Technical Field

This invention concerns a method for removing cholesterol from organic or biological materials, such as lipid containing foodstuffs of animal origin, and especially from milk and dairy products.

Background Art

It is widely accepted that serious health risks attach to high plasma cholesterol levels. In Australia, coronary heart disease is responsible for more than 50,000 deaths every year, and death from coronary heart disease is twice as frequent as death from cancer. Dairy products, in particular, are perceived as contributing significantly to dietary cholesterol butterfat, for example, contains approximately 3mg cholesterol per gram, and consequently there is considerable interest internationally in reducing the cholesterol level of dairy products.

Disclosure of Invention

This invention is based on the fact that certain steroids or triterpenes and cyclodextrins, have an affinity for cholesterol and bile acids, and we have shown that such agents on binding to selected solid supports form very effective and convenient adsorbents for cholesterol.

Accordingly, this invention provides a method for removing cholesterol from organic or biological substances of animal origin wherein such substances are contacted, in emulsion form, with a cholesterol, adsorbent comprising a steroid, a

triterpene or a cyclodextrin and/or mixtures thereof chemically bonded to a support.

In instances where the biological material is not in emulsion form it should be dispersed in a suitable liquid to form an emulsion prior to contacting with the adsorbent. However biological materials already in such form, such as milk or cream, may be contacted directly with the adsorbent without any pre- reatment.

The term "emulsion" as used herein is intended to include miscellar solutions or cholesterol-containing fat associated with protein as in lipoproteins.

Typical steroids for use in the practise of this invention include diosgenin, digitonin and tomatidine, and a particularly preferred compound is diosgenin. Suitable triterpenes are medicagenic acid and hederagenin. Cyclodextrins include % , β- and ϊ-cyclodextrin, or their modified forms.

Suitable support materials will

i) not significantly impair the cholesterol affinity of the aforesaid steroids etc; ϋ) be essentially inert to cholesterol and other components of the material to be treated; iii) bind to the steroid etc in such a way that the bond is, not readily disrupted by processes for separation of cholesterol from the adsorbent.

Silica has proved to be a suitable support material. Examples of other materials include, polystyrene, polymethylmethacrylate and cellulose.

The adsorbent may itself be the support if it can be presented in the form of relatively insoluble particles. This may be achieved by bonding the adsorbent to itself in such a way as to have active groups on the surface of the particles. The adsorbent may be cross linked to promote insolubility.

A further advantageous feature of the aforementioned adsorbents is that they can readily and economically be regenerated for further use by washing with solvents, such as acetic acid, or mixtures of butanol and acetic acid, which remove the cholesterol.

Surprisingly it has been found that adsorbents used in the method of the invention can work ^' quite effectively at lower temperatures even though the conventional method would suggest that higher temperatures should be required to obtain a satisfactory degree and ratio of adsorption. The cholesterol containing fats in many biological materials are solid at low temperature. It is therefore to be expected that the solid fats will reduce the rate of adsorption of cholesterol by the adsorbent in comparison with the liquid form of the fats at higher temperatures. It is believed that the size of cholesterol containing globules of fats in the emulsions is a factor in ensuring that a satisfactory adsorption rate is realised. When the fat globules are very small, as in the case of milk, the cholesterol tends to accumulate at the surface of the globules with the result that it can readily transfer to the adsorbent even though the fat globules are solid.

The low temperature capability of a preferred method of the invention is particularly useful in relation to biological materials, such as with milk or cream, which spoil if they are not chilled.

Thus in a preferred aspect of the invention the temperature at which the absorption is carried out is below 18 C. More preferably a temperature range of 0 to 8 C is appropriate. Where such low adsorption temperatures are used it is preferred that the fats in the emulsion be of sufficiently small size to ensure that at least 30% of the cholesterol is adsorbed within 20 minutes of being contacted with the adsorbent.

The average time the biological material will need to be in contact with the adsorbent to achieve an effective degree of adsorption will vary, depending on a number of factors, such as, quantity of adsorbent material and nature of the biological material being treated. However, generally speaking an average contact period of from 1/2 to 20 minutes more preferably 2 to 20 minutes will give satisfactory results.

