ACTIVE INGREDIENTS,

THEIR BIOAVAILABILITY

AND THE HEALTH BENEFITS OF THE

PUNICA GRANATUM LINN

(POMEGRANATE)

A Research Review

Dirk Budka, M.Sc. Senior Biomedical Scientist, MSML Research Unit

© Front picture: Cleanfoods Ltd, Bangalore, India

CONTENT

Preface page 01

The Claims page 02

Scientific Classification page 04

Nutritional Value page 04

Compact page 05

History page 08

Cultivars page 12Pollination page 14Climate page 14Soil page 15Propagation page 15Culture page 15Harvesting and Yield page 12Keeping Quality & Storages page 16Pest and Disease page 18Food Uses page 18

Introduction to Health Benefits of Pomegranate page 20

Active Ingredients of the Pomegranate page 22

Antioxidants page 23Phenolics page 25TANNINS page 27

Tannin Table I page 29Punicalagin page 29Ellagic Acid page 30Pelagonidin, Cyanidin page 32Gallic Acid page 33Quercetin page 33

Pomegranate and Cancer page 34

Pomegranate and Atherosclerosis page 45

… ff CONTENT …

Chronic Obstructive Pulmonary Disease page 54

Osteoarthritis page 56

Increase in Sperm Quality page 58

Erectile Dysfunction page 59

Alzheimer’s Disease page 61

Menopausal Symptoms page 62

Cosmeceutical page 63

Antimicrobial page 67

Reference List page 77

List of Research Centers page 84

Correspondence Address: MSML c/o Hale Clinic,7 Park Crescent, London W1B 1PFSenior biomedical Scientist: Dirk BudkaSenior Practitioner: Dr. Peter Gruenewald, MDdirk.budka@ntlworld.com

www.immuneclinic.com www.bacteriaclinic.com

www.virusmedicalclinic.com www.parasiteclinic.com

www.fungusandyeast.com www.nutritionlondon.net

www.ageless-technologies.com www.ibsforum.co.uk

www.stop-readymeals.com

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PREFACE

Plants are a valuable source of natural products for maintaining human health and the

use of plant compounds for pharmaceutical purposes has increased. According to the

WHO (World Health Organization), medical plants would be the best source to obtain

a variety of drugs.

The Pomegranate was once named the “most medicinal fruit in the world” and you

will read in this paper about the many health benefits of this fruit.

Of course, a lot has still to be done – a lot of research is necessary to understand the

interactions between the many active ingredients and the interactions between

pharmaceuticals and the active ingredients in pomegranate.

Virologist, bacteriologist, mycologist and parasitologist are just at the beginning of

the understanding of the microbial world.

Novel treatments are essential… and what’s wrong in using natural products,

inexpensive and available in the next shop, to treat ailments/diseases?

Let’s start with the pomegranate.

Dirk Budka

June 2008

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ACTIVE INGREDIENTS, THEIR BIOAVAILABILITY AND THE HEALTH

BENEFITS OF THE PUNICA GRANATUM LINN (POMEGRANATE)

Many claims have been made over the past decade regarding the health benefits of the

Pomegranate, once named “the most medicinal fruit in the world”. This study is

looking at the research, scientific studies and the proven and non-proven claims.

The claims:

- Pomegranate has antimicrobial properties and can therefore be protective and/or

fights off bacteria, viruses, fungi and parasites.

- It can protect/is protective against

- Prostate Cancer

- Lung Cancer

- Colon Cancer

- Skin Cancer

- Eosophageal Cancer

- Diabetes

- Osteoarthritis

- Hypercholesterolemia

- Atherosclerosis

- Obstructive Pulmonary Disease

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- Alzheimer’s Disease

- Tuberculosis

- Macular Degeneration and Vision Loss

- Erectile Dysfunction

- Chronic inflammation (arthritis and cystic fibrosis)

- Menopausal Symptoms

- Anti-histaminic

- Pomegranate helps to stabilize/increase sperm quality - it has anti-malarial

properties, it protects the neonatal brain against hypoxic-ischemic injury and is

classified within the cosmetic industry as a Cosmeceutical (combining a feature of

both cosmetic and pharmaceutical).

