WO2007039040A1

WO2007039040A1 - Food composition

Info

Publication number: WO2007039040A1
Application number: PCT/EP2006/008846
Authority: WO
Inventors: Yuguang Lin; Mario A Vermeer
Original assignee: Hindustan Lever Ltd; Hindustan Unilever Ltd; Unilever NV
Current assignee: Hindustan Unilever Ltd; Unilever NV
Priority date: 2005-09-30
Filing date: 2006-09-08
Publication date: 2007-04-12
Anticipated expiration: 2008-03-30
Also published as: WO2007039041A1

Abstract

A food product comprising (a) one or more sterols selected from the group of fatty acid ester of ß-sitosterol, ß-sitostanol, campesterol, campestanol, stigmasterol, stigmastanol and mixtures thereof; and (b) an extract from hawthorn (c) wherein the weight ratio of sterols to the total amount of oleanolic acid and ursolic acid is from 1 : 10 to 50 : 1 and wherein the amount of sterols is from 0.1 to 20 wt%.

Description

Food composition

Description

Field of the invention

The invention relates to a composition, specifically to a functional food composition having one or more health effects in humans. In particular, such health effect is related to a decreased risk of cardiovascular disorders.

Background to the invention

Plant sterols and their analogues and derivatives [hereafter referred to as sterols] enjoy a considerable scientific interest for their health promoting properties. For example sterols and fatty esters thereof are known for their LDL cholesterol lowering properties, LDL cholesterol being an established risk factor for atherosclerotic vascular diseases. However, the maximal cholesterol lowering effect of plant sterols/stanols is about 10-15%. For many subjects, more than 15% of cholesterol lowering is desired, there is a need to enhance the cholesterol lowering effect of sterols via combining them with other, preferably food or natural, ingredients .

One of the new trends is the combination of sterols with other health promoting ingredients, to target specific medical conditions or to achieve improved performance of the sterols. For example US 2002/0068095 discloses the combination of (a) one or more phytosterols or phytostanols with (b) (b) a composition capable of inhibiting cholesterol absorption and (c) (c) a composition capable of increasing cholesterol metabolism, wherein at least one of the compositions (b) and (c) is derived from plants.

This patent document mentions a great number of potentially suitable plant sources to obtain the desired compositions (b) and (c) . A great number of these plants however are medical plants which would not be suitable for the use in food products .

It is an object of the present invention to provide a compositions comprising a combination of sterols and a specific plant derived material, wherein the source, form and amount of said sterols and plant materials are such that improved effects can be obtained in lowering cholesterol levels. The composition of the invention can suitably be used in a variety of products, especially food products as described herein.

Fruit and vegetable intake has been associated with a lower plasma cholesterol concentration and a lower mortality of coronary heart disease. We have found a method to extract active ingredients from food plants, particularly hawthorn to lower plasma cholesterol via reducing cholesterol absorption. Hawthorn might be considered to be a food plant since it has a long consumption history in China and other Asian countries.

Surprisingly, we found that the combination of these active extracts with sterols in specific weight ratios leads to a synergistic cholesterol lowering effect, which is much higher than the maximal effect (15% as above described) of sterols alone. Accordingly, the present invention provides food compositions comprising combinations of active ingredients to reduce the risk of cardiovascular diseases beyond what is obtainable by using only one of the active ingredients. Surprisingly it has been found that if sterols are combined with active materials from hawthorn in compositions comprising these ingredients in specific weight ratios, this leads to unexpectedly good lowering of plasma lipid concentrations in mammals .

Accordingly in a first aspect of the invention there is provided a food product comprising (a) one or more sterols selected from the group of fatty acid ester of β-sitosterol, β-sitostanol, campesterol, campestanol, stigmasterol, stigmastanol and mixtures thereof ; and (b) an extract from hawthorn (c) wherein the weight ratio of sterols to the total amount of oleanolic acid and ursolic acid is from 1 : 10 to 50 : 1 and wherein the amount of sterols is from 0.1 to 20 wt%.

