WO2000056177A2 - Pomegranate extracts and methods of using thereof - Google Patents

Abstract

Pomegranate extracts and methods of use thereof are provided. Particularly, an antioxidative composition comprising an extract from pomegranate is provided. A method of reducing lipid peroxidation, aggregation or retention, HDL oxidation in a sample and a method of alleviating atherosclerosis in a patient are also provided.

Description

Pomegranate Extracts and Methods of Using Thereof

Background of the Invention Area of the Art

The invention relates generally to Pomegranate Extracts and Methods of Using

Thereof.

Description of the Prior Art

Throughout this application, various references are referred to within parentheses.

Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains. Full bibliographic citation for these references may be found at the end of this application, preceding the claims.

Major risk factors for atherosclerosis include increased plasma low density lipoprotein (LDL) levels, as well as LDL modifications such as its retention, oxidation

and aggregation (1-5). Blood platelets activation also contribute to accelerated atherosclerosis (6-8). Oxidative modification of LDL is thought to play a key role during

early atherogenesis. Oxidized LDL (Ox-LDL) is taken up by macrophages at enhanced

rates via their scavenger receptors (9), leading to the formation of lipid-laden foam cells,

the hallmark of early atherosclerosis (10). Cells of the arterial wall (including

endothelial cells, smooth muscle cells and macrophages) can oxidize LDL in vitro in the

presence of catalytic amounts of transition metal ions (11-13).

Although increased resistance of LDL to oxidation was observed after treatment

with various synthetic pharmaceutical agents (14-17), an effort is made to identify

natural food products which can offer antioxidant protection against LDL oxidation. The previous study has demonstrated the beneficial effects against LDL oxidation of dietary

supplementation of β-carotene (18, 19), lycopene (20), vitamin E (21) and flavonoids from red wine (22, 23), licorice (24) or olive oil (25).

The pomegranate tree, which is said to have flourished in the Garden of Eden, has

been extensively used as a folk medicine in many cultures. In ancient Greek mythology,

pomegranates are known as the "fruit of the dead," and in the ancient Hebrew tradition,

pomegranates adorned the vestments of the high priest. The Babylonians regarded its

seeds as an agent of resurrection, the Persians as conferring invincibility on the battlefield, and for ancient Chinese it symbolized longevity and immortality.

Edible parts of pomegranate fruits (about 50% of total fruit weight) comprise

80% juice and 20% seeds. Fresh juice contains 85% moisture, 10% total sugars, 1.5% pectin, ascorbic acid and polyphenolic flavonoids. Pomegranate seeds are a rich source

of lipids, proteins, crude fibers, pectin and sugars.

The dried pomegranate seeds contain the steroidal estrogen estrone (26, 27), the

isoflavonic phytoestrogens genistein and daidzein and the phytoestrogenic coumestrol (28). In pomegranate juice, fructose and glucose are present in similar quantities,

calcium is 50% of its ash content and the principal amino acids are glutamic and aspartic

acid (29, 30). Content of soluble polyphenols in pomegranate juice varied within the

limits of 0.2% to 1.0%, depending on variety, and include mainly anthocyanins (such as

cyanidin-3 -glycoside, cyanidin-3, 3-diglycoside and delphindin-3-glucosid), catechins,

ellagic tannins, and gallic and ellagic acids (31).

Constitutes of pomegranate have been studied for their antiviral and antifungal effects. For example, U.S Patent No. 5,840,308 (61) describes an antiviral and antifungal composition comprising a mixture of a ferrous salt and an extract of a plant including,

inter alia, pomegranate rind. U.S. Patent No. 5,411,733 (32) describes an antiviral agent containing a crude drug from, mter alia, the root bark and fruit peel of pomegranate.

Prior to the present invention, however, no one has studied the effects of

pomegranate extracts on LDL atherogenic modifications, including its retention,

oxidation and aggregation. No one has used pomegranate extracts for the purpose of treating or ameliorating atherosclerosis.

Summary of the Invention

It is an object of the present invention to study any effects of pomegranate

extract, particularly the effect as an antioxidant. It is also an object of the present

invention to provide methods of using pomegranate extract, for example, as an

antioxidant. It is further an object of the present invention to provide a method of

preventing or ameliorating atherosclerosis.

Accordingly, one aspect of the present invention provides a composition, the

biologically active component of the composition consisting essentially of an extract

from pomegranate, and the composition comprising a carrier. The composition may be used as nutritional supplements, pharmaceutical preparations, vitamin supplements, food additives or foods supplements. The composition may be used in a dosage unit as

tablets, suspensions, implants, solutions, emulsions, capsules, powders, syrups, liquid compositions, ointments, lotions, creams, pastes, gels, and the like. According to embodiments of the invention, the extract may be an extract of juice or the inner or outer

peel of pomegranate, or a mixture thereof.

-Another aspect of the present invention provides an antioxidative composition for

treating disorders associated with conditions including lipoprotein oxidation,

aggregation, retention; macrophage atherogenicity, platelet activation and atheroscleorosis. The biologically active component of the composition consists

essentially of an effective amount of an extract from pomegranate. The examples of

disorders include arteriosclerotic heart disease and its associated complications,

including myocardial infarction; cerebral vascular disease (including cerebral

insufficiency or stroke); peripheral vascular disease (including peripheral vascular disease in the aorta and femoral and corotid arteries); abdominal aortic aneurysms; renal

artery stenosis; arteriosclerotic disease, disorders associated with transplant complications; disorders associated with post-operative heart valve replacement;

disorders associated with the complications of diabetes mellitus; thrombophlebitis; and

other disorders associated with increased platelets and increased platelet activation.

A further aspect of the present invention provides a method of ameliorating

disorders associated with conditions including lipoprotein oxidation, aggregation, retention; macrophage atherogenicity, platelet activation and atheroscleorosis. The

method comprises administering to a subject in need thereof a therapeutically effective amount of a composition comprising an extract from pomegranate.

Yet another aspect of the present invention provides a method of ameliorating in

a sample conditions including lipoprotein oxidation, aggregation, retention; macrophage atherogenicity and platelet activation. The method comprises a step of contacting the sample with a sufficient amount of an extract from pomegranate.

The invention is defined in its fullest scope in the appended claims and is

described below in its preferred embodiments.

Description of the Figures

The above-mentioned and other features of this invention and the manner of

obtaining them will become more apparent, and will be best understood, by reference to

the following description, taken in conjunction with the accompanying drawings. These

drawings depict only a typical embodiment of the invention and do not therefore limit its scope. They serve to add specificity and detail, in which:

Figure 1 shows the effect of pomegranate juice on AAPH- induced plasma lipid

peroxidation in a juice concentration study. Figure 1(A) shows the extent of plasma lipid peroxidation measured by the TBARS assay. Figure 1(B) shows the extent of plasma lipid peroxidation measured by the lipid peroxides assay.

Figure 2 shows the effect of pomegranate juice on LDL susceptibility to oxidation: concentration study.

Figure 3 shows the mechanisms for pomegranate juice protection against LDL oxidation.

Figure 4 shows the capacity of pomegranate constituents (juice, peels and seeds) to inhibit copper ion-induced LDL oxidation.

Figure 5 shows the effect of pomegranate juice supplementation on human plasma oxidative state in ex vivo studies.

Figure 6 shows the effect of pomegranate juice supplementation to humans on

the susceptibility of their HDL to oxidation in ex-vivo studies.

Figure 7 shows the effect of pomegranate juice supplementation to E° mice on their plasma oxidative stress during aging. Figure 8 shows the effect of pomegranate juice supplementation to humans on

the susceptibility of their LDL to oxidation in ex-vivo studies.

Figure 9 shows the effect of pomegranate juice supplementation to E° mice on

their LDL susceptibility to copper ions-induced oxidation in ex-vivo studies.

Figure 10 shows the effect of pomegranate juice on the susceptibility of LDL to

aggregation in in vitro and ex-vivo studies.

Figure 11 shows the effect of pomegranate juice on "LDL retention," analyzed

by LDL capacity to bind chondroitin sulfate.

Figure 12 shows the effect of pomegranate juice consumption by E° mice on their peritoneal macrophage lipids peroxidation and their ability to oxidize LDL.

Figure 13 shows the effect of pomegranate juice consumption by E° mice on their macrophage uptake of native or oxidized LDL.

Figure 14 shows the effect of pomegranate juice on human platelet aggregation in in vitro and ex-vivo studies.

Detailed Description of the Invention

The present invention demonstrated, for the first time, antiatherogenic properties of pomegranate juice (PJ) or pomegranate extract as related to its inhibitory effect on

lipid peroxidation in plasma, in lipoproteins and in macrophages. In addition,

pomegranate extract also possesses inhibitory effects on atherogenic modifications of

lipoprotein, including its retention, oxidation and aggregation. Furthermore,

antiatherogenicity of pomegranate extract could be also related to its ability to attenuate

platelet activation, an additional important risk factor for atherosclerosis.

Accordingly, one aspect of the present invention provides a composition, the biologically active component of the composition consisting essentially of an extract from pomegranate. The composition also comprises a carrier.

For the purpose of the present invention, an extract from pomegranate may be an

extract from the whole pomegranate or from any constituents of pomegranate. Examples of constituents of pomegranate that may be used to make the extract of the present

invention include, but are not limited to, juice, seed, and the inner and outer peel of pomegranate. In one embodiment of the present invention, the extract is juice extract of

whole pomegranate. In another embodiment of the present invention, the extract is from

the inner or outer peel of pomegranate. In a further embodiment of the present invention,

the extract may be a mixture of two or more extracts of the whole pomegranate or any constituents of pomegranate.

Methods of making juice extract of whole pomegranates are commonly known in

the art, and need not be repeated here. In general, any methods that may produce

pomegranate juice that naturally occurs in pomegranate may be used. For the purpose of the present invention, the juice may be concentrated or diluted from its natural concentration. The juice may also be mixed with extracts of other constituents of

pomegranate.

