites cannot adsorb molecules that are too large to fit into their pores and canal systems.

SUMMARY OF THE INVENTION

Despite the size limits to selective molecular adsorption, it has been surprisingly found that hydrophobic dealuminised zeolite Y exhibits a high adsorption capacity for anthocyans from different plant materials. Being relatively large in size, anthocyans would not be expected to fit into zeolite pores.

The present invention provides a new method in which hydrophobic dealuminised zeolites are used for the extraction and purification of anthocyans from plant materials. The method enables the possibility to extract and purify large amounts of anthocyans without any undesirable changes in the purified products, for example, an accompanying loss of antioxidant activity. Additionally, the zeolites may be used over and over again.

Accordingly, in a first aspect the invention relates to a method for the extraction of anthocyans from plant materials comprising the steps of providing plant material(s) comprising anthocyans, providing one or more hydrophobic dealuminised zeolite(s), adding the plant material(s) to one or more hydrophobic dealuminised zeolite(s), allowing adsorption of the anthocyans to one or more hydrophobic zeolite(s), eluting the anthocyans from the hydrophobic zeolite(s), and obtaining the anthocyans which have been extracted and purified from the plant materials.

According to another aspect the invention relates to a kit, comprising hydrophobic zeolite particles to extract anthocyans from plant material.

According to a third aspect the invention relates to products obtained by the invented method and/or by the kit.

The invention provides a new improved method for the purification of anthocyans from plant material(s). A method which enables the possibility both to obtain large amounts of purified anthocyans as well as to reduce and/or eliminate the risk for the residual presence of toxic compounds within the purified anthocyans.

Additionally, the present invention provides the ability to separate different anthocyans. A possibility to separate the compounds would allow better usage of these compounds in, for example, food or pharmaceutical products.

DESCRIPTION OF THE DRAWINGS

Figure 1: Figure 1 shows the chemical structure for several different anthocyanidines where R₃ and R shown in the figure are composed of hydrogen (H), and for anthocyanins where R₃ and R₄ are hydrogen (H) or carbohydrates. Figure 1 also lists the substituent groups for different anthocyanidines. Figure 2: Figure 2 shows that hydrophobic dealuminised zeolite Y adsorbs more anthocyans than aluminium-containing zeolite Y.

Figure 3: Figure 3 shows that elution from a column containing hydrophobic dealuminised zeolite Y particles is effective for the extraction of anthocyans from bilberry juice (Vaccinium myrtillus). Figure 3 also shows that a fractionated elution of the anthocyans is obtained when the column is first eluted with pure ethanol and then as a second elution with acidified ethanol, 75 % ethanol (vol/vol) in 0.1 M HC1.

Figure 4A: Figure 4A shows the HPLC analysis of blueberry juice diluted 200 times with water. Effectively, Figure 4 A shows an HPLC profile at 503 nm of anthocyans comprising anthocyanidines and anthocyanins in blueberry juice.

Figure 4B: Figure 4B shows the HPLC analysis profile at 503 nm of the eluted fraction obtained using pure ethanol from a zeolite column, which is effectively the anthocyanidine fraction.

Figure 4C: Figure 4C shows the HPLC analysis profile at 503 nm of the eluted fraction obtained using acidified ethanol from a zeolite column, which is effectively the anthocyanin fraction.

Figure 4D: Figure 4D shows a HPLC analysis profile at 503 nm of the hydrolysed material from Figure 4C, which is effectively the anthocyanidine fraction. Figure 4D also confirms that the fraction shown in Figure 4B is the anthocyanidine fraction.

Figure 5: Figure 5 shows that a continuous elution process to extract anthocyans through a column packed with hydrophobic dealuminised zeolite particles is both possible and effective, allowing a yield value of almost 80%.

Figure 6A: Figure 6A shows that a column of hydrophobic dealuminised zeolite Y particles iseffective for the extraction of anthocyans from juice of another plant, namely, blackberry juice, Rubusfruticosus.

Figure 6B: Figure 6B shows that a column of hydrophobic dealuminised zeolite Y particles is effective for the extraction of anthocyans from the juice of another plant, namely, chokeberry juice, Aronia melanocarpa.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

The term "anthocyans"is intended to mean a subgroup belonging to the flavonoids class of plant phenolics and comprising anthocyanidines and anthocyanins.