Detailed description of the invention: Discussion of prior art documents

KR 2004 027 829 (Aimsphil Co LTD) discloses nanoliposome compositions suitable for treating skin diseases. These compositions comprise a combination of pentacyclic triterpenic acid (selected from ursolic acid, ursolic acid derivates and ursolic isomers) and sterols (selected from cholesterol, cholesterol acetate, cholesterol isostearate and cholesteryl macadamiate) . Dl does not disclose food products or the use of specific fatty acid esterified steols as described above.

US 2002/0068095 discloses a composition comprising one or more phytosterols or phytostanols . D2 does not disclose the specific esterified sterols of the current invention. Also although D2 mentions several possible plants as a suitable ingredient for the composition, the specific combination of hawthorn extract and sterols in the ratios as currently claimed do not seem to be suggested or disclosed.

US 5,665,359 discloses the administration of the use of Shanzha fruit to obtain a hypocholesterolemic effect . D3 does not seem to disclose the combined use of hawthorn extract and specific esterified sterols in specific ratios in accordance to the present invention.

The hawthorn extract

The term hawthorn refers to the plan of Crateaegus species or its fruits. There are more than 250 species of hawthorn all over the world, consisting of small trees and scrubs.

For the purpose of the invention the term hawthorn refers to the Crataegus Oxyacantha especially to the Chinese variant of hawthorn, Crataegus pinnatifada. The term hawthorn extract refers to extracts of hawthorn or parts thereof, in particular extracts of the fruits of hawthorn. For the purpose of the invention the term hawthorn extract refers both to extracts of hawthorn still comprising one or more solvents or concentrated or dried or powdered versions thereof.

For the application in functional food products, hawthorn in itself or hawthorn juice are less suitable, since in hawthorn or its juice the concentration of active ingredients is relatively low. Preferred hawthorn extracts can be obtained by using organic solvents, for example dichloromethane (DCM) , ethylacetate, acetone, ethanol or heptane. Particularly preferred is the use of edible organic solvents. Particularly preferred is the use of ethanol . The extract can for example be obtained by contacting dried hawthorn berries with the solvent e.g for a period of 1 to 24 at ambient temperature (Soxhlett extraction) . After extraction, the solvent may be partially or fully removed e.g to obtain a concentrated extract or a dried, e.g powdered or syrup hawthorn extract .

It is believed that these organic solvents will extract a great variety of components from the hawthorn berry. Applicants by no means want to be bound by any theory as to which of these components are actually responsible for the beneficial effects of the hawthorn extract. It is known that hawthorn comprises ursolic acid (UA) and oleanolic acid (OA) . Equally it is known from US 20040132816 that hawthorn extracts comprises bioflavonoids. Without wanting to be bound by any theory, applicants believe that especially the hawthorn components which are soluble in organic solvents such as ethanol , may provide beneficial effects when used in combination with sterols. Examples of potentially beneficial components of hawthorn are oligomeric procyanidins and flavans such as catechins and epicatechin, triterpene derivatives such as OA and UA and phenolics such as chlorogenic acid and caffeic acid. UA and OA are sufficiently soluble in organic solvents, especially ethanol and for this reason applicants believe that the presence of UA and OA in the extract can be seen as a marker for the presence of possible yet unidentified beneficial ingredients in the extract .

Dried hawthorn fruit typically comprises about 3 g/kg of UA and OA. Also typically the weight ratio of OA to UA in dried hawthorn fruit is about 1 : 6. Compositions of the invention comprise sterols and a hawthorn extract. The amount and concentration of the extract should be chosen such that the weight ratio of sterols to the total of Oleanolic acid and Ursolic acid is from 1 : 10 to 50 : 1, more preferred from 5 : 1 to 30 : 1, most preferred from 10 : 1 to 20 : 1.

A preferred extract of Hawthorn comprises at least 10 wt% of materials extracted from hawthorn and up to 90 wt% of solvent. Preferably the amount of materials from hawthorn is at least 25 wt%, more preferred 50 wt%, while the solvent level in the extract preferably is from 0 to 50 wt%, for example from 10 to 40 wt%. Especially preferred are hawthorn extracts which are substantially free from solvent, most preferably are hawthorn extracts which after extraction have been dried to form a hawthorn extract in powder form.