Extracts from constituents of pomegranate, i.e., seed, inner or outer peel, may be

made by methods commonly known in the art. For example, the seed, inner or outer peel

of pomegranate may be diluted in water and the extract may be made by crushing,

squeezing, or extensive vortexing. The insoluble materials of the extract may be

separated from the soluble supernatant of the extract. Perferably, the supernatant of the extract is used for the purpose of the present invention, although any oily, lipidic fraction of the extract may also be used. The extract from constituents of pomegranate may be concentrated or diluted, or mixed with each other or with pomegranate juice extract.

The extract of pomegranate of the present invention may be in a liquid or solid form. In accordance with one embodiment of the present invention, a solid form of extract may be made by lyophilizing the liquid extract of the present invention. Other

methods that are commonly known in the art may also be used.

Compositions of the present invention may be a variety of kinds, including, but not limited to, nutritional supplements, pharmaceutical preparations, vitamin

supplements, food additives or foods supplements. Compositions of the present

invention may be in convenient dosage forms, including, but not limited to, tablets,

suspensions, implants, solutions, emulsions, capsules, powders, syrups, liquid

compositions, ointments, lotions, creams, pastes, gels, or the like.

Compositions of the present invention include a carrier. Depending on the kind

of compositions of the present invention, a carrier may be a dietary suitable carrier or a pharmaceutically acceptable carrier, as long as it is compatible with the particular kind of

compositions of the present invention. Examples of a dietary suitable carrier include, but are not limited to, dietary suitable excipients, diluents and carriers. Examples of a

pharmaceutically acceptable carrier include, but are not limited to, biocompatible

vehicles, adjuvants, additives and diluents to achieve a composition usable as a dosage

form. As used herein, the terms "pharmaceutically acceptable," "physiologically

tolerable" and grammatical variations thereof, as they refer to compositions, carriers,

diluents and reagents, are used interchangeably and represent that the materials are capable of administration to or upon a mammal without the production of undesirable physiological effects.

The compositions of the present invention may be used alone or in combination

with other biologically active ingredients. A composition of the present invention, alone or in combination with other active ingredients, may be administered to a subject in a

single dose or multiple doses over a period of time, generally by oral administration. Various administration patterns will be apparent to those skilled in the art. The dosage ranges for the administration of the compositions of the present invention are those large

enough to produce the desired effect. The dosage should not be so large as to cause any

adverse side effects, such as unwanted cross-reactions and the like. Generally, the

dosage will vary with the age, weight, sex, condition and extent of a condition in a

subject, and the intended purpose. The dosage can be determined by one of skill in the

art without undue experimentation. The dosage can be adjusted in the event of any

counter indications, tolerance, or similar conditions. Those of skill in the art can readily

evaluate such factors and, based on this information, determine the particular effective concentration of a composition of the present invention to be used for an intended

purpose.

In one embodiment of the present invention, a composition contains the extract of

pomegranate in a dosage unit in an amount that contains at least 30 to 3000 μmols per

dosage unit of polyphenols. For the purpose of the present invention, polyphenols are

those naturally present in the extract of pomegranate. It should be appreciated that

polyphenols are used herein as a measurement for the amount of extract that need to be

used in each dosage unit. They are not used herein as an indication that they are the active, or the only active, ingredients of the extract. In fact, it is possible that something else, or the synergy of polyphenols and other components of an extract of the present invention, may be responsible for the activities of the extract.

The term "dosage unit" as used herein refers to physically discrete units suitable as unitary dosages for animals, each unit containing a predetermined quantity of active

material calculated to produce the desired therapeutic effect in association with the required diluent, e.g., a carrier or vehicle. The specifications for the unit dose of this

invention are dictated by and are directly dependent on (a) the unique characteristics of

the active material and (b) the limitations inherent in the art of compounding such active

material for therapeutical use in animals.

In accordance with one aspect of the present invention, compositions of the

present invention may be used as an antioxidant for treating a disorder associated with a

condition including, but not limited to, lipoprotein oxidation, aggregation, retention;

macrophage atherogenicity, platelet activation and atheroscleorosis. Therefore, one aspect of the present invention provides an antioxidative composition for preventing or ameliorating disorders associated with a condition selected from a group consisting of

lipoprotein oxidation, aggregation, retention; macrophage atherogenicity, platelet activation and atheroscleorosis. The biologically active component of the composition

consists essentially of an effective amount of an extract from pomegranate.

The term "an effective amount" as used herein means that the amount of the

extract of the present invention contained in an antioxidative composition of the present invention is of sufficient quantity which, upon administration to a subject, may produce

some beneficial effect for a subject against a disorder associated with a condition

including, but not limited to, lipoprotein oxidation, aggregation, retention; macrophage atherogenicity, platelet activation and atheroscleorosis. Examples of such a disorder include, but are not limited to, arteriosclerotic heart disease and its associated

complications including myocardial infarction; cerebral vascular disease (including cerebral insufficiency or stroke); peripheral vascular disease (including peripheral

vascular disease in the aorta and femoral and corotid arteries); abdominal aortic aneurysms; renal artery stenosis; arteriosclerotic disease, disorders associated with

transplant complications; disorders associated with post-operative heart valve replacement; disorders associated with the complications of diabetes mellitus;

thrombophlebitis; and other disorders associated with increased platelets and increased

platelet activation.

The antioxidative composition of the present invention may be used alone or in

combination with other desirable biological active ingredients. The antioxidative

composition of the present invention may also be used in combination with a

pharmaceutically acceptable carrier of the present invention. In accordance with embodiments of the present invention, an antioxidative

composition of the present invention may contain the extract of pomegranate in a dosage

unit in an amount that contains at least 30 to 3000 μmols per dosage unit of polyphenols.

Preferably, the extract is an extract of juice, seed, inner peel or outer peel of

pomegranate. Most preferably, the extract is an extract of inner or outer peel of

pomegranate.

The present invention also provides a method of ameliorating disorders

associated with a condition including lipoprotein oxidation, aggregation, retention; macrophage atherogenicity, platelet activation and atheroscleorosis. The method comprises administering to a subject in need thereof a therapeutically effective amount of a composition comprising an extract from pomegranate.

The term "therapeutically effective amount" as used herein means that the amount of a composition of the present invention administered is of sufficient quantity to

prevent or ameliorate to some beneficial degree a disorder associated with a condition including, but not limited to, lipoprotein oxidation, aggregation, retention; macrophage

atherogenicity, platelet activation and atheroscleorosis. The amount must be large

enough to produce the desired effect, but not so large as to cause any adverse side effects. Generally, the therapeutically effective amount will vary with the subject's, age, weight,

sex, condition, the extent of a condition in the subject, and the potency of the

composition, and can be determined by one of skill in the art without undue

experimentation. In one embodiment of the present invention, in one dosage unit, a

composition may contain an extract of pomegranate in an amount that contains at least

30 to 3000 μmols of polyphenols per dosage unit. One or more doses may be administered daily, for one or several days or indefinitely. In one embodiment,

compositions that contain an extract of pomegranate in an amount that contains at least

300 to 3000 μmols of polyphenols per dosage unit may be orally administered once daily

for a period of at least two weeks. For example, two glasses of pomegranate juice/per

day may be consumed by a human for a period of at least two weeks. If the composition

is administered by injection, the composition may contain an extract in an amount that

contains at least 30 to 600 μmols of polyphenols per dosage unit. The amount in each

dosage should be sufficient to result in a serum level of at least 1.5 μmols to 10 μmols of

polyphenols. Compositions that contain an extract of pomegranate may be administered to a subject orally or parentally by injection. The compositions are administered in a

manner compatible with the dosage formulation and in a therapeutically effective amount. The quantity to be administered and timing of administration depends on the subject to be treated, capacity of the subject's system to utilize the active ingredient, and

degree of therapeutic effect desired. Precise amounts of the active ingredient required to be administered depend on the judgement of the practitioner and are peculiar to each

individual. However, suitable dosage ranges for systematic application are disclosed herein and depend on the route of administration. Suitable regimens for administration

are also variable but are typified by an initial administration followed by repeated doses

at intervals by subsequent oral administration, injection or other administration.

The composition of the present invention may also be administered with other

active ingredients or a pharmaceutically acceptable carrier. Preparations for oral or

parental administration of a composition of the invention include sterile aqueous or

non-aqueous solutions, suspensions and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable

organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parental

vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium

chloride, dextrose and water, lactated Ringer's, or fixed oils. Intravenous vehicles

include fluid and nutrient replenishers, electrolyte replenishers (such as those based on

Ringer's dextrose), and the like. Preservatives and other additives may also be present

such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like.

For the purpose of the present invention, a subject may be any subject that is in need of the treatment. Preferably the subject is a mammal. Examples of mammals

include, but are not limited to, mice, dogs, cats, hamsters, sheep, goats, cows, pigs, rabbits, humans, and the like. More preferably, the subject is human.

The present invention also provides a method of ameliorating in a sample a condition including lipoprotein oxidation, aggregation, retention; macrophage atherogenicity, and platelet activation. The method comprises a step of contacting the

sample with a sufficient amount of an extract from pomegranate.

For the purpose of the present invention, a sample may be a sample from a

mammal, particularly from humans. Examples of a sample include, but are not limited

to, serum, plasma, urine, lipoproteins such as LDL and HDL, blood, peritoneal

macrophages and aortas, and the like. The sample may be contacted with an extract from

pomegranate by directly adding the extract to the sample, or by administering the extract

to a mammal wherefrom the sample is derived, as described above. The amount of the extract is sufficiently effective if the condition (such as

lipoprotein oxidation, aggregation, retention; macrophage atherogenicity, and platelet

activation) may be ameliorated. This amount may vary, depending on the particularity of a sample, the contacting condition, and the invented purpose. One skilled in the art can

readily determine the sufficient amount of abstract to be used in view of the disclosure of

this invention.

The following examples are intended to illustrate, but not to limit, the scope of

the invention. Indeed, those of ordinary skill in the art can readily envision and produce

further embodiments, based on the teachings herein, without undue experimentation.