In order to ensure a high level of adsorption, the molar ratio of adsorbent to cholesterol in the biological material should not be lower than 0.5, more preferably it should exceed 50. o ^Tne ratio may be such as to ensure removal of at least 35% of the cholesterol in the biological material, more preferably 60%.

In one form, a process according to this invention will 5 involve the following steps:-

i) where necessary, rendering the material to be treated to liquid form, eg by dispersion in a solvent or emulsion, 0 ϋ) bringing the liquid material into intimate contact with the adsorbent, eg by flow through a column packed with the adsorbent, or by agitation with the adsorbent, iii) separating the cholesterol-reduced material from c the adsorbent,

iv) regenerating the adsorbent by elution of the adsorbed cholesterol.

The invention also covers biological materials which have been reduced in cholesterol in accordance with the method of the - 05 invention.

Best Modes for Carrying Out the Invention

The invention will now be described with reference to the 10 following examples:

Example 1

Butterfat containing cholesterol isotopically labelled with

14 15 C was dissolved in hexane and passed through a column packed with a steroid, (25R)-spirost-5en-38-ol

(C ₂ _H ₄₂ 0 ₃ ), covalently linked to silica. The radioactivity from the labelled cholesterol was reduced by

42.6% as shown in Table 1.

20

TABLE 1 - Removal of cholesterol from butterfat by (25R)-spirost-5en-38-ol (C^H.-O.,) covalently linked to silica.

25

Activity of labelled cholesterol (DPM)

Untreated butterfat 4440

Butterfat after passage through column 2550 Material recovered from column by

30 extraction with acetic acid 1884

Chemical estimation of cholesterol in these samples further confirmed that cholesterol had been removed from the Butterfat.

EXAMPLE 2

Cholesterol labelled with 14C was emulsified by using a sonicator with oleic acid, nomo-olein and taurocholic acid in phosphate buffer (pH7.0). This emulsion was shaken with (25R)-spirost-5en-38-ol (C^H^-O.,) bonded to silica gel. The adsorbent removed 82% of the cholesterol from the emulsion. (As shown in Table 2.) Ordinary, untreated silica had no significant effect on the . cholesterol content of the emulsion.

TABLE 2. Removal of cholesterol from an emulsion by the (25R)-spirost-5en-38-ol ' ^C 27 ^H 42°3' covalently linked to silica.

Activity of labelled cholesterol (DPM)

Untreated emulsion 455

After exposure to untreated silica 449 After exposure to the steroid linked to silica 80

Measurement of Cholesterol Adsorption

in examples 3 to 6 standard emulsions containing H-labelled cholesterol were used for routine screening of adsorbents.

The emulsions were prepared in isotonic phosphate buffer and contained 14C-labelled Cr-EDTA added to provide a means of monitoring any uptake of water by the adsorbent. Two emulsions were used:

* "Weak emulsion" (WE) containing cholesterol (0.1 mM), oleic acid (1.2 mM) , monolein (6.0 mM) and sodium taurocholare (10.0 mM) .

* "Strong emulsion" (SE) containing cholesterol (0.25 mM), oleic acid (1.2 mM), monolein (6.0 mM) and sodium taurocholate (10.0 mM) . (The same cholesterol concentration as milk)

EXAMPLE 3

Cyclodextrins and Sapogenins Attached to So1id Supports

A variety of solid adsorbents can be prepared. The choice of chemistry for the attachment procedure will depend on the nature of the solid support.

It is obviously essential not to destroy in the attachment process the ability of the cyclodextrin or saponin to form a stable complex with cholesterol, (e.g. the cyclodextrin cavity can easily be closed and rendered inaccessible to cholesterol. )

Adsorption of cholesterol was determined by shaking 4 ml of milk or emulsion with (typically) 0.1 g of the solid adsorbent.

(1) Silica

β-cyclodextrin: Silicic acid (10 g) and glycidoxypropyltrimethoxysilane (30 g) were made into a slurry with dry dimethyl formamide (600 ml). Dry β-cyclodextrin (30 g) was dissolved in dry dimethyl formamide (375 ml) and sodium hydride (9 g) added with stirring, allowed to react for 15 min. and the solution filtered, taking precautions to exclude water. The slurry and the solution were mixed and then refluxed under dry conditions for 4 hours. The solid product was collected by filtration and washed with dimethyl formamide, methanol and water. The product was finally

dried with gentle heat under vacuum. The effectiveness of the product is shown in Table 3.