Typically the amount of extract to be used in the composition of the invention will depend on a number of factors. Depending on for example the amount of sterol in the composition and the amount of UA and OA in the extract, the amount of hawthorn extract in food compositions or of the invention may vary in a broad range provided the specific weight ratio of sterols to the total of UA and OA is obtained which is within the range as specified above. Generally however the amount of hawthorn extract will be from 0.01 to 20 wt% of the food composition, more general from 0.1 to 10 wt% such as for example 1 to 5 wt%.

If the hawthorn extract comprises ethanol as the solvent, then preferably the amount of extract in the food composition is such that the level of ethanol in the food is less than 0.05 wt%, more preferred less than 0.03 wt% such as from 0.003 to 0.03 wt%. This lowering of the amount of ethanol without unduly affecting the beneficial properties of the extract may for example be achieved by using concentrated versions or powdered versions of the extract. The Sterols

For the purpose of the invention the term sterols refers to phytosterols, phytostanols, analogues, esters and derivatives thereof. The term sterol esters also refers to fatty acid esters of phytosterols or phytostanols

Typically, the phytosterols, phytostanols and their analogues and derivatives may be selected from one or more of phytosterols, phytostanols, synthetic analogues of phytosterols and phytostanols and esterified derivatives of any of the foregoing, and mixtures of any of these. The total amount of such substances in a food product of the inveniton is preferably from 0.01% to 20%, more preferably from 0.1% to 15%, still more preferably from 0.2% to 8%, and most preferably from 0.3% to 8% by weight of the food product composition.

Preferably, the daily intake of such sterol-type component of the combination is from 0. Ig to 3g, more preferably from 1.5g to 2.5g, especially from 2g to 2.25g per day.

Sterols can be classified in three groups, 4-desmethylsterols, 4-monomethylsterols and 4 , 4 ' -dimethylsterols . In oils they mainly exists as free sterols and sterol esters of fatty acids although sterol glucosides and acylated sterol glucosides are also present. There are three major phytosterols namely beta-sitosterol , stigmasterol and campesterol . The phytostanols are the respective 5α- saturated derivatives of phytosterols such as sitostanol, campestanol and their derivatives . Synthetic analogues of any of the sterols or stanols (which include chemically modified natural components) may also be used.

Preferably the sterol is selected from the group comprising fatty acid ester of β-sitosterol , β-sitostanol , campesterol, campestanol, stigmasterol , stigmastanol and mixtures thereof.

Fatty Acid Esters

The sterol materials recited above may optionally be provided in the form of one or more fatty acid esters thereof. Mixtures of esterified and non-esterified materials may also be used.

Thus, any of the sterols and their synthetic analogues used in the present invention are preferably esterified with a fatty acid. Preferably, they are esterified with one or more C2-22 fatty acids. For the purpose of the invention the term C2-22 fatty acid refers to any molecule comprising a C2-2₂ main chain and at least one acid group. Although not preferred within the present context the C₂-₂₂ main chain may contain 1-6 double bonds, be partially substituted or side chains may be present. Preferably, however the C₂-₂2 fatty acids are linear molecules comprising one or two acid group (s) as end group (s) . Most preferred are linear C₈-2₂ fatty acids as occur in natural liquid oils.

Suitable examples of any such fatty acids are for example lauric acid, palmitic acid, stearic acid, arachidic acid, behenic acid, oleic acid, cetoleic acid, erucic acid, elaidic acid, linoleic acid and linolenic acid and mixtures thereof. When desired a mixture of fatty acids may be used for esterification of the sterols. For example, it is possible to use a naturally occurring fat or oil as a source of the fatty acid and to carry out the esterification via an interesterification reaction. Use of a natural source nearly always results in a mixture of fatty acids.

In a particular embodiment, the fatty acid mixture contains a high amount (>50%, preferably >70%, further preferred >80%) of unsaturates, being either monounsaturated fatty acids (MUFA) and/or polyunsaturated fatty acids (PUFA) . This does not only provide the advantage of e.g. PUFA itself having good blood cholesterol lowering capacity, but also of the sterols esters prepared with such fatty acids.