EXAMPLES

METHODS Human studies

Thirteen healthy men, aged 20-35, nonsmokers and under no medication, were

included in the study. Subjects were students or laboratory staff from the Technion

Faculty of Medicine. They received 50ml/day of pomegranate juice (1.5 mmoles of total polyphenols/day) for a period of two weeks.

The compliance with the pomegranate juice supplementation in all subjects was satisfactory, as assessed by a daily contact with the subjects. The subject's body mass

index (BMI) was 23.0±1.5 and it did not change during the study. All subjects continued with their habitual diet during the study. The study was approved by the Helsinki

Committee of the Rambam Medical Center, Israel Ministry of Health (No-912). Blood samples were drawn after 12 hours of fast, before study entry, and after one and two

weeks of pomegranate juice consumption. Mice study

Apolipoprotein E-deficient (E°) mice were generously provided by Dr. Jan Breslow, the Rockefeller University, New York. Gene targeting in mouse embryonic

stem cells was used to create mice that lack apolipoprotein E (33). Thirty E° mice, aged

six weeks, were divided into three groups, 10 mice in each group. The three groups

received in their drinking water 0, 6.25 or 12.5 μL of pomegranate juice (equivalent to 0,

0.175 and 0.350 μmoles of total polyphenols) per mouse per day.

Blood was taken at 6, 9 and 14 weeks of age for plasma and LDL analyses. Peritoneal macrophages and aortas were obtained at the end of the study.

Pomegranate processing

Pomegranates were picked by hand, washed, chilled to 32°C and stored in tanks.

Then the fniit was crushed, squeezed and enzymatically treated with pectinase to yield

the pomegranate juice and the by-products, which include the inner and outer peels and

the seeds. Pectinase hydrolyzes alpha- 1.4 galacturonide bonds in pectin and thus it

improves extraction and filtration, and prevents formation of pectin gels. The juice was filtered, pasteurized, concentrated and stored at -18°C.

Peels and seeds extracts

One gram of inner or outer peels/seeds was diluted in 5ml of water followed by crushing, squeezing and extensive vortexing. Then the extract was centrifuged to remove any water insoluble materials and the supernatant was used for LDL oxidation analyses (Fig. 4A).

In the seeds extraction, an upper oily, lipidic fraction appeared which was not used in the study of aqueous extracts, but may contain also some active compounds.

Therefore, the oily, lipidic fraction may also be used as an extract of the present invention.

As ingredients other than polyphenols may also act as potent antioxidants, the effect of the extract was also studied on the basis of weight (Fig. 4B).

For this purpose, the extracts were lyophilized to remove the aqueous part. Now

the pomegranate juice contained about 30-fold more weight material than the peels and

thus, when we compared them on the basis of weight, the polyphenols concentration in

the pomegranate juice was extremely low and did not act at this low polyphenols concentration as an antioxidant.

In contrast, the peels were active antioxidants, as they contain both polyphenols at high enough concentration to act as antioxidants and additional non-polyphenols antioxidants. Polyphenols determination

Total polyphenols concentration in pomegranate juice was determined

spectrophotometrically with the phosphomolybdic phosphotungstic acid reagents (31). Plasma livid peroxidation

Human plasma obtained from a healthy volunteer was diluted (x2) with phosphate buffered saline (PBS). In the in vitro studies, increased concentrations of

pomegranate juice polyphenols (0-1.5 μmol/L) were added to the plasma, whereas in the in vivo studies plasma was obtained from the subjects that participated in the study

before and after two weeks of PJ consumption, and from E° mice at 0, 9 and 14 weeks of PJ consumption.

The plasma was incubated in the absence or presence of lOOmmol/L of the free radical generator 2,2'-Azobis 2-amidinopropane hydrochloride (AAPH, Wako Chemical

Industries Ltd., Japan) for two hours at 37°C. AAPH is a water-soluble azo compound

that thermally decomposes to produce peroxyl radicals at a constant rate. Plasma lipid

peroxidation was determined by measuring the amount of generated thiobarbituric acid reactive substances (TBARS) (34) and lipid peroxides (35). Serum paraoxonase (arylesterase activity)

Arylesterase activity was measured using phenylacetate as the substrate. Initial

rates of hydrolysis were determined spectrophotometrically at 270nm. The assay

mixture included 5 μL of serum, 1.0 mmol/L phenylacetate, and 0.9 mmol/L CaCl₂ in 20

mmol/L Tris HCl, pH 8.0. Nonenzymatic hydrolysis of phenylacetate was substracted

from the total rate of hydrolysis. The E₂₇₀ for the reaction was 1,310 M^"1 cm^"1. One unit

of arylesterase activity is equal tol μmol of phenylacetate hydrolyzeαV min /mL (36). In

the in vitro study, increasing concentrations of pomegranate juice were incubated with normal serum for 10 minutes prior to the analysis of arylesterase activity. Total plasma antioxidant status

Total antioxidant status was measured in plasma by a commercially available kit (Randox Laboratories Limited, UK, Cat No. NX 2332) applicable for COBAS MIRA. Plasma was incubated with ABTS (2,2'-Azino-di-[3-ethylbenzthiazoline sulphonate])

and a peroxidase (metmyoglobin) and H₂O₂, to produce a radical cation. The resulting product has a relatively stable blue-green color, which is measured at 600nm.

-Antioxidants in the added sample cause suppression of this color production to a degree

which is proportional to their concentration (37).

Lipoproteins isolation

For the in vitro studies, LDL was isolated from plasma derived from healthy

normohpidemic volunteers. For the ex vivo studies, human plasma was collected before

study entry (baseline), and after one and two weeks of pomegranate juice administration.

In the mice study, LDL was isolated from blood samples drawn from E° mice before and

after 9 weeks and 14 weeks of pomegranate juice administration. Plasma samples were stored at 4°C for two weeks until all three samples were

collected. Then, LDL and HDL were isolated from the plasma samples. No significant differences could be measured in the basal oxidative state (no oxidant added) of the

lipoproteins. The lipoproteins (LDL and HDL) were prepared by discontinuous density

gradient ultracentrifugation as previously described (38). The lipoproteins were washed

at d= 1.063 g/mL, dialyzed against 150 mmol/L NaCl, 1 mmol/L Na₂ EDTA (pH 7.4) at

4°C. The LDL and HDL fractions were then sterilized by filtration (0.45 μm), kept

under nitrogen in the dark at 4°C and used within two weeks. The lipoproteins protein concentration was determined with the Folin phenol reagent (39). Prior to oxidation,

LDL and HDL were dialyzed against EDTA-free, phosphate buffered saline (PBS) solution at pH7.4, and at 4°C. Lipoprotein oxidation

LDL or HDL (100 μg of protein/mL) were incubated with 5 μmol/L of CuSO₄for three hours at room temperature. Formation of conjugated dienes was continuously monitored by measuring the increase in absorbance at 234 nm (40). Incubations were

carried out in the spectrophotometer cuvette (Ultraspec 3000, Pharmacia, LKB,

Biochrom Ltd., Cambridge, UK). The initial backround of the samples ranged between

0.1-0.2 O.D. as recorded at 234 nm. After initial absorbance was recorded, the

spectrophotometer was set to zero against blank, and the increase in absorbance during

LDL or HDL oxidation was recorded every 10 minutes. The lag time required for the

initiation of lipoprotein oxidation was calculated from the oxidation curve. LDL aggregation

LDL (100 μg of protein/mL) was mixed by vortex at a fixed strength, and the absorbance at 680 nm was monitored every 10 seconds against a blank solution (41).

LDL retention

Human LDL isolated before and after one or two weeks of pomegranate juice

supplementation was used in the ex vivo studies, whereas in the in vitro studies LDL was

preincubated with increasing concentrations of pomegranate juice (up to 3.5 μmol/L of

polyphenols) for one hour at 37°C. LDL. 200 μg of lipoprotein protein mL were then incubated with chondroitin sulfate (CS, 100 μg/mL) for 30 minutes at room temperature.

The lipoprotein was precipitated with a commercial kit for HDL cholesterol reagent

(phosphotungstic acid / MgCl₂, Sigma Co, St. Louis, MO) that precipitated all the LDL present in the samples followed by a 10-minute centriftigation at 2000xg (42). After

discarding the supernatant, the LDL in the precipitate was dissolved in 0.1 N NaOH and analyzed for its glycosaminoglycans (GAGs) content, using the 1 ,9-dimethylmethylene blue (DMMB) spectrophotometric assay for sulfated glycosaminoglycans (43). Briefly,

2.5mL of ice-cold DMMB working solution (46 μmol/L DMMB, 40mmol/L glycine,

40mmol/L NaCl in 5% ethanol, adjusted to pH 3.0) was added to 500 μL of the dissolved

precipitate.

The absorbance at 525nm was then immediately measured. Chondroitin sulfate

was used as a standard and was included within each series of assays. A similar

preparation of LDL, with no chondroitin sulfate added, was used in parallel as a control.

GAGs content obtained in the control was subtracted from the GAGs content in LDL preparations that were incubated with chondroitin sulfate (CS). Mouse peritoneal macrophages

Mouse peritoneal macrophages (MPM) were harvested from the peritoneal fluid, four days after intraperitoneal injection of 3 mL of thioglycolate (24 g/L, in saline) into

each mouse (44). The harvested cells (10-20χl0⁶ /mouse) were washed and centrifuged

three times with PBS at lOOOxg for 10 minutes. Then the cells were resuspended to

10⁹/L in DMEM containing 10% horse serum (heat-inactivated at 56°C for 30 minutes),

lOOU/mL penicillin, 100 μg/mL streptomycin, and 2 mmol/L of glutamine.

The cell suspension was dispensed into 35 mm plastic Petri dishes and incubated in a humidified incubator (5% CO₂, 95% air) for two hours. The dishes were washed

once with 5 mL of DMEM to remove nonadherent cells, and the monolayer was further incubated under similar conditions for 18 hours, prior to analyses of various macrophage functions.