Scientific Classification

Kingdom Plantae

Division Magnoliophyta

Class Magnoliopsida

Subclass Rosidae

Order Myrtales

Family Lythraceae

Genus Punica

Species P. granatum

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The Nutritional Value per 100 g

of the Pomegranate (aril only)

Carbohydrates 17.17 g

-sugars 16.57 g

- dietary fibre 0.60 g

Fat 0.30 g

Protein 0.95 g

Thiamin (B1) 0.030 mg

Riboflavin (B2) 0.063 mg

Niacin (B3) 0.300 mg

Pantothenic acid (B5) 0.596 mg

Vitamin B6 0.105 mg

Folate (B9) 6 µg

Vitamin C 3 mg

Calcium 3 mg

Iron 0.30 mg

Magnesium 3 mg

Phosphorus 8 mg

Potassium 259 mg

Zinc 0.12 mg

Source: USDA Nutrient Database

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COMPACT

a) ANTI ATHEROGENIC: Pomegranate significantly reduces oxidative stress

by inhibiting the formation of oxidized LDL lipoproteins and macrophage

lipid peroxidation. The pomegranate juice decreases LDL susceptibility to

aggregation and retention, increases the activity of serum paraoxinase (a HDL

esterase), and suppresses oxidized LDL degration and cholesterol biosynthesis

in macrophages, that can lead to reduced cellular cholesterol accumulation and

foam cell formation.

b) CORONARY HEART DISEASE: The effects of pomegranate have been

studied in patients suffering from CHD and myocardial ischemia. They were

randomly assigned into two groups. One group was given 240 ml pomegranate

juice daily for three months, while the other group drank a beverage of similar

caloric content, amount, flavour and colour. After three months, the extent of

stress induced ischemia decreased in the pomegranate group and increased in

the control-group.

c) CAROTID ARTERY STENOSIS: Consumption for three years of

pomegranate juice by atherosclerotic patients with carotid artery stenosis

reduced blood pressure and LDSL oxidation.

d) HYPERTENSION: Pomegranate juice consumption (50 ml/1.5 mmol of total

polyphenols per day for two weeks) by hypertensive patients showed a 36%

decrease in serum angiotensin converting enzyme (ACE) activity and a 5%

reduction in systolic blood pressure.

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e) DIABETES: Pomegranate juice consumption (50 ml/day for three months) by

diabetic patients did not worsen the diabetic parameters such as serum

glucose, cholesterol and triglyceride levels, but resulted in antioxidative

effects on serum and macrophages. The effect of concentrated pomegranate

juice consumption (40g/daily for eight weeks) on lipid profiles of type II

diabetic patients (14 female/8male) with hyperlipidemia was also evaluated.

Pomegranate juice significantly reduced the level of total cholesterol, LDL-

cholesterol.

f) SKIN: Pomegranate aqueous extract promotes regeneration of the dermis, and

lipophilic fractions prepared from pomegranate seed promoted regeneration of

epidermis in human skin cells in laboratory conditions.

g) CANCER. Antioxidant compounds – present in the diet – are considered

chemo-preventative and chemotherapeutic agents. PROSTATE CANCER:

Ellagic acid, Caffeic acid, Luteolin and Punicic acid were tested in vitro as

inhibitors on the invasion of human PC-3 prostate cancer cells. All compounds

significantly inhibited the cell-invasion when they were employed

individually. BREAST CANCER: In laboratory conditions, pomegranate

extract had significant cytotoxic and growth inhibition effects on human breast

cancer cells, inhibiting the growth of the cells through induction of apoptosis.