Preferably fatty acid mixtures of sunflower, safflower, rapeseed, linseed, olive oil, linola and/or soybean are used. These are typical sources of high PUFA and/or low SAFA.

Suitable esterification conditions are for example described in WO 92/19640.

Food products

It is a further object of the invention to provide a food composition having one or more health effects in humans, in particular such health effect is related to a decreased risk of cardiovascular disorders. Another object is to provide such food composition having a acceptable taste, preferably a good taste. Still a further object is to provide a food composition, that upon consumption by a human subject is effective in causing in the human subject a reduction of level of total blood cholesterol and/or a reduction of level of LDL cholesterol .

Accordingly the invention also relates to a food composition comprising sterols and hawthorn extract wherein the weight ratio of sterols to the total of Oleanolic acid and Ursolic acid is from 1 : 10 to 50 : 1, more preferred from 5 : 1 to 30 : 1, most preferred from 10 : 1 to 20 : 1.

The food composition according to the invention may preferably be used as a physiologically functional food product. According to a preferred embodiment, the consumption of the food composition by a human subject is effective in causing in the human subject a reduction of level of total blood cholesterol and/or a reduction of level of LDL cholesterol .

The food products according to the invention may be of any food type. They may comprise common food ingredients in addition to the sterol and the hawthorn extract, such as flavour, sugar, fruits, minerals, vitamins, stabilisers, thickeners, etc. in appropriate amounts. Preferably the food products are fruit juice products, dairy type products or frozen confectionery products. These preferred types of food products are described in some detail below and in the examples. These preferred food products may advantageously be used as part of a fat containing meal, or be used in conjuction with such a meal.

Examples of food products according to the invention are:

• Fruit juice products

Examples of fruit juice products according to the invention are juices derived from citrus fruit like orange and grapefruit, tropical fruits, banana, peach, peer, strawberry or berry in which the sterols and the hawthorn extract extract are dissolved or suspended optionally in the presence of an appropriate emulsifier The average serving size for the fruit juice products is 250 ml. Typically the amount of sterol in 250 ml juice will be from 0.2 to 2 grams.

• Dairy type products Examples of dairy products according to the invention are milk, dairy spreads, cream cheese, milk type drinks and yoghurt, wherein the the sterols and hawthorn extract extract are dissolved or suspended optionally in the presence of an appropriate emulsifier. Optionally flavour or other additives may be added, also optionally the product could be homogenized by a high speed mixer. The average serving size for the dairy type product is 100 ml. Typically the amount of sterol in 100 ml dairy product will be from 0.2 to 2 grams.

An average serving size for a yoghurt type product could be from 50 to 250 g, generally from 80 to 200 g. Typically the amount of sterol in such a serving will be from 0.2 to 2 grams.

* Frozen Confectionery Products For the purpose of the invention the term frozen confectionery product includes milk containing frozen confections such as icecream, frozen yoghurt, sherbet, sorbet, ice milk and frozen custard, water-ices, granitas and frozen fruit purees.

Preferably the level of solids in the frozen confection e.g. sugar, fat, flavouring etc) is more than 3 wt.%, more preferred from 10 to 70 wt.%, for example 40 to 70 wt.%. Ice cream will typically comprise 0 to 20 wt . % of fat, 0.1 to 5 wt.%sterols, 0.1 to 5wt . % hawthorn extract, sweeteners, 0 to 10 wt. % of non-fat milk components and optional components such as emulsifiers, stabilisers, preservatives, flavouring ingredients, vitamins, minerals, etc, the balance being water. Typically ice cream will be aerated e.g. to an overrun of 20 to 400 %, more specific 40 to 200 % and frozen to a temperature of from -2 to - 200 °C, more specific -10 to -30 ⁰C. Ice cream normally comprises calcium at a level of about 0.1 wt%.

The average serving size of a frozen confectionery product is 85g.

• Nutrition bars

As example a composition of a granola bar is given. Granola bars consists of a binder syrup which binds the granola and fruit ingredients into a solid bar. E.g. nuts, pieces of chocolate, or a low moisture yoghurt filling can be added as well. The bar can be enrobed with a coating of e.g. chocolate or a low moisture yoghurt layer.