Macrophage glutathione content

Cells (2xl0⁶/lmL of PBS) were sonicated twice, for 20 seconds each time, at 80

watts. Cellular protein content was determined using the Folin phenol reagent method (39). For total glutathione analysis, 5% sulfosalicylic acid was added to the supernatant

of the sonicated cells (1 :2, v:v), followed by cell centrifugation at 20,000xg. Glutathione

content in all samples was measured in the supernatant by the 5,5-dithiobis-2-

nitrobenzoic acid (DTNB)-GSSG reductase recycling assay (45). Superoxide anion release

The production of superoxide anion (O₂ ^") by mouse peritoneal macrophages was

measured as the superoxide dismutase inhibitable reduction of acetyl ferricytochrome C (46). Cells (2xl0⁶/well) were suspended in lmL of Hanks' Balanced Salts Solution (HBSS) containing acetyl ferricytochrome C (150 μmol/L). Superoxide production by

the cells was stimulated by the addition of LDL (100 μg protein/mL) and 5 μmol/L CuSO₄, for one hour.

To some control samples, superoxide dismutase (SOD, 30 μg/mL) was added.

The amount of superoxide release was determined in the medium and was expressed as

nmoles of superoxides /mg cell protein, using an extinction coefficient of E₅₅₀=21 mM^"1

cm^"1.

LDL oxidation by macrophages

Mouse peritoneal macrophages (MPM, 2xl0⁶/35mm dish) were incubated with

LDL (100 μg of protein/mL) in RPMI medium (phenol-free) in the presence of 2 μmol/L of CuSO₄ for six hours. LDL was also incubated under similar conditions in the absence of cells. The extent of LDL oxidation was measured directly in the medium (after

centrifugation at lOOOxg for 10 minutes in order to spin down detached cells) by the TBARS assay (34). Macrophage-mediated oxidation of LDL was calculated by

subtraction of the oxidation rate in the absence of cells from that obtained in the presence of macrophages (47).

Cellular uptake of lipoproteins by macrophages

LDL was radioiodinated by the iodine monochloride method, as modified for

lipoproteins (48). Radioiodinated oxidized LDL (^l25I-Ox-LDL) was prepared from ¹²⁵ 1-

LDL that was dialysed against PBS, followed by incubation with 5 μmol/L of CuSO₄, at

37°C for 24 hours. ¹²⁵I-LDL or ¹²⁵I-Ox-LDL (10 μg of protein mL) was incubated with

the cells at 37°C for five hours. Lipoproteins cellular degradation was measured in the

collected medium as the trichloroacetic acid (TCA)-soluble, non-lipid radioactivity, which was not due to free iodide (49). Lipoprotein degradation in a cell-free system, measured under identical conditions, was minimal (less than 10%) and was subtracted

from the total degradation. The remaining cells were washed three times with cold PBS

and dissolved in 0.1 N NaOH for protein and cell-associated lipoproteins determination.

Cellular binding of ¹²⁵I-Ox-LDL was determined after incubation of the cells with

increasing concentrations of ¹²⁵I-Ox-LDL or ¹²⁵I-LDL at 4°C for four hours. Then the

cells were washed with cold PBS on ice, dissolved in 0.1N NaOH, and samples were taken to measure radioactivity. Platelet aggregation

For platelet studies, venous blood (10 mL) was collected through siliconized syringes into 3.8% sodium citrate, at a ratio of 9:1 (v:v). Platelet-rich plasma (PRP) was

prepared by low-speed centriftigation (lOOxg for 10 minutes) at 25°C, and the remaining

sample was recentrifuged at lOOOxg for 10 minutes to obtain platelet-poor plasma (PPP) (50). Collagen (Nycomed Arzneimittel, Munchen, Germany) was used as the

aggregating agent at a concentration of 2 μg/mL, as this concentration caused up to 75%

aggregation amplitude. Platelet aggregation was performed at 37°C in a PAP-4

computerized aggregometer, using platelet poor plasma (PPP) as a reference system for

PRP. In the in vitro studies, increasing concentrations of pomegranate juice were

incubated for 10 minutes with lmL of PRP prior to analyses of platelet aggregation.

Results were expressed as the slope of the aggregation curve and are given as cm min.

Eree radical scavenging capacity

DPPH (l,l-diphenyl-2-picryl-hydrazyl) is a radical-generating substance which is widely used to monitor free radical scavenging abilities of various antioxidants (51). To

analyze free radical scavenging capacity, increasing concentrations of pomegranate juice

(0-14 μmol/L of polyphenols) were mixed with 3 mL of 0.1 mmol DPPH/L in ethanol.

The time course for the change in optical density at 517 nm was kinetically monitored

(51).

Stfltt-yt/αs

Student's paired test was performed for all statistical analyses. Results are given

as mean ± S.D. for the in vitro studies and as mean ± S.E.M. for the in vivo studies. For

the in vitro experiments described in Figs IA, 2 A and 3, only one experiment, representative of three different experiments performed, is shown. The degree of variation between the three experiments ranged between 7%-9%. The computer software

program STATEASE (version 1.00; Data Plus Systems Inc., New York) was used for computation.

RESULTS The present invention analyzed the antioxidative capacity against lipid peroxidation and the antiatherogenicity of pomegranate juice, which is rich in some

specific polyphenols, in vitro and ex vivo, in healthy human volunteers and in the

atherosclerotic apolipoprotein E deficient mice, which are under oxidative stress. The results are set forth below.

I. In vitro studies

To study the effect of pomegranate juice in vitro on the susceptibility of plasma

to lipid peroxidation, plasma from healthy volunteers was incubated with 100 mmol/L of

the free radical generator AAPH for two hours at 37°C in the presence of increasing concentrations of pomegranate juice.

Figure 1 shows the effect of pomegranate juice on AAPH-induced plasma lipid

peroxidation. Normal human plasma (diluted x2 in PBS) was incubated with increasing

pomegranate juice concentrations (0-1.5 μmol of polyphenols/L), in the absence or

presence of lOOmmol/L of AAPH for two hours at 37°C. The extent of plasma lipid

peroxidation was measured by the TBARS assay (A) and by the lipid peroxides assay

(B). Values obtained in the absence of AAPH were subtracted from those obtained in the

presence of AAPH. Results are given as mean ± S.D. (n=3).

Figure 1 demonstrates that pomegranate juice inhibits AAPH-induced plasma lipid peroxidation in a dose-dependent manner. A 46% inhibition in thiobarbituric acid reactive substances (TBARS) formation (Fig IA) and a 21% inhibition in lipid peroxides formation (Fig IB) was obtained upon using a concentration of 0.17 mL/L of

pomegranate juice, which is equivalent to 0.5 μmol of total polyphenols/L.

Figure 2 shows the effect of pomegranate juice on LDL susceptibility to

oxidation: concentration study. LDL (100 μg of protein/mL) was incubated with

increasing concentrations of pomegranate juice (0-3.5 μmol/L of polyphenols). LDL

oxidation was induced by its incubation with 5 μmol/L CuSO₄, and was measured as

TBARS formation after two hours of incubation (A), or as conjugated dienes formation,

kinetically monitored at 234 nm (B). LDL oxidation was also induced by 5mmol/L of

AAPH (C) or by J-774 A.1 macrophages in the presence of 2 μmol/L CuSO₄ (D) and

measured as TBARS formation. Results are given as mean ± S.D. (n=3). PJ,

pomegranate juice. The susceptibility of isolated LDL to oxidation induced by copper ions was

substantially inhibited by pomegranate juice in a dose-dependent manner, as demonstrated by a reduction in TBARS formation (Figure 2 A) and a prolongation of the

lag time required for the initiation of LDL oxidation by 40 minutes (Figure 2B), upon

using of 0.24 mL pomegranate juice / L (equivalent to 0.7 μmol polyphenols/L). On

using 3.5 mL of pomegranate juice /L (equivalent to 1 μmol of polyphenols/L), the

initiation of LDL oxidation was not achieved even after 180 minutes. Similarly, pomegranate juice dose-dependently inhibited LDL oxidation induced either by the

radical generator AAPH (Figure 2C) or by J-774 A.l macrophages (Figure 2D). To study the mechanism responsible for the antioxidative capacity of pomegranate juice in vitro, the present invention analyzed the potency of pomegranate

juice to scavenge free radicals, to chelate transition metal ions, or to increase serum paraoxonase activity. Figure 3 shows the mechanisms for pomegranate juice protection against LDL oxidation. Figure 3 (A) shows the free radical scavenging capacity of

pomegranate juice. 1 , 1 -diphenyl-2-picrylhdrazyl (DPPH) ethanolic solution at a final

concentration of 100 μmol/L was mixed with increasing concentrations (0-14 μmol of

polyphenols/L) of pomegranate juice (PJ), or with 50 μmol/L of vitamin E. The time

course of the changes in absorbance was continuously monitored at 517nm. Figure 3 (B)

shows the copper-ions chelating capacity of pomegranate juice. LDL (100 μg of

protein/mL) was incubated with increasing concentrations of CuSO₄ (0-100 μmol/L) in

the absence (Control) or presence of Na₂ EDTA (EDTA) and 0.25 μmol/L or 0.56 μmol

of pomegranate juice (PJ), polyphenols/L for two hours at 37°C. The extent of LDL

oxidation was measured by the TBARS assay. Figure 3 (C) shows the effect of pomegranate juice on serum paraoxonase activity. Increasing concentrations of

pomegranate juice (0-0.5 μmol of polyphenols/L) were added to human serum (obtained

from normolipidemic volunteers) and incubated for 10 minutes at 37°C before measuring

arylesterase activity. Results represent mean ± S.D. (n=3).