COLON: Pomegranate juice suppressed inflammatory cell signalling in the

HT-29 human colon cancer cell line.

h) ANTIMICROBIAL: Pomegranate seeds have potent antimicrobial activities

against bacteria and fungi/yeasts. The interaction between pomegranate

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i) (methanolic extract) and the antibiotics chloramphenicol, gentamicin,

ampicillin, tetracycline, and oxacillin against 30 clinical isolates of

methicillin-resistant and methicillin-sensitive Staphylococcus aureus,

demonstrates that pomegranate extract dramatically enhances the activity of

all antibiotics tested. Not only was bacterial growth tested, but also bacterial

enterotoxin production and it was shown that staphylococcal enterotoxin

production was inhibited. Pomegranate juice was also screened for inhibitory

activity against HIV-1 IIIB using CD4 and CXCR4 as cell receptors.

j) ADVERSE REACTION: One person developed a late-onset tongue-oedema

due to pomegranate intake as proven by a double blind oral challenge test.

Another case relates to a 7-year-old asthmatic child who showed clinical

conditions of bronchospasm, moments after ingesting several pomegranate

seeds. (Salbutamol inhaler brought on an immediate positive response).

Pomegranate may increase the risk of rhabdomyolysis during rosuvastatin

treatment for McArdle disease (serious muscle damage). In a study of 15

patients allergic to pomegranate, 13 had sensitivity to pollen, 10 to nuts and 8

to peaches. One case was very interesting, because the female patient stated

that she never ate pomegranate before. Nevertheless, her mother had

frequently eaten this fruit while breastfeeding the daughter. In this case,

maternal milk could have been the source of the child’s sensitization.

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HISTORY

Photo: Courtesy of OZ Granate PTY Ltd.

Moving way back in time, we find the Pomegranate celebrated in Egyptian papyri,

cited in the Old Testament as ‘rimmon’, and appearing in Greek mythology, in Roman

history and in the Koran. Well before the Christian era, they were introduced into

China from Samarkhand. The Pomegranate has appeared throughout history in some

of the greatest documents and art and architecture, from Homer and Chaucer, to

Shakespeare and Raphael, and to Cezanne in more modern times.

Tree of Life Symbolism of the Pomegranate

The fruit is mentioned by various cultures and religions. The pomegranate tree is said

to have flourished in the Garden of Eden and is very likely the “apple” of the Adam

and Eve story in Genesis, from the mysterious ‘Tree of Life’. Greek and Persian

mythology mentions the fruit as representing life, regeneration, and marriage. The

ancient Chinese believed the seeds symbolized longevity and immortality. In Judaism,

pomegranates appear in many contexts, cultural and religious.

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The fruit is also a symbol of resurrection and life in Christianity, and it is one of the

three “blessed fruits” in Buddhism.

The Pomegranate was used as a decoration in the Temple of Solomon, as a regal

symbol for Kings and Queens, and as a decoration on the robes of priests. In fact, a

thimble-sized, ivory pomegranate bearing an ancient Hebrew inscription is the only

relic ever recovered from the Solomon’s Temple.

It has always been a symbol of many virtues, including love and fertility in particular,

and health and abundance. The abundance of seeds suggested all these virtues, and a

spiritual dimension as well. The juice has been compared to blood, and the obvious

‘crown’ has led to royal connections, as well as a likeness to female breasts.

While the pomegranate originated generally from Persia (Iran) and has been

cultivated in Central Asia, Georgia, Armenia and the Mediterranean region for several

millennia.

In Georgia, and Armenia to the east of the Black Sea, there are wild pomegranate

groves outside of ancient abandoned settlements. The cultivation of the pomegranate

has a long history in Armenia; decayed remains of pomegranates dating back to 1000

BC have been found in the country.

Carbonized pips and pieces of the peel of the fruit have been identified in Early

Bronze Age levels of Jericho, as well as Late Bronze Age levels of Hala Sultan Tekke

on Cyprus and Tiryns. A large, dry pomegranate was found in the tomb of Djehuty,

the butler of Queen Hatshepsut; Mesopotamian cuneiform records mention

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pomegranates from the mid-Third millennium BC onwards. It is also extensively

grown in South China and in Southeast Asia, whether originally spread along the

route of the Silk Road or brought by sea traders.

The ancient city of Granada in Spain was renamed after the fruit during the Moorish

period. Spanish colonists later introduced the fruit to the Caribbean and Latin

America, but in the English colonies it was less at home: “Don’t use the pomegranate

inhospitably, a stranger that has come so far to pay his respects to thee” the English

Quaker Peter Collinson wrote to the botanizing John Bartram in Philadelphia, 1762.