Granola bar example (weight per cent)

0-40 % granola base (oat flakes, wheat flakes, rice crispies) 0-60 % binder syrup (corn syrup, sugar, salt, lecithine, vegetable oil)

0-40 % fruit filling (low moisture jelly, dried fruits 0.1 to 5 wt% of sterol and hawthorn extract.

Moisture content of a nutrition bar is typically below 15%.

• Food emulsions, (fruit) spreads The food product may be an oil and water containing emulsion, for instance a spread or a margarine. Oil and water emulsion is herein defined as an emulsion comprising oil and water and includes oil in water (0/W) emulsions and water in oil emulsions (W/0) and more complex emulsions for instance water- in-oil-in-water (W/O/W/O/W) emulsions. Oil is herein defined as including fat .

Preferably the food product is a spread, frozen confection, a drink or a sauce. Preferably a spread according to the invention comprises 30-90 wt . % vegetable oil. Advantageously a spread has a pH of 4.2-6.0.

Other food product according to the invention can be prepared by the skilled person based on common general knowledge, using the hawthorn extract the sterols as an ingredient in suitable amounts. Examples of such food products are baked goods, dairy type foods, snacks, etc.

Description of figures:

Figure 1: Plasma cholesterol -lowering effect of hawthorn extract, sterols (PSE) as well as recipe of these two ingredients.

Figure 2: Liver free cholesterol (CH) and cholesterol ester concentrations of hamsters fed different diets as indicated. Data are presented as mean±SE. No statistical difference was observed among groups with regard to CH concentrations. * indicates P<0.01 compared to control. Figure 3. Cholesterol absorption-inhibiting effect of hawthorn extract, sterols (PSE) as well as combination of these two ingredients.

The invention will now be illustrated by means of the following examples

Example I

Example IA: preparation of hawthorn extract in ethanol Non-heated dried sliced hawthorn berries were obtained. The hawthorn berries were milled in a cooled milling machine. 425 grams of the powdered hawthorn berries were extracted by the Soxhlett method with 2X liters of ethanol. The extraction was executed for approximately 12 hours (40 minutes per Soxhlett cycle) .

The total ethanol extract was dried by using a RotaVap. The yield of the extraction was 167 grams (39%) of hawthorn extract .

Example IB: preparation of hawthorn extract in different solvents Extractions as in example IA with 5 different solvents at room temperature provided the following results: solvent OA UA OA UA

%-extract %-extract yield yield

DiChloroMethane 9.8% 32 .1% 0.06% 0.19%

(DCM-hawthorn)

EthylAcetate 8.8% 31 .2% 0.07% 0.23%

Aceton 6.1% 19 .8% 0.08% 0.26%

Ethanol 1.7% 6. 4% 0.08% 0.31%

Heptane 1.2% 1. 7% 0.00% 0.00% ursolic acid and oleanolic acid were analysed by Ci₈-HPLC or GC- TOF. Comparable results were obtained by both methods. Ethanol is the extraction solvent providing the highest yield but with a low purity for oleanolic and ursolic acid. DiChloroMethane and EthylAcetate provide higher purities but with a lower yield. The impurities in the ethanol extract will be mainly sugars originating from the hawthorn fruit.

Example II

Investigation of cholesterol-lowering effect of hawthorn extract in combination with sterol esters.

Study methods

Male LVG Golden Syrian hamsters (aged 8 weeks, approximately 70-80 g of body weight at arrival) were acclimated to individual stainless steel cages and fed a basal semi purified diet (4) before initiation of the experimental diet treatment. These animals were randomly assigned to five diet groups (denoted A to E) based on their body weight. Each group consisted of 20 hamsters. Animals were housed individually in cages in a temperature-controlled environment under a 12 h light-dark cycle (lights on 18:00 h) . The experimental diets were semi purified diets modified from AIN-93 rodent diet. The four diets were:

A) Control diet

B) Hawthorn extract diet

C) Plant sterol ester (PSE) diet D) Hawthorn extract + PSE diet

Table 1. Nutrient composition of the control and experimental diets

The details of the nutrient composition of each diet are shown in Table 1. The diets contained 30 energy % as fat (15.6 en% SAFA, 8,9 en% MUFA and 4,8 en% PUFA, resembling to a typical Western fat intake) . PSE was β-sitosterol which had been esterified with fatty acids from sunflower oil to an esterification degree of >92%. Animals were fed experimental diets for 4 weeks. Hamsters had free access to food and water.