Figure 3 A shows that the addition of 4.9 mL of pomegranate juice /L (14 μmol of

polyphenols/L) to a DPPH solution induced a dose-dependent decrease in the absorbance

at 517nm, which reached a plateau within 7 minutes of incubation. This is a pattern

similar to that obtained with 50 μmol/L of vitamin E, which is a potent free radical

scavenger (Fig 3 A). In order to examine whether pomegranate juice inhibits LDL oxidation by chelation of metal ions, LDL (100 mg of protein/L) was incubated with 0.2 mL of pomegranate juice/L (0.56 μmol of polyphenols/L) in the presence of increasing

concentrations of copper ions. Incubation of LDL with 25 μmol/L of Na₂ EDTA served

as a positive control, as EDTA is a potent chelator of metal ions. Figure 3B demonstrates the 25 μmol/L of EDTA inhibited copper ion-induced LDL oxidation upon

using up to 40 μmol/L of CuSO₄. At higher copper ion concentrations, EDTA could no

longer overcome the pro-oxidative effect of CuSO₄. In contrast, 0.2 mL/L of

pomegranate juice (0.56 μmol/L of polyphenols) inhibited CuSO₄-induced LDL

oxidation even at a CuSO₄ concentration as high as 80 μmol/L (Fig 3B), suggesting that

pomegranate juice does not chelate copper ions.

HDL-associated paraoxonase (PON 1) activity in serum is related to protection of

LDL against oxidation. Upon incubation of human serum with increasing concentrations

of pomegranate juice for 10 minutes at 37°C, pomegranate juice dose-dependently

increased serum PON 1 activity by up to 33% (Fig 3C). These results suggest that pomegranate juice inhibits plasma LDL lipid peroxidation in vitro, and this effect is associated with its capacity to scavenge free radicals, as well as to increase serum PON 1

activity.

Analyses of antioxidant properties against LDL oxidation of pomegranate

constituents other than the juice include the pomegranate outer and inner peels and its

seeds. We thus prepared aqueous solutions of the peels and the crushed seeds.

Polyphenols analysis of the aqueous solutions of the concentrated pomegranate juice, the inner and outer peels and the seeds revealed that they contain: 22,830, 10,320, 6314 and 630 μM of total polyphenols, respectively.

On comparing the inhibitory effects of these pomegranate constituents, based on equal polyphenols concentration, it was demonstrated that the aqueous extracts of the

inner and outer peels were more powerful antioxidants, in comparison to the juice. This data suggests that they may contain more potent antioxidant polyphenols. Figure 4

shows the capacity of pomegranate constitutents ()^ui^ce _> peels and seeds) to inhibit copper ion-induced LDL oxidation. In Figure 4 (A), aqueous extracts of pomegranate juice (crushed seeds, inner and outer peels) were prepared. The amount of total polyphenols

was determined as described under "Methods". LDL (100 μg of protein/ml) was

incubated with increasing polyphenols concentration (0-1.5 μM) of pomegranate juice, or

aqueous extracts of crushed seeds, inner and outer peels, for two hours at 37°C in the

presence of 5 μM CuSO₄. In Figure 4(B), the pomegranate juice and the aqueous

extracts of crushed seeds, inner and outer peels were lyophylized and their dry weight

measured. All samples were dissolved in water and diluted to lmg of weight/ml. LDL

(100 μg of protein/ml) was then incubated with increasing concentrations (0-100 μg weight ml) of the pomegranate fractions for two hours at 37°C in the presence of 5 μM

CuSO₄. LDL oxidation was analyzed by the TBARS assay. Results represent mean ±

S.D. (n=3).

Figure 4(A) shows that the concentrations of polyphenols, which were required to

inhibit LDL oxidation by 50% (IC₅₀), were 0.56 and 0.66 μM for the inner peel and outer

peel, respectively, compared to 1.00 μM that was obtained for the juice (Fig 4A). The

aqueous extract obtained from the crushed seeds was found to be a weak antioxidant against LDL oxidation (Fig 4A).

It is possible that other substances except polyphenols can contribute to the antioxidant activity of the pomegranate constituents; thus the present invention analyzed

the effect of increasing weight concentrations (0-100 μg/ml, Fig 4B), rather than that of the total polyphenols content (Fig 4A) on copper ions-induced LDL oxidation. The inner

and outer peels contain potent antioxidants in comparison to the crushed seeds and the pomegranate juice, which showed no inhibitory effects at the concentrations used (Fig

4B). Per lmg of weight, the inner and outer peels contain 20-30-fold more polyphenols than the aqueous fractions of the seeds and the pomegranate juice (566 and 739 nmole of

polyphenols per mg weight vs. 22 and 25 nmol of polyphenols per mg weight, respectively).

The ineffectiveness of the pomegranate juice to inhibit LDL oxidation in this

study (Fig 4B), in comparison to its potency shown in the previous study (Fig 4A), can

possibly be related to the much lower total polyphenols concentrations (about 900-fold)

of PJ that was used for the study presented in Fig 4, compared to that used for the study shown in Fig 4A. II. Ex vivo studies

Antioxidative effects of pomegranate juice were tested ex-vivo in two systems: healthy humans and atherosclerotic mice. For the human study, 13 healthy, non-smoking

men were supplemented with 50mL/day of pomegranate juice (contains 1.5 mmoles of

total polyphenols) for a period of two weeks. The studies on the atherosclerotic

apolipoprotein E deficient (E°) mice included dietary supplementation with 6.25 or 12.5

μL of pomegranate juice/mouse/day (equivalent to 0.175 or 0.350 μmoles of total polyphenols, respectively), and were analyzed in comparison to a control placebo-treated

group (water consumption with no pomegranate juice added). A. Plasma lipid pattern

Pomegranate juice administration to healthy men for a period of two weeks had no significant effect on plasma lipid profiles, including total cholesterol concentration,

LDL-cholesterol, VLDL-cholesterol, HDL-cholesterol and triglycerides (Table 1). Postprandial samples obtained from three of the volunteers after two or four hours of pomegranate juice consumption revealed no significant effect on all major routine

chemistry, hematology and coagulation assays (data not shown). Similarly, no

significant effect could be demonstrated on plasma lipids concentrations in the E° mice

that consumed pomegranate juice, in comparison to control mice that consumed only water (data not shown).

TABLE 1 :

Effect of pomegranate juice supplementation on plasma lipids and lipoproteins pattern.

Time after Pomegranate juice supplementation

0 (Before) 1 week 2 weeks Total cholesterol 194±14 200±14 204±12

LDL - cholesterol 121±10 122±9 136+11

VLDL - cholesterol 27 ± 4 30 ± 5 28 ± 2

HDL - cholesterol 43 ± 3 42 ± 3 39 ± 2

Triglycerides 136±21 149±22 144±12

Results are given as mg/dl and expressed as mean ± S.D. (n=13).

B. Plasma lipid peroxidation

Human plasma, obtained after two weeks of pomegranate juice consumption, demonstrated a small but significant (p<0.01) 6% decreased susceptibility to AAPH-

induced lipid peroxidation, in comparison to plasma obtained prior to pomegranate juice consumption, as measured by lipid peroxides formation.

Figure 5 shows the effect of pomegranate juice supplementation on human plasma oxidative state in ex vivo studies. Blood was obtained from 13 subjects before or after two weeks of pomegranate juice supplementation. Figure 5 (A) shows the susceptibility of plasma to AAPH-induced lipid peroxidation. Plasma samples were

diluted (x2) in PBS and lipid peroxidation was induced by incubation with lOOmmol/L

of AAPH for two hours at 37°C. The extent of plasma lipid peroxidation was determined

by the lipid peroxides assay. In Figure 5 (B), plasma total antioxidant status was

measured by a commercially available kit, as described under "Methods". In Figure 5 (C), serum paraoxonase was determined by measuring arylesterase activity. Results

represent mean ± S.D. (n=3) *p<0.01, ** p<0.05 (After vs. Before). Figure 5A shows a significant (p<0.05) 9% increment in plasma total antioxidant status two 2 weeks of pomegranate juice consumption, in comparison to plasma derived before juice consumption (Fig 5B).

Plasma oxidative state studied in three of the volunteers was not affected after

two and four hours of pomegranate juice consumption as determined by total antioxidant

status (TAS) and AAPH-induced lipid peroxidation (data not shown). A significant

(p<0.01) 18% increase in serum paraoxonase (PON 1) activity was monitored after

pomegranate juice consumption for a period of two weeks (Fig 5C). As serum paraoxonase is bound to HDL, it was questioned whether the increased serum

paraoxonase activity following pomegranate juice consumption is associated with increased resistance of HDL to oxidation.

Figure 6 shows the effect of pomegranate juice supplementation to humans on the

susceptibility of their HDL to oxidation in ex-vivo studies. Figure 6 (A) shows the susceptibility to oxidation of HDL obtained from 12 healthy volunteers before (0) or

after one week or two weeks of pomegranate juice supplementation. HDL (100 μg of

protein/mL) was incubated with 5 μmol/L CuSO₄ for three hours at room temperature.

The formation of conjugated dienes was kinetically monitored at 234 nm and the lag

time was measured. Results represent the mean ± S.D . (n= 12). Figure 6 (B) is a

representative figure of HDL oxidation before and after one week or two weeks of

pomegranate juice supplementation.

Pomegranate juice consumption (50 mL/day) for a period of two weeks gradually

increased the resistance of HDL to copper ion-induced oxidation as shown by a

prolongation in the lag time required for the initiation of HDL oxidation (Fig 6A) from 37+2 minutes to 45±6 minutes before and two weeks after PJ consumption, respectively. Figure 6B shows a representative kinetic analysis of copper ion-induced oxidation of

HDL derived before study entry, and after one and two weeks consumption of pomegranate juice.

Pomegranate juice consumption exhibited antioxidative effects also when

administered to E° mice. Figure 7 shows the effect of pomegranate juice

supplementation to E° mice on their plasma oxidative stress during aging. E° mice (10 mice in each group) at the age of 6 weeks were supplemented with placebo (Control) or

with 6.25 or 12.5 μL (equivalent to 0.175 or 0.350 μmols of total polyphenols, respectively) of pomegranate juice (PJ). Blood samples were drawn at the age of 6, 9

and 14 weeks. In Figure 7 (A), the plasma oxidative state was determined by measuring the levels of lipid peroxides in plasma samples. In Figure 7 (B), the antioxidant status of

the plasma samples was measured using a commercial kit. Results represent the mean ± S.D. (n=3). PJ, pomegranate juice.