The term "anthocyanidines" refers to a name for the molecules with the following general chemical structure shown in Figure 1.

The term " anthocyanins" refers to molecules having a molecular structure similar to anthocyanidines but possessing glycoside substituent groups at one, or both, of positions (R₃) and (R ). This is shown more clearly in Figure 1 and Table 1.

The term "plant material(s)" is intended to mean all extracts obtainable from all parts of plants, such as fruits, for example berries, leaves, flowers, stems, roots, and so on. The terms "hydrophobic" and "dealuminised" zeolites is intended to mean zeolites possessing a high Si/Al ratio and carrying thus less framework charge; the degree of hydrophobicity being dictated by the Si/Al ratio.

Description of the invention

The present invention provides a new application for hydrophobic dealuminised zeolites, as a method to extract anthocyans from plant materials. Anthocyans are known antioxidants which may be used, directly or indirectly, for human consumption, or in the production of food or pharmaceuticals. The use of the hydrophobic dealuminised zeolites involves contacting the plant material(s) with the zeolites, in such quantity or concentration as to allow the required adsorption of anthocyans present within the plant material to occur.

The employment of the invention may take place in a continuous or semicontinuous manner. One method entails directly contacting the hydrophobic dealuminised zeolite(s) with the plant material(s), while yet another method may involve packing a column, filter or similar device, with the hydrophobic zeolite(s), through or over which the plant material(s) is passed. Other methods commonly known to the person skilled in the art of separation and extraction techniques can also be employed. The hydrophobic zeolites cannot adsorb unlimited amounts of anthocyans, which implies that the former must periodically be eluted and regenerated. Treatment with organic solvents is one method of disrupting the chemical bonds between zeolites and the adsorbed molecules, and in this way, enforce an elution of adsorbed material from a zeolite column. Adsorbed anthocyans may be eluted from the dealuminised zeolites using an organic solvent such as ethanol or, for example, acidified ethanol, 75 % ethanol (volume/volume) from about 0.01 to about 0.4 M HC1, or, from about 0.01 to about 0.1 M HC1.

The hydrophobic zeolites may have the fundamental structure normally observed in these materials, i.e., where the atoms are predominantly Al and Si. However, hydrophobic zeolites possessing a structure where the atoms B, P, Fe, Ga, Ge, have, to some extent, replaced Al and Si, may also be envisaged.

The hydrophobic dealuinised zeolites may have the molecular formula, [(Al₂0₃)_x(Si0₂)_y] where x and y are integers, which may have the ratio y/x from about 15, such as from about 100 or from about 200 or often from about 1000. A higher ratio of silicon to aluminium confers stronger hydrophobic properties, gives stability in waterbased systems within a broad pH-region and makes the zeolites insensitive to oxidative and reductive agents. Additionally, these hydrophobic dealuminised zeolites can withstand high pressure and high temperatures without being altered in their properties. Also, according to the invention, at least one type of hydrophobic dealuminised zeolite is to be used as the adsorbent, but a combination of different zeolite types can also be used depending on the application.

The word "combination" may be first defined to mean a mixture of hydrophobic zeolites comprising, for example zeolite Y and mordenite, zeolite Y and silicalite, or zeolite Y and mordenite and silicalite. In this context, the combination of hydrophobic zeolites is chosen from the group consisting of silicalite, mordenite and zeolite Y. Of these three, zeolite Y is expected to have a dominant role in the adsorbent function. Secondly, the word "combination" may also be defined to mean a distribution of hydrophobic zeolites, all possessing the fundamental structure comprising predominantly Al and Si atoms and having the molecular formula, [(Al₂θ₃)_x(Si0₂)_y] where x and y are integers, where the y/x ratios are different in each hydrophobic zeolite. Hydrophobic zeolites can be used untreated, or, in the form of sintered crystals, or, in the form of crystals included or suspended in materials which are not zeolites. This can additionally be adapted to, or, combined in some other way, with one or more, permeable materials which are not zeolites.