“Plant it against the side of thy house, nail it close to the wall. In this manner it thrives

wonderfully with us, and flowers beautifully, and bears fruit this hot year. I have

twenty-four on one tree… Doctor Fothergill says, of all trees this is most salutiferous

to mankind.”The pomegranate had been introduced as an exotic to England the

previous century, by John Tradescant the elder, but the disappointment that it did not

set fruit there led to its repeated introduction to the American colonies, even New

England. It succeeded in the South: Bartram received a barrel of pomegranates and

oranges from a correspondent in Charleston, South Carolina, 1764. Thomas Jefferson

planted pomegranates at Monticello in 1771: he had them from George Wythe of

Williamsburg

In the Mesopotamian region, the pomegranate is still prized both as a medication and

as a symbol of beauty, longevity, fertility and wisdom worldwide.

Because of its role in the Greek legend of Persephone, the pomegranate came to

symbolize fertility, death, and eternity and was an emblem of the Eleusinian

Mysteries. In Christian art, it is a symbol of hope.

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The pomegranate tree is native from Iran to the Himalayas in northern India and has

been cultivated since ancient times throughout the Mediterranean region of Asia,

Africa and Europe. The fruit was used in many ways as it is today and was featured in

Egyptian mythology and art, praised in the Old Testament of the Bible and in the

Babylonian Talmud, and it was carried by desert caravans for the sake of its thirst-

quenching juice. It travelled to central and southern India from Iran about the first

century A.D. and was reported growing in Indonesia in 1416. It has been widely

cultivated throughout India and drier parts of southeast Asia, Malaya, the East Indies

and tropical Africa. The most important growing regions are Egypt, China,

Afghanistan, Pakistan, Bangladesh, Iran, Iraq, India, Burma and Saudi Arabia. There

are some commercial orchards in Israel on the coastal plain and in the Jordan Valley.

It is rather commonly planted and has become naturalized in Bermuda where it was

first recorded in 1621, but only occasionally seen in the Bahamas, West Indies and

warm areas of South and Central America. Many people grow it at cool altitudes in

the interior of Honduras. In Mexico it is frequently planted, and it is sometimes found

in gardens in Hawaii. The tree was introduced in California by Spanish settlers in

1769. It is grown for its fruit mostly in the dry zones of that state and Arizona. In

California, commercial pomegranate cultivation is concentrated in Tulare, Fresno and

Kern counties, with small plantings in Imperial and Riverside counties. There were

2,000 acres (810 ha) of hearing trees in these areas in the 1920's. Production declined

from lack of demand in the 1930's but new plantings were made when demand

increased in the 1960's.

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Cultivars

There is little information available on the types grown in the Near East, except that

the cultivars 'Ahmar', 'Aswad', 'Halwa' are important in Iraq, and 'Mangulati' in Saudi

Arabia. 'Wonderful' and 'Red Loufani' are often grown in the Jewish sector of Israel,

while the sweeter, less tangy 'Malissi' and 'Ras el Baghl', are favored in the Arab

sector.

In India there are several named cultivars. Preference is usually given those with

fleshy, juicy pulp around the seeds. Types with relatively soft seeds are often classed

as "seedless". Among the best are 'Bedana' and 'Kandhari'. 'Bedana' is medium to

large, with brownish or whitish rind, pulp pinkish-white, sweet, seeds soft. 'Kandhari'

is large, deep-red, with deep-pink or blood-red, subacid pulp and hard seeds. Others

include:

'Alandi' ('Vadki')–medium-sized, with fleshy red or pink, subacid pulp, very hard

seeds.

'Dholka'–large, yellow-red, with patches of dark-pink and purple at base, or all-over

greenish-white; thick rind, fleshy, purplish-white or white, sweet, pulp; hard seeds.

The plant is evergreen, non-suckering, desirable for commercial purposes in Delhi.

'Kabul'–large, with dark-red and pale-yellow rind; fleshy, dark-red, sweet, slightly

bitter pulp.