The basal oxidative state, measured as lipid peroxides in plasma of control E°

mice (that did not consume pomegranate juice), increased gradually during aging from

260 nmol/mL of plasma at 6 weeks of age, to 309 and 535 nmol/mL of plasma at 9 and 14 weeks of age, respectively (Fig 7A). Following pomegranate juice consumption,

plasma lipid peroxidation was markedly reduced, and this effect was pomegranate juice

concentration-dependent (Fig 7A). Similarly, serum total antioxidant status was higher

in E° mice that consumed pomegranate juice in comparison to control mice, and this

effect was again juice concentration-dependent (Fig 7B). Serum paraoxonase activity

decrement during aging in the atherosclerotic E° mice, which are under excess oxidative stress (23), however, was not protected by pomegranate juice consumption (data not

shown).

C. LDL modifications (oxidation, aggregation, retention) a. LDL oxidation

Figure 8 shows the effect of pomegranate juice supplementation to humans on the

susceptibility of their LDL to oxidation in ex-vivo studies. In Figure 8 (A), LDL (100 μg

of protein/ml) obtained from 12 healthy volunteers before (0) or after one week or two

weeks of pomegranate juice supplementation was incubated with 5 μmol/L CuSO₄ for

three hours at room temperature. The formation of conjugated dienes was kinetically monitored at 234 nm and the lag time was measured. Results are given for each individual, as well as the mean ± S.D. (n=12). In Figure (B), a representative experiment

of LDL oxidation, before and after one week or two weeks of pomegranate juice supplementation, is shown.

The susceptibility of LDL (derived from healthy volunteers after consumption of pomegranate juice for one and two weeks) to copper ions-induced oxidation was found to be gradually reduced, as shown by a prolongation of the lag time required for the

initiation of LDL oxidation by 29% and 43%, in comparison to LDL obtained prior to

juice consumption (from 35±6 minutes before juice consumption to 44±6 minutes and 50

±6 minutes after consumption of pomegranate juice for one and two weeks, respectively,

Fig 8A). A representative kinetic analysis of copper ion-induced LDL oxidation from

this study is shown in Fig 8B.

Pomegranate juice consumption also reduced the propensity of E° mice-derived

LDL to copper ion-induced oxidation. In E° mice that consumed 6.25 μL/day or 12.5 μ L/day of pomegranate juice for a period of two months, LDL oxidation was delayed by

100 minutes and by 120 minutes, respectively, in comparison to LDL obtained before juice administration (data not shown). Figure 9 shows the effect of pomegranate juice

supplementation to E° mice on their LDL susceptibility to copper ions-induced oxidation

in ex vivo studies. LDLs were isolated from plasma samples that were collected from E°

mice (10 mice in each group) that received placebo (Control), or 6.25 μL or 12.5 μL of

pomegranate juice (PJ) (equivalent to 0.175 or 0.350 μmoles of total polyphenols,

respectively), at the age of 6, 9 or 14 weeks. The LDLs (100 μg of protein mL) were

incubated with 5 μmol/L CuSO₄, for two hours at 37°C. The extent of LDL oxidation was measured by the TBARS (A) or lipid peroxides (B) assays. Results represent mean ± S.D. (n=3).

The progressive increase with age in the susceptibility of the mice LDL to oxidation was significantly attenuated by pomegranate juice consumption, in a dose-

dependent manner, as shown for both TBARS (Fig 9A) and lipid peroxides (Fig 9B) formation.

b. LDL aggregation

Atherogenicity of LDL is attributed not only to its oxidative modification, but also to its aggregation (4). It was previously shown that LDL oxidation leads to its

subsequent aggregation (21), and it has been recently reported that polyphenols from red

wine can reduce LDL aggregation in vitro and in vivo (23).

Figure 10 shows the effect of pomegranate juice on the susceptibility of LDL to

aggregation in in vitro and ex-vivo studies. In Figure 10 (A), LDL (100 μg of

protein/mL) was incubated without (Control) or with 7 μmol/L or 14 μmol/L of polyphenols of pomegranate juice (PJ) for 10 minutes at room temperature, before

measuring LDL aggregation. LDL aggregation (by vortexing) was kinetically monitored at 680nm. A representative experiment out of three similar studies is given. In Figure 10

(B), the extent of LDL aggregation was measured in 13 healthy volunteers before (0) and

after one week or two weeks of pomegranate juice supplementation. Results are given

for each individual as well as the mean ± S.D. (n=13).

The addition of increasing concentrations of pomegranate juice to LDL reduced its susceptibility to aggregation (by vortexing) in a dose-dependent fashion (Fig 10A).

Upon analyzing the susceptibility to aggregation of LDL isolated from individual subjects that consumed pomegranate juice for one and two weeks, in 7 out of the 13 subjects studied, a pattern of reduction in LDL aggregation was observed, although the mean value showed no significant changes (Fig 10B). c. LDL retention

Extracellular matrix (ECM) proteoglycans (PGs) can bind LDL through their glycosaminoglycans (GAGs) moieties, and such interaction can lead to the entrapment of

LDL in the arterial wall, a phenomenon called "LDL retention" (5). Figure 11 shows the effect of pomegranate juice on "LDL retention" analyzed by LDL capacity to bind

chondroitin sulfate. In Figure 11 (A), LDL (200 μg of lipoprotein protein/mL) was

incubated with increasing concentrations of pomegranate juice (0-3.5 μmol of

polyphenols/L) for one hour at 37°C, followed by the addition of chondroitin sulfate (CS,

100 μg/mL) and a further incubation for 30 minutes at room temperature. LDL was then

precipitated, and the LDL-associated GAGs content was analyzed in the precipitate using

the DMMB assay as described under "Methods". Results are presented as mean ± S.D. In Figure 11 (B), chondroitin sulfate (CS, 100 μg/mL) was incubated for 30 minutes at

room temperature with LDL (200 μg of lipoprotein protein/mL) isolated from plasma of

the studied individuals, before and after one or two weeks of pomegranate juice

consumption. Results of "LDL retention" for each individual, as well as the mean ±

S.D., are presented.

According to Figure 11, addition of increasing concentrations of pomegranate

juice (0-3.5 μmoL of polyphenols/L) to LDL (200 μg of lipoprotein protein/mL) induced

a substantial dose-dependent reduction in the capacity of LDL to bind chondroitin sulfate

(CS, 100 μg/mL). LDL binding to chondroitin sulfate decreased by up to 75%,

following its incubation with 3.5 μmol/L of pomegranate juice polyphenols (Fig 11 A). The capacity of LDLs, obtained from subjects that were supplemented with pomegranate juice, to bind chondroitin sulfate (CS) was also determined, as an indication for their

"LDL retention."

The mean value for LDL-associated GAGs was not significantly affected after pomegranate juice consumption (Fig 1 IB). Pomegranate juice supplementation for one

week and for two weeks affected "LDL retention" only in some of the volunteers with no

significant effect on the mean value for all study participants. In 69% and 54% of the

cases, a decrease in LDL capacity to bind CS was observed after one week and two

weeks of pomegranate juice supplementation, respectively, compared to the results

obtained before juice administration (Fig 1 IB).

III. Macrophage atherogenicity It has been recently shown that macrophages can undergo lipid peroxidation

under oxidative stress and, subsequently, these cells can oxidize LDL (52, 53). LDL oxidation by macrophages is considered to be a major event during early atherogenesis, and it is associated with cellular uptake of the modified lipoprotein, leading to

macrophage cholesterol accumulation and foam cell formation (1). The present

invention has thus studied the effect of dietary consumption of pomegranate juice in E°

mice on macrophage lipid peroxidation and, subsequently, on macrophage activities

related to foam cell formation, including cell-mediated oxidation of LDL and cellular

uptake of lipoproteins.

A. Macrophage-mediated oxidation of LDL

Mouse peritoneal macrophages (MPM) were isolated from the peritoneal cavity of control E° mice, as well as from E° mice that consumed pomegranate juice (12.5 μ

L/mouse/day, equivalent to 0.35 μmoles of total polyphenols) for a period of two

months. Figure 12 shows the effect of pomegranate juice consumption by E° mice on their peritoneal macrophage lipids peroxidation and their ability to oxidize LDL. In

Figure 12(A), macrophage lipid peroxidation: Lipid peroxides content was assayed in cell sonicate of the MPM. Figure 12 (B) shows macrophage-mediated oxidation of LDL.

In (B), MPM were incubated for six hours at 37°C with LDL (100 μg protein mL), in the

presence of 2 μmol/L of CuSO₄. At the end of the incubation, LDL oxidation was

measured directly in the medium by the TBARS assay. Figure 12 (C) shows macrophage

superoxide anion release. In (C), cells were stimulated by the addition of LDL (100 μg

protein/mL) and CuSO₄ (5 μmol/L) to MPM for one hour at 37°C. The amount of

superoxide release was measured as described under "Methods". Figure 12 (D) shows

macrophage total glutathione content. In (D), cell sonicate was used for this assay as

described in "Methods". Results are given as mean ± S.D. (n=3), * p<0.01 (vs. Control). PJ, Pomegranate juice.

Figure 12A demonstrates that MPM isolated from E° mice after consumption of

pomegranate juice contained 53% less lipid peroxides, in comparison to MPM from

control E° mice. Incubation of these cells with LDL (100 μg of protein/mL) for 18

hours, under oxidative stress (in the presence of 2 μmol/L of CuSO₄), revealed that

pomegranate juice consumption resulted in a 82% inhibition in macrophage-mediated

oxidation of LDL, as measured by the TBARS assay (Fig 12B).

Macrophage-mediated oxidation of LDL was shown to involve activation of NADPH oxidase and superoxide anion release (12), and it depends on the balance

between cellular oxidants and antioxidants, including the glutathione system (13, 47).

Figure 12C indeed shows that pomegranate juice consumption significantly reduced (by 49%) superoxide anion release from macrophages that were activated by

incubation with LDL in the presence of copper ion. In parallel, the cellular content of glutathione increased by 25% in macrophages derived from E° mice that consumed

pomegranate juice, in comparison to MPM from control E° mice (Fig. 12D).