Hydrophobic dealuminised zeolites cannot adsorb unlimited amounts of anthocyans.The adsorption capacity of zeolites can, however, be regenerated through alternately washing at low and high pH, from about pH 1 to about pH 8.5. After washing, the hydrophobic dealuminised zeolites are equilibrated to the desired pH where adsorption of anthocyans is to be achieved. Hydrophobic dealuminised zeolites are able to withstand heating, and if their absorption capacity worsens due to blockage by organic material or microbiological growths, this property can also be improved through heating. One suggested method includes heating up to a temperature above 700°C, such as above 800 °C or 900-1100 °C. If there is a wish to eliminate risks for chemical contamination and microbial infection before the start of an extraction process, one may conduct a similar heating process as a pretreatment of the hydrophobic dealuminised zeolites.

Both the high bonding strength and bonding rates of anthocyans to the zeolites allows the possibility to limit the retention time between the antocyan- containing sample and the zeolite in a column or filter. Thus, a high pressure stream of sample may be used in the setup, allowing a short retention time of the sample in the column or filter, such as from about several tens of seconds to 20-30 minutes, or, from about 10 seconds to 15 minutes, or, from about 20 seconds to 10 minutes. EXAMPLES

Examples 1-5 given below illustrate the invention. These examples are present to exemplify the invention; they are not, however, intended to limit in any way the invention as covered by the claims.

Example 1 Objective

The object of the experiment was to compare adsorption of anthocyans using respectively dealuminised and aluminium-containing hydrophobic zeolite(s) after batch incubation of bilberry juice (Vaccinum myrtillus).

Experimental

A portion of bilberry juice (5 ml) was incubated on a rocking table with 20 mg/ml hydrophobic dealuminised zeolite Y (•) or with aluminium-containing zeolite Y (■). After 15 minutes, the samples were centrifuged. The supernatants were removed and a new portion of zeolite (20 mg/ml) was added to the bilberry juice. After an additional 15 minutes, the samples were centrifuged and a further portion of zeolite (20 mg/ml) was added to the bilberry juice. In total, 5 portions of zeolite were added to the bilberry juice.

Results and conclusions

Figure 2 shows that hydrophobic dealuminised zeolite Y adsorbs more anthocyans than aluminium-containing zeolite Y.

Example 2 Objective

The object of the experment was to illustrate the amounts of anthocyans extracted from bilberry juice (Vaccinium myrtillus) using hydrophobic dealuminised zeolite Y particles, at different stages of the elution process.

Experimental

A column (25 mm diameter) was packed with hydrophobic dealuminised zeolite Y particles which were initially degassed and washed with water. Bilberry juice, diluted with water in the ratio (1 :5), to make a total of 10 ml, was added to the zeolite column. The column was washed firstly with 20 ml water (see fractions 2-3 in figure 3) and then with 20 ml 50 mM NaCl (see fractions 4-5 in figure 3). To elute the adsorbed anthocyans, the column was washed with 20 ml 96 % ethanol (see fractions 6-7), 20 ml 75 % ethanol (v/v) in 0.1 M HC1 (see fractions 8-9), or 20 ml 75 % ethanol (v/v) in 0.4 M HC1 (see fractions 10-11). Fractions of 10 ml were collected.

Results and conclusions

Figure 3 shows that elution from a column containing hydrophobic dealuminised zeolite Y particles is effective for the extraction of anthocyans from bilberry juice (Vaccinium myrtillus). The figure also shows that a fractionated elution of the anthocyans is obtained when the column is first eluted with pure ethanol and then as a second elution with acidified ethanol, 75 % ethanol (v/v) in 0.1 M HC1.

Example 3 Objective

The object of the experiment was to show the results of the high pressure liquid chromatography (HPLC) analysis of blueberry juice {Vaccinium myrtillus) and anthocyans eluted from a zeolite column.

Experimental

The analysis was performed using an ODS Hypersil column (50 x 3,00 mm, 3 μm) with an elution gradient starting with; 0 to 2 minutes 100 % mobile phase A and then from 2 to 11 minutes a gradient from 0 to 30 % of the mobile phase B. The mobile phase, A, consisted of 1 % phosphoric acid, 10 % acetic acid in water. The mobile phase B used was 100% acetonitrile. The flow rate was 0.5 ml/minute and the anthocyans were identified at an absorbance value of 503 nm.

Results and conclusions

Figure 4 A shows the HPLC analysis of blueberry juice diluted 200 times with water. Effectively, Figure 4 A shows an HPLC profile at 503 nm of the anthocyans (comprising anthocyanidines and anthocyanins) content in blueberry juice. Figure 4B shows the HPLC analysis of the eluted fraction obtained using pure ethanol from a zeolite column. (In this case, the blueberry juice was first added to the column, and after washing, the column was eluted with pure ethanol. The eluate is equivalent to that obtained in fraction 6 in Example 2.) Figure 4B effectively shows the HPLC profile at 503 nm of the anthocyans eluted with pure ethanol, which is the anthocyanidine fraction.