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'Muscat Red'–small to medium, with thin or fairly thick rind, fleshy, juicy, medium-

sweet pulp, soft or medium-hard seeds. The plant is a moderately prolific bearer.

'Paper Shell'–round, medium to large, pale-yellow blushed with pink; with very thin

rind, fleshy, reddish or pink, sweet, very juicy pulp and soft seeds. Bears heavily.

'Poona'–large, with dark-red, gray or grayish-green rind, sometimes spotted, and

orange-red or pink-and-red pulp.

'Spanish Ruby'–round, small to medium or large; bright-red, with thin rind, fleshy,

rose-colored, sweet, aromatic pulp, and small to medium, fairly soft seeds. Considered

medium in quality.

'Vellodu'–medium to large, with medium-thick rind, fleshy, juicy pulp and medium-

hard seeds.

'Muscat White'–large, creamy-white tinged with pink; thin rind; fleshy, cream-

colored, sweet pulp; seeds medium-hard. Bears well. Desirable for commercial

planting in Delhi.

'Wonderful'–originated as a cutting in Florida and propagated in California in 1896.

The fruit is oblate, very large, dark purple-red, with medium-thick rind; deep-red,

juicy, winey pulp; medium-hard seeds. Plant is vigorous and productive.

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In California, 'Spanish Ruby' and 'Sweet Fruited' were the leading cultivars in the past

century, but were superseded by 'Wonderful'. In recent years 'Wonderful' is losing

ground to the more colorful 'Grenada'.

Mexicans take especial pride in the pomegranates of Tehuacan, Puebla. Many

cultivars are grown, including 'Granada de China' and 'Granada Agria'.

The Japanese dwarf pomegranate, P. granatum var. nana, is especially hardy and

widely grown as an ornamental in pots. The flowers are scarlet, the fruit only 2 in (5

cm) wide but borne abundantly. Among other ornamental cultivars are 'Multiplex'

with double, creamy white blooms; 'Chico', double, orange-red; 'Pleniflora', double,

red; 'Rubra Plena', double, red; 'Mme. Legrelle' and 'Variegata', double, scarlet

bordered and streaked with yellowish-white.

Pollination

The pomegranate is both self-pollinated and cross-pollinated by insects. There is very

little wind dispersal of pollen. Self-pollination of bagged flowers has resulted in 45%

fruit set. Cross-pollination has increased yield to 68%. In hermaphrodite flowers, 6 to

20% of the pollen may be infertile; in male, 14 to 28%. The size and fertility of the

pollen vary with the cultivar and season.

Climate

The species is primarily mild-temperate to subtropical and naturally adapted to

regions with cool winters and hot summers, but certain types are grown in home

dooryards in tropical areas, such as various islands of the Bahamas and West Indies.

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In southern Florida, fruit development is enhanced after a cold winter. Elsewhere in

the United States, the pomegranate can be grown outdoors as far north as Washington

County, Utah, and Washington, D.C., though it doesn't fruit in the latter locations. It

can be severely injured by temperatures below 12º F (-11.11º C). The plant favors a

semi-arid climate and is extremely drought -tolerant.

Soil

The pomegranate thrives on calcareous, alkaline soil and on deep, acidic loam and a

wide range of soils in between these extremes. In northern India, it is spontaneous on

rockstrewn gravel.

Propagation

Pomegranate seeds germinate readily even when merely thrown onto the surface of

loose soil and the seedlings spring up with vigor. However, to avoid seedling

variation, selected cultivars are usually reproduced by means of hardwood cuttings 10

to 20 in (25-50 cm) long. Treatment with 50 ppm. indole-butyric acid and planting at

a moisture level of 15.95% greatly enhances root development and survival. The

cuttings are set in beds with 1 or 2 buds above the soil for 1 year, and then

transplanted to the field. Grafting has never been successful but branches may be air-

layered and suckers from a parent plant can be taken up and transplanted.