B. Macrophage uptake of oxidized LDL and native LDL

The present invention also studied the effect of pomegranate juice consumption

on macrophage uptake of oxidized-LDL (Ox-LDL) and native LDL. Figure 13 shows

the effect of pomegranate juice consumption by E° mice on their macrophage uptake of

native or oxidized LDL. Mouse peritoneal macrophages (MPM) were isolated from the

peritoneal fluid of control E° mice and E° mice that consumed 12.5 μL of pomegranate

juice (PJ) for a period of two months. MPM were incubated with 10 μg of ¹²⁵I-Ox-LDL

or ^I25I-LDL (10 μg of protein/mL) at 4°C for two hours, followed by determination of lipoprotein binding (A, D), or at 37°C for five hours for determination of lipoprotein cell-

association (B, E) or lipoprotein degradation (C, F). Results are given as mean ± S.D. (n=3), *p<0.01 (vs. Control). PJ, pomegranate juice.

Incubation of MPM, derived from E° mice that consumed 12.5 μL of

pomegranate juice/mouse/day for a period of two months, with ¹²⁵I-labeled oxidized LDL

(10 μg of protein/ml) resulted in a significant reduction in cellular lipoprotein binding

(Fig. 13 A), cell-association (Fig. 13B) and degradation (Fig. 13C) by 16%, 22% and

15%, respectively, in comparison to Ox-LDL binding, cell-association and degradation

by MPM from control E° mice. Similarly, pomegranate juice consumption also reduced

macrophage binding, cell-association and degradation of native LDL by 31 %, 19% and by 27%, respectively (Figs. 13 D, E, F).

IV. Platelet aggregation

Circulating human platelets play an important role in the development of atherosclerosis, and increased platelet aggregation is associated with enhanced atherogenicity (6-8). To study whether pomegranate juice can inhibit platelet

aggregation, platelet rich plasma (PRP) was incubated for 30 minutes at 37°C with

increasing concentrations of pomegranate juice, after which aggregation was induced by

the addition of collagen (2 μg/mL). A pomegranate juice dose-dependent inhibition, by

up to 90%, of collagen-induced platelet aggregation was observed (Fig 14A).

Analysis of PRP aggregation was also studied ex vivo. Following two weeks of

pomegranate juice consumption, a significant (p<0.02) 11% reduction in collagen-

induced platelet aggregation was noted, in comparison to platelet aggregation prior to PJ consumption at the beginning of the study (Fig 14B). Figure 14 shows the effect of pomegranate juice on human platelet aggregation in

both in vitro and ex-vivo studies. In Figure 14 (A), platelet-rich plasma (PRP) was

incubated with increasing concentrations of pomegranate juice (0 - 220 μmol of

polyphenols/ L) for 10 minutes at 37°C, prior to analysis of platelet aggregation in

response to 2 μg/mL of collagen. The extent of platelets aggregation is expressed as the

slope (cm/min) of the aggregation curve. In Figure 14(B), PRP was prepared from 11

healthy volunteers before (0) or after two weeks of pomegranate juice supplementation.

Results are given for each individual and also as mean ± S.D. (n=l 1).

SUMMARY DISCUSSION

The present invention analyzed the effect of pomegranate juice (PJ) on lipoprotein oxidation, aggregation and retention; on macrophage atherogenicity and on

platelet aggregation in vitro and ex vivo in healthy male volunteers and in the atherosclerotic apolipoprotein E deficient (E°) mice. The in vitro studies demonstrated a

significant dose-dependent antioxidant capability of PJ against lipid peroxidation in plasma (by up to 33%), in low density lipoprotein (LDL, by up to 43%), and in high density lipoprotein (HDL, by up to 22%). The water soluble fractions of pomegranate's

inner and outer peels, but not the seeds, were even stronger antioxidants against LDL

oxidation than the juice. The antioxidative effects of PJ against lipid peroxidation in whole plasma and in isolated lipoproteins were clearly shown, ex vivo, in humans and in

the atherosclerotic mice following consumption of PJ for up to 2 and 14 weeks,

respectively. The mechanisms for the antioxidative effects of PJ against lipid

peroxidation could be related to its capacity to scavenge free radicals. Furthermore, PJ consumption by humans increased the activity of serum paraoxonase, which is an HDL-

associated esterase that acts as a potent protector against lipid peroxidation.

Pomegranate juice not only inhibited LDL oxidation but also reduced two other

related modifications of the lipoprotein, i.e., its retention to proteoglycan (as analyzed by

LDL binding to chondroitin sulfate) and its susceptibility to aggregation (induced by LDL vortexing).

Macrophage atherogenicity was studied in mouse peritoneal macrophages

(MPMs) from E° mice. Following PJ consumption, macrophage-mediated oxidation of

LDL was reduced by 88% and this effect was associated with reduced cellular lipid peroxidation, reduced superoxide anion release and elevated content of macrophage glutathione. Furthermore, the uptake of oxidized LDL and that of native LDL, by MPMs that were obtained after PJ administration, were significantly reduced by about 20%.

The above inhibitory effects of PJ consumption on the macrophage ability to oxidize LDL, from one hand, and on the uptake of oxidized LDL, from the other hand, can

substantially contribute to attenuation of cellular cholesterol accumulation and foam cell formation.

Finally, PJ consumption by humans significantly inhibited, by up to 11%, their

blood platelet aggregation, induced by collagen.

Taken together, the results of the present study clearly demonstrated a potent

antiatherogenicity of pomegranate juice consumption in healthy humans and in

atherosclerotic mice, and these characteristics could be associated mainly with PJ

antioxidative properties.

The lipid peroxidation hypothesis of atherosclerosis (1-3) is supported by evidence for the occurrence of oxidized lipoproteins in the atherosclerotic lesion (54), by the increased oxidizability of LDL from atherosclerotic patients (55) and by the

antiatherogenicity of some antioxidants against LDL oxidation (13, 56). The impressive

ability of pomegranate extract to inhibit in vitro and ex vivo lipid peroxidation in plasma,

as well as in isolated LDL and HDL, was shown in several different oxidative systems

including transition metal ions, free radical generator and arterial cells. By more than

one assay (TBARS, lipid peroxides and conjugated dienes formation) the present

invention was able to demonstrate the substantial antioxidative capacity of pomegranate to scavenge free radicals, a major mechanism for the action of some potent natural

antioxidants, including vitamin E and flavonoids (57, 58). Pomegranate juice is rich in specific polyphenolic flavonoids, such as anthocyanines, which possess potent free radical scavenging capabilities.

While not wanting to be bound by the theory, it is believed that chelation of

copper ion, in contrast, was probably not related to the inhibitory effect of pomegranate extract on LDL oxidation, as relatively high concentrations of copper ion did not

overcome the inhibition of LDL oxidation by pomegranate juice. Similarly, other

polyphenols-rich nutrients, such as licorice (24), were also not able to chelate copper

ions.

It is worth noting that in the present study the inhibitory effect of pomegranate

juice against LDL oxidation was shared also by aqueous extracts obtained from the outer

and inner peels of pomegranates. When compared per total polyphenols content (or per

weight), the peels were more potent antioxidants against LDL oxidation than the juice.

These fractions may contain different flavonoid compositions from that present in the pomegranate juice, with a more potent antioxidative capacity.

Paraoxonase is an HDL-associated esterase, which was shown to protect the

HDL, as well as LDL, from oxidation. This protection is probably the result of

paraoxonase ability to hydrolyze specific oxidized lipids in oxidized lipoproteins (59,

60). The present invention demonstrated that pomegranate juice significantly increased

serum arylesterase activity (a major activity of the enzyme paraoxonase), as

demonstrated both in vitro (during plasma lipid peroxidation) and ex vivo in the human

study. Paraoxonase is inactivated by lipid peroxides (61), and it has been recently demonstrated the ability of red wine flavonoids (23) and that of licorice derived glabridin (61) to preserve paraoxonase activity during lipoprotein oxidation. The present invention, however, showed not only preservation but also enhancement of paraoxonase

activity.

While not wanting to be bound by the theory, it is believed that this latter effect

may be partly related to the presence of calcium ion in P J (29), as this ion is a co-factor for the enzyme arylesterase activity (36). The elevation in serum paraoxonase activity

after pomegranate extract consumption was associated with increased plasma antioxidant

status and with reduction in plasma oxidation. Furthermore, the susceptibility of HDL (the carrier of paraoxonase in serum) to oxidation was significantly reduced following PJ

consumption. These results further strengthen the inverse association between serum

paraoxonase activity and lipid peroxidation (59).

In the atherosclerotic, apolipoprotein E deficient (E°) mice, which are under

oxidative stress (62), upon pomegranate juice consumption, a substantial reduction in the plasma lipid peroxidation state, as well as in the susceptibility of their LDL to copper ions-induced oxidation, was shown. These inhibitory effects were higher in the

atherosclerotic mice than the effects shown in healthy human volunteers. This

phenomenon may be related to the high initial oxidative stress, which exists in E° mice, that was substantially reduced by the pomegranate juice antioxidant capability.

Atherosclerosis is a multifactorial disease, and factors other than LDL oxidation

can accelerate atherogenesis independently, or in association with lipid peroxidation.

Such factors include LDL retention (5) and LDL aggregation (3).

LDL oxidation is thought to occur in the arterial wall after lipoprotein binding to extracellular matrix proteoglycans. The present invention has developed a simple assay

to determine LDL binding to chondroitin sulfate as an indicator for "LDL retention." While in vitro, an impressive inhibitory effect of PJ on "LDL retention" could be observed; the ex vivo study revealed that in only 50-60% of the volunteers inhibition of "LDL retention" was achieved. This may be related to additional factors that affect

"LDL retention" in vivo, such as the LDL density, charge, and its sialic acid content (63,

64).

LDL retention can predispose the lipoprotein to oxidation, and LDL oxidation can lead to an additional atherogenic modification-lipoprotein aggregation (21).