TABLE 3. Adsorption of cholesterol from emulsion or milk by β-cyclodextrin attached to silica.

Adsorbent Emulsion % Cholesterol Support Removed

Coarse silica ^e WE 82 Fine Silica WE 80 Coarse silica M 85

0.1 adsorbent with 4 ml emulsion - coars silica particle size 70-230μ diameter - fine silica has a surface area of 200± 30 ~ per gram, lg adsorbent wites 10ml milk.

EXAMPLE 4

Diosgenin: the procedure for attachment was the same as that described for cyclodextrin. Butterfat containing

14 C-labelled cholesterol was passed through a column packed with the adsorbent. The results are shown in Table 4.

TABLE 4 Adsorption of cholesterol from butterfat by diosgenin attached to silica.

Adsorbent Emulsion % Cholesterol Support Removed

Coarse silica Butterfat 42 . 6

EXAMPLE 5

Polymethylmethacrylate β-cyclodextrin : Polymethylmethacrylate (4. 1 g) was dissolved in pyridine (100 ml ) and

_ _

β-cyclodextrin (3.25 g) was added and dissolved. To this was added 1 ml of a methanolic solution of sodium methoxide (200 g/1) and the mixture refluxed for 30 min. The solid product that formed was filtered, washed with water and dried under vacuum.

TABLE 5 Adsorption of cholesterol from emulsion or milk by β-cyclodextrin attached to polymethylmethacrylate.

Adsorbent Emulsion/Milk % Cholesterol Support (SE or WE) (M) Removed

Polymethyl WE; 91 methylacrylate SE' 97 M ¹ 93

0.1 of adsorbent added to 4.0 ml emulsion and held at

200° f:or 15 min. 0.1 g adsorbent added to 3.0 ml milk.

EXAMPLE 6

CROSS-LINKED ADSORBENTS

Tomatine: Tomatine (1 g) was dissolved in dry dimethyl formamide (5 ml) and sodium hydride (0.5 g) added. The reaction was allowed to proceed for 10 min. Epichlorohydrin (20 ml) was then added and the solution heated at 120 for 4 hour. The excess epichlorohydrin was removed by distillation. The solid precipitate thus obtained was washed with dimethyl formamide and ethanol and dried under vacuum.

β-cyclodextrin: β-cyclodextrin (5 g) was wetted with water ( 2ml) and dissolved in 50% aqueous sodium hydroxide (6ml). Epichlorohydrin (50ml) was added and allowed to react at room temperature (ca 20 ) for

12hr. The solid product was washed with methanol, hot water and cold water.

TABLE 6 Adsorption of cholesterol from emulsion or milk by cross-linked tomatine or β-cyclodextrin.

Adεorbant Emulsion/Milk % Cholesterol

(SE) (M) Removed

Tomatine SE 30

M 41 β-cyclodextrin SE 75

EXAMPLE 7 β-Cyclodextrin Linked to Diosgenin

Diosgenin (3g) was dissolved in pyridine (25ml) and acidified with sulphuric acid to approximately pH=4. Cyclodextrin

(8.2g) in pyridine (75ml) was added at 85°C with stirring over 15 minutes. After 45 minutes, the mixture was poured into water (600ml). The solid product was collected by filtration and washed with water, followed by acetone.

TABLE 7 Adsorption of cholesterol from emulsion by β-cyclodextrin linked to diosgenin.

Adsorbent Emulsion % Cholesterol

Removed

β-cyclodextrin linked to WE 57 diosgenin

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In addition to their value in the treatment of cholesterol-containing foodstuffs, it is envisaged that adsorbents according to this invention might form the basis of pharmaceutical products or dietary supplements for reducing intestinal absorption of cholesterol or bile acids.

Also, the adsorbents of this invention might be used to extract cholesterol and bile acids from source material, such as bile (an abattoir waste). The adsorbed cholesterol or bile acids could then be selectively released by washing with suitable solvents, and used as precursors for the synthesis of steroid-based drugs.