Culture

Rooted cuttings or seedlings are set out in pre-fertilized pits 2 ft (60 cm) deep and

wide and are spaced 12 to 18 ft (3.5-5.5 m) apart, depending on the fertility of the

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soil. Initially, the plants are cut back to 24 to 30 in (60-75 cm) in height and after they

branch out the lower branches are pruned to provide a clear main stem. Inasmuch as

fruits are borne only at the tips of new growth, it is recommended that, for the first 3

years, the branches be judiciously shortened annually to encourage the maximum

number of new shoots on all sides, prevent straggly development, and achieve a

strong, well-framed plant. After the 3rd year, only suckers and dead branches are

removed.

For good fruit production, the plant must be irrigated. In Israel, brackish water is

utilized with no adverse effect. In California, irrigation water is supplied by overhead

sprinklers which also provide frost protection during cold spells. The pomegranate

may begin to bear in 1 year after planting out, but 2 1/2 to 3 years is more common.

Harvesting and Yield

The fruits ripen 6 to 7 months after flowering. In Israel, cultivar 'Wonderful' is

deemed ready for harvest when the soluble solids (SSC) reach 15%. In California,

maturity has been equated with 1.8% titratable acidity (TA) and SSC of 17% or more.

The fruit cannot be ripened off the tree even with ethylene treatment. Growers

generally consider the fruit ready for harvest if it makes a metallic sound when

tapped. The fruit must be picked before over maturity when it tends to crack open if

rained upon or under certain conditions of atmospheric humidity, dehydration by

winds, or insufficient irrigation. Of course, one might assume that ultimate splitting is

the natural means of seed release and dispersal.

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The fruits should not be pulled off but clipped close to the base so as to leave no stem

to cause damage in handling and shipping. Appearance is important, especially in the

United States where pomegranates may be purchased primarily to enhance table

arrangements and other fall (harvest-time) decorations. Too much sun exposure

causes sunscald–brown, russeted blemishes and roughening of the rind.

The fruit ships well, cushioned with paper or straw, in wooden crates or, for nearby

markets, in baskets. Commercial California growers grade the fruits into 8 sizes, pack

in layers, unwrapped but topped with shredded plastic, in covered wood boxes,

precool rapidly, and ship in refrigerated trucks.

Keeping Quality and Storage

The pomegranate is equal to the apple in having a long storage life. It is best

maintained at a temperature of 32º to 41º F (0º-5º C). The fruits improve in storage,

become juicier and more flavorful; may be kept for a period of 7 months within this

temperature range and at 80 to 85% relative humidity, without shrinking or spoiling.

At 95% relative humidity, the fruit can be kept only 2 months at 41º F (5º C); for

longer periods at 50º F (10º C). After prolonged storage, internal breakdown is

evidenced by faded, streaky pulp of flat flavor. 'Wonderful' pomegranates, stored in

Israel for Christmas shipment to Europe, are subject to superficial browning ("husk

scald"). Control has been achieved by delaying harvest and storing in 2% O2 at 35.6º

F (2º C). Subsequent transfer to 68º F (20º C) dispels off-flavour from ethanol

accumulation.

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Pests and Diseases

The pomegranate butterfly, Virachola isocrates, lays eggs on flower-buds and the

calyx of developing fruits; in a few days the caterpillars enter the fruit by way of the

calyx. These fruit borers may cause loss of an entire crop unless the flowers are

sprayed 2 times 30 days apart. A stem borer sometimes makes holes right through the

branches. Twig dieback may be caused by either Pleuroplaconema or Ceuthospora

Phyllosticta. Discoloration of fruits and seeds results from infestation by Aspergillus

castaneus. The fruits may be sometimes disfigured by Sphaceloma punicae. Dry rot

from Phomopsis sp. or Zythia versoniana may destroy as much as 80% of the crop

unless these organisms are controlled by appropriate spraying measures. Excessive

rain during the ripening season may induce soft rot. A post-harvest rot caused by

Alternaria solani was observed in India in 1974. It is particularly prevalent in cracked

fruits.

Minor problems are leaf and fruit spot caused by Cercospora, Gloeosporium and

Pestalotia sp.; also foliar damage by whitefly, thrips, mealybugs and scale insects;

and defoliation by Euproctis spp. and