Figure 4C shows the HPLC analysis of the eluted fraction obtained using acidified ethanol from a zeolite column. (In this case, the blueberry juice was first added to the column, and after washing, the column was eluted firstly with pure ethanol, followed by acidified ethanol. The eluate is equivalent to that obtained in fraction 9 in Example 2.)

Figure 4C effectively shows the HPLC profile at 503 nm of the anthocyans eluted with acidified ethanol, which is the anthocyanin fraction. Figure 4D shows a HPLC analysis of the hydrolysed material from Figure

4C. Here, the hydrolysis process was performed by incubating the eluted fraction obtained from the zeolite column using acidified ethanol (i.e., the anthocyanin fraction), for 60 minutes in 4 M HC1 in a boiling water bath, i.e., at 100 °C. Deglycosylation of anthocyanin was expected to occur on hydrolysis, producing anthocyanidines. Figure 4D effectively shows the HPLC profile at 503 nm of the anthocyanidine fraction. It was observed that the same or very similar HPLC profile was given in Figure 4B (i.e., the fraction obtained from the eluted fraction of anthocyans from the zeolite column using pure ethanol). Figure 4D thus confirms that the fraction shown in Figure 4B is the anthocyanidine fraction. To summarise, Figures 4A-4D show that "fractionated elution" intended herein to mean the separation of anthocyanidines (deglycosylated) and anthocyanins (glycosylated), may be effected by first eluting the anthocyans adsorbed on a zeolite column with pure ethanol, which elutes the anthocyanidines, followed by acidified ethanol, which in this second step elutes the anthocyanins.

Example 4 Objective

The objective of this experiment was to show that a continuous process to extract anthocyans through a column packed hydrophobic dealuminised zeolite Y particles is a feasible process.

Experimental

A column was packed with hydrophobic dealuminised zeolite Y particles which were initially degassed and washed with water. The bilberry juice, diluted with water in the ratio (1 :5), to form a total of 10 ml, was added to the column, then washed with 13 ml water (see fraction 2, Figure 5), followed by 32 ml 50 mM NaCl (see fraction 3, Figure 5). To elute the adsorbed anthocyans, 19.5 ml 96% ethanol (see fractions 4-6, Figure 5), 22.5 ml 75% ethanol (v/v) in 0.1 M HC1 (see fractions 7-8, Figure 5) and finally, 14 ml 75% ethanol (v/v) in 0.4 M HC1 (see fractions 9- 10, Figure 5) were pumped progressively through the column. By visually monitering the washing and elution process, the different amounts of fractional volumns were collected. In total, 5.1 mg anthocyans with an antioxidant capacity equivalent to 584 nmol trolox, a known antioxidant, was applied to the column. Out of this, a total of 4,0 mg anthocyans with an antioxidant capacity equivalent to 473 nmol trolox was eluted and collected, giving a yield value of ca. 78.4 wt. % for the extraction process.

Results and conclusions Figure 5 shows that a continuous elution process to extract anthocyans through a column packed with hydrophobic dealuminised zeolite particles is both possible and effective, allowing a yield value of almost 80%.

Example 5 Objective

The objective of this experiment was to illustrate the extraction of anthocyans from two other plant materials, blackberry juice, Rubus fruticosus (see Figure 6A), and chokeberry juice Aronia melanocarpa (Figure 6B) respectively, using a column packed with hydrophobic dealuminised zeolite Y particles.

Experimental

In the experiment involving blackberry juice, a column was packed with hydrophobic dealuminised zeolite Y particles which were initially washed with 50 ml 50 mlvl phosphate buffer pH 6.4; blackberry juice diluted with water (in a 1 :5 ratio), to form a total of 10 ml, was then applied to the zeolite column. The column was washed firstly with 20 ml 5 mM phosphate buffer pH 6.4 (see fractions 2-3, Figure 6A) and then with 20 ml 50 mM NaCl in 5 mM phosphate buffer pH 6.4 (see fractions 4-5, Figure 6A). To elute the adsorbed anthocyans, the column was eluted with 20 ml 96 % ethanol (see fractions 6-7, Figure 6A), 20 ml 75 % ethanol (v/v) in 0.1 M HCl ("acidified ethanol"; see fractions 8-9, Figure 6A) and finally with 20 ml 75 % ethanol (v/v) in 0.4 M HCl (see fractions 10-11, same figure). Fractions of 10 ml were collected.