Blood and autopsy-sampling procedures

At termination of the study, hamsters were deprived for food overnight (approximately 16 hours) . Blood samples were drawn into EDTA-wetted syringes by aorta puncture under halothane narcosis. The animals then were killed by decapitation, and liver samples were collected. Plasma was collected after centrifugation at 1500 x g at room temperature for 10 min. Liver sample collection: The bile in the gallbladder was collected through puncture, and then the gallbladder was excised. The complete liver was excised and weighed. About 1 g of liver was sampled and frozen at -20⁰C for hepatic lipid analysis .

Plasma lipid and lipoprotein analysis Plasma total cholesterol (TC) and triacylglycerol (TAG) concentrations were determined by enzymatic assays (Sigma Chemicals, Deisenhofen, Germany). Plasma lipoproteins were isolated by sequential ultracentrifugation. Three fractions were isolated based on the following densities: VLDL (d<1.006 kg/L) , LDL (1.006 <d <1.055 kg/L) , HDL (1.055 <d < 1.21 kg/L) . TC and TAG concentrations in the fractions were determined using enzymatic assays.

Hepatic cholesterolmeasurement . A 300-mg portion of liver was homogenised. Lipids in liver samples were extracted with dichloromethane (DCM) . TC and free cholesterol (FC) were determined as described above. The concentration of cholesterol esters (CE) was calculated as the difference between TC and FC.

Study results

Plasma cholesterol lowering effect (Figure 1) Hamsters administered hawthorn extract, PSE or hawthorn extract + PSE had significantly lower plasma non-HDL cholesterol (i.e. VLDL+LDL) concentration than the control group (P<0.05). Only hawthorn extract + PSE diet lowered plasma total cholesterol compared to control diet (P<0.05) . In addition, the combination of hawthorn extract and sterols has stronger plasma TAG lowering effect compared to control, hawthorn extract or sterol treatments individually. These data demonstrate that the combination of hawthorn extract and sterols has synergistic plasma cholesterol- and TAG-lowering effects.

Hepatic cholesterol lowering effect (Figure 2) : The hawthorn extract alone has no hepatic cholesterol lowering effect, while the combination of hawthorn extract and sterols has extra hepatic cholesterol (ester) lowering effect compared to sterol treatment alone. These data further indicate that the combination of these two ingredients has synergistic cholesterol lowering effect.

Cholesterol absorption -inhibiting effect (figure 3) . Hawthorn extract and sterols (PSE) individually reduced cholesterol absorption as indicated by an increased fecal neutral sterol (beta-cholestanol , cholesterol and coprostanol) excretion. The combination of hawthorn extract and sterols leads to an extra fecal neutral sterol excretion compared to hawthorn extract or sterol treatment alone. These data further support that the combination of these two ingredients has synergistic action on inhibiting cholesterol absorption.

Claims

1. A food product comprising

(a) one or more sterols selected from the group of fatty acid ester of β-sitosterol, β-sitostanol, campesterol, campestanol, stigmasterol, stigmastanol and mixtures thereof ; and (b) an extract from hawthorn

(c) wherein the weight ratio of sterols to the total amount of oleanolic acid and ursolic acid is from 1 : 10 to 50 : 1 and wherein the amount of sterol is from 0.1 to 20 wt%.

2. A composition according to claim 1 which is a food product .

3. A composition according to claim 1, wherein the hawthorn extract comprises ethanol as the solvent material.

4. A composition according to claim 1, wherein the hawthorn extract is a powdered extract .

5. A composition according to claim 1 wherein the amount of sterols is from 0.2 to 8 wt%.

6. A composition according to claim 1, wherein the amount of hawthorn extract is 0.01 to 20 wt% of the composition, preferably from 0.1 to 10 wt%, more preferably from 1 to

5 wt%.