Aggregated LDL is taken up by macrophages at an enhanced rate, leading to cellular

cholesterol accumulation and foam cell formation (4).

Macrophages can also cause LDL aggregation, independently of its oxidation,

following the secretion of proteoglycans from the cells under certain conditions (65).

The present invention demonstrated that LDL aggregation was also inhibited in vitro by

PJ and this may be related to hydrophobic interactions between PJ constituents and the lipoprotein (66). In the human study, however, in only 54% of the volunteers was LDL aggregation inhibited after PJ consumption. The inability of PJ to affect LDL

aggregation ex vivo in all subjects may be related to LDL composition differences or

dose and duration of administration. Such differences can affect LDL interaction with PJ

constituents and, hence, can change lipoprotein-lipoprotein association (64) and LDL

susceptibility to oxidation, which can then also affect lipoprotein aggregation. The

observation that some subjects were refractory to LDL modifications may be related to the short time of pomegranate juice consumption.

Arterial wall macrophages play a major role during early atherogenesis. The present invention has demonstrated that, under oxidative stress, lipid peroxidation affects not only lipoproteins but also cellular lipids (52). Furthermore, cell-mediated oxidation

of LDL can be achieved following incubation of lipid peroxidized macrophages with LDL, even in the absence of transition metal ion (52, 53). Macrophage-mediated

oxidation of LDL is associated with activation of cellular NADPH oxidase which produces superoxide anions (12). Superoxide ions can be converted under certain

conditions into a more potent reactive oxygen species (67), which can then convert native LDL to atherogenic oxidized LDL. Macrophage-mediated oxidation of LDL is

substantially increased in glutathione-depleted cells and cellular lipid peroxides are

formed under these conditions (13, 47).

The present study clearly showed that macrophage-mediated oxidation of LDL

was substantially reduced by macrophages derived from E° mice after PJ consumption.

This anti-atherogenic effect was associated with increased cellular glutathione content,

reduced macrophage superoxide anion release and reduced macrophage lipid peroxidation. These observations further support a key role for cellular lipid peroxidation in macrophage-mediated oxidation of LDL (52, 53).

Polyphenolic flavonoids, which can accumulate in the cell plasma membrane and in the cytosol, as well as other constituents of pomegranate extract, can affect not only

cellular oxygenases, such as NADPH oxidase (12) and macrophage antioxidants (such as

the glutathione system (46)), but they can also cause conformational changes in plasma

membrane constituents, such as cellular receptors for lipoproteins. The present invention thus analyzed the uptake of oxidized LDL, as well as native LDL, by peritoneal

macrophages from E° mice, following PJ consumption. The present invention was able to demonstrate reduced cellular degradation, cell-association and cellular binding of both lipoproteins, in comparison to their interaction with cells from control mice. These

results suggest that PJ constituents, which probably accumulate in the macrophage plasma membrane, may affect cellular receptors for lipoproteins by a stearic modification. It may be also that PJ constituents which accumulate intracellularly can

affect lipoprotein receptors synthesis. Consumption of pomegranate juice by the atherosclerotic E° mice thus reduced oxidative stress in the cells (which was associated

with reduced cell-mediated oxidation of LDL), and also reduced the uptake of oxidized

LDL. Both of these processes contribute to attenuation of macrophage cholesterol

accumulation and foam cell formation.

Finally, platelet activation, an additional risk factor for atherosclerosis (6), is also

associated with oxidative stress (7). The ability of PJ consumption to reduce platelet

activation in humans was supported by a direct effect of PJ on platelet aggregation as

shown in vitro. This effect may be related to an interaction of PJ constituents with the platelet surface binding sites for collagen. It may also be that the antioxidative properties

of PJ constituents, as demonstrated by their ability to scavenge free radicals, can attenuate oxidative stress-induced platelet activation.

In summary, the present invention was able to demonstrate impressive

antiatherogenic capabilities of pomegranate juice or pomegranate extract in three related

components of atherosclerosis, plasma lipoproteins, arterial macrophages and blood platelets. While not wanting to be bound by the theory, it is believed that the potent

antioxidative capacity of pomegranate extract against lipid peroxidation may be the central link for the anti-atherogenic effects of pomegranate juice on lipoproteins, macrophages and platelets.

The present invention may be embodied in other specific forms without departing from its essential characteristics. The described embodiment is to be considered in all respects only as illustrative and not as restrictive. The scope of the invention is,

therefore, indicated by the appended claims rather than by the foregoing description. All changes which come within the meaning and range of the equivalence of the claims are

to be embraced within their scope.

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Claims

WHAT IS CLAIMED IS:

1. A composition, the biologically active component of the composition

consisting essentially of an extract from pomegranate, and the composition comprising a carrier.

2. The composition of claim 1, wherein the composition is in a form selected

from a group consisting of nutritional supplements, pharmaceutical preparations, vitamin supplements, food additives or foods supplements.

3. The composition of claim 2, wherein the composition is a pharmaceutical preparation, and the carrier is a pharmaceutically acceptable carrier.

4. The composition of claim 2, wherein the composition is in a form selected from a group consisting of nutritional supplements, vitamin supplements, food additives

or foods supplements, and the carrier is a dietary suitable carrier.

5. The composition of claim 1 , wherein the composition is in a dosage unit

form selected from a group consisting of tablets, suspensions, implants, solutions, emulsions, capsules, powders, syrups, liquid compositions, ointments, lotions, creams,

pastes, gels, and the like.

6. The composition of claim 1 , wherein the extract is the extract of

pomegranate juice, pomegranate inner peel, pomegranate outer peel, or a mixture thereof.

7. The composition of claim 1 , wherein the extract is present in a dosage unit

in an amount that contains at least 30 to 3000 μmols per dosage unit of polyphenols,

wherein the polyphenol are those naturally present in the extract of pomegranate.

8. An antioxidative composition for treating disorders associated with a

condition selected from a group consisting of lipoprotein oxidation, aggregation,

retention; macrophage atherogenicity; platelet activation; and atheroscleorosis; the

biologically active component of the composition consisting essentially of an effective amount of an extract from pomegranate.

9. The antioxidative composition of claim 8, wherein the composition is in a dosage unit form selected from a group consisting of tablets, suspensions, implants, solutions, emulsions, capsules, powders, syrups, liquid compositions, ointments, lotions, creams, pastes, gels, and the like.

10. The antioxidative composition of claim 8, wherein the extract is the extract of pomegranate juice, pomegranate inner peel or pomegranate outer peel, or a mixture thereof.

11. The antioxidative composition of claim 8, wherein the extract is present in

a dosage unit in an amount that contains at least 30 to 3000 μmols per dosage unit of

polyphenols, wherein the polyphenols are those naturally present in the extract of pomegranate.

12. The antioxidative composition of claim 8, wherein the disorders are

selected from a group consisting of arteriosclerotic heart disease and its associated

complications (including myocardial infarction); cerebral vascular disease (including

cerebral insufficiency or stroke); peripheral vascular disease (including peripheral

vascular disease in the aorta and femoral and corotid arteries); abdominal aortic

aneurysms; renal artery stenosis; arteriosclerotic disease, disorders associated with transplant complications; disorders associated with post-operative heart valve replacement; disorders associated with the complications of diabetes mellitus;

thrombophlebitis; and other disorders associated with increased platelets and increased

platelet activation.

13. A method of ameliorating disorders associated with a condition selected

from a group consisting of lipoprotein oxidation, aggregation, retention; macrophage atherogenicity; platelet aggregation; and atheroscleorosis; the method comprising

administering to a subject in need thereof a therapeutically effective amount of a composition comprising an extract from pomegranate.

14. The method of claim 13, wherein the subject is a mammal.

15. The method of claim 13 , wherein the subj ect is human.

16. The method of claim 13, wherein the composition is a juice extract of pomegranate.

17. The method of claim 13, wherein the extract is from inner or outer peel of pomegranate.

18. The method of claim 13, wherein the composition is further comprising a

pharmaceutically acceptable carrier.

19. The method of claim 13, wherein the composition is administered orally.

20. The method of claim 13, wherein the composition is administered in a

form selected from a group consisting of tablets, suspensions, implants, solutions,

emulsions, capsules, powders, syrups, liquid compositions, ointments, lotions, creams,

pastes, gels, and the like.

21. The method of claim 16, wherein the pomegranate juice extraction is

administered orally to a human per day in an amount that contains at least 30 to 3000

μmol of polyphenols, wherein the polyphenols are polyphenols naturally occurring in the

extract of pomegranate.

22. The method of claim 17, wherein the composition is administered per day

in an amount that contains an amount of polyphenols from about 30 μmol to about 3000

μmol, wherein the polyphenols are polyphenols naturally occurring in the extract of

pomegranate.

23. The method of claim 13, wherein the disorders are selected from a group consisting of arteriosclerotic heart disease and its associated complications (including myocardial infarction); cerebral vascular disease (including cerebral insufficiency or stroke); peripheral vascular disease (including peripheral vascular disease in the aorta and femoral and corotid arteries); abdominal aortic aneurysms; renal artery stenosis;

arteriosclerotic disease, disorders associated with transplant complications; disorders

associated with post-operative heart valve replacement; disorders associated with the complications of diabetes mellitus; thrombophlebitis; and other disorders associated with

increased platelets and increased platelet activation.

24. A method of ameliorating in a sample a condition selected from a group

consisting of lipoprotein oxidation, aggregation, retention; macrophage atherogenicity;

and platelet aggregation, wherein the method comprises a step of contacting the sample

with a sufficient amount of an extract from pomegranate.

25. The method of claim 24, wherein the sample is from a mammal.

26. The method of claim 25, wherein the sample is from a human.

27. The method of claim 24, wherein the extract is a juice extract of

pomegranate.

28. The method of claim 24, wherein the extract is from inner or outer peel of

pomegranate.

29. The method of claim 24, wherein the extract is in an amount that contains

at least 30 to 3000 μmol of polyphenols, wherein the polyphenols are polyphenols

naturally occurring in the extract of pomegranate.