In the experiment involving chokeberry juice, a column was packed with hydrophobic dealuminised zeolite Y particles which were initially washed with 50 ml 50 mM phoshate buffer pH 6.4; chokeberry juice diluted (1 :5) with water to form a total of 10 ml, was then applied to the zeolite column. The column was washed firstly with 20 ml 5 mM phosphate buffer pH 6.4 (see fractions 2-3) and then with 20 ml 50 mM NaCl in 5 mM phosphate buffer pH 6.4 (see fractions 4-5). To elute the adsorbed anthocyans, the column was eluted with 20 ml 96 % ethanol (see fractions 6-7), 20 ml 75 % ethanol (v/v) in 0.1 M HCl (see fractions 8-9) and finally with 20 ml 75 % ethanol (v/v) in 0.4 M HCl (see fractions 10-11). Fractions of 10 ml were collected. Results and conclusions

Figures 5 A and 5B show that a column of hydrophobic dealuminised zeolite Y may effectively be used for the extraction of anthocyans from juice of two other plant materials, namely, blackberry juice (Rubus fruticosus), and chokeberry juice (Aronia melanocarpa).

Claims

1. A method for the extraction of anthocyans from plant material(s) comprising the steps of a) providing plant material(s) comprising anthocyans, b) providing one or more hydrophobic dealuminised zeolite(s), c) adding the plant material(s) to one or more hydrophobic dealuminised zeolite(s), d) allowing adsorption of the anthocyans to one or more hydrophobic zeolite(s), e) eluting the anthocyans from the hydrophobic zeolite(s), and f) obtaining the anthocyans which have been extracted and purified from the plant material(s).

2. The method according to claim 1, wherein the hydrophobic dealuminised zeolite(s) provided in step (b) has the chemical structural formula [(Al₂0₃)_x(Si0₂)_y] where x and y are whole integers and y/x is from about 15.

3. The method according to claim 2, wherein y/x is from about 100.

4. The method according to claim 3, wherein y/x is from about 200.

5. The method according to claim 4, wherein y/x is from about 1000.

6. The method according to any of the preceding claims, wherein the hydrophobic dealuminised zeolite(s) is selected from the group consisting of silicalite, mordenite and zeolite Y.

7. The method according to any of the preceding claims, wherein the hydrophobic dealuminised zeolite(s) are a combination of zeolites, selected from the group consisting of zeolite Y, mordenite and silicalite.

8. The method according to any of the preceding claims, wherein the hydrophobic dealuminised zeolite(s) comprise not less than 10% zeolite Y.

9. The method according to any of the preceding claims, wherein the hydrophobic dealuminised zeolite(s) comprises a distribution of hydrophobic zeolites, having the molecular formula, [(Al₂0₃)_x(Siθ₂)_y] where x and y are integers, and where the y/x ratio for each hydrophobic zeolite may be different but is not less than 15.

10. The method according to any of the preceding claims, wherein the hydrophobic dealuminised zeolite(s) are in the form of sintered zeolite crystals or zeolite crystals included in or coated with or suspended in one or more non-zeolite materials.

11. The method according to any of the preceding claims, wherein the hydrophobic dealuminised zeolite(s) provided in step (b) are attached to a surface, or packed in a column or filter or formed to a column or filter.

12. The method according to any of the preceding claims, wherein ethanol and/or acidified ethanol are used to elute the anthocyans.

13. The method according to any of the preceding claims, wherein the elution in step(e) comprises at least two separate steps using ethanol followed by acidified ethanol.

14. The method according to claim 13, for the analysis of the content and/or distribution of the anthocyanins and anthocyanidines.

15. A kit, comprising hydrophobic zeolite particles to extract anthocyans from plant material(s).

16. The kit according to claim 15 to be used in the method according to claims 1-14.

17. Product(s) obtained by any of the preceding claims .

18. The product(s) according to claim 17 wherein the product is anthocyans.

19. Use of the products according to claims 17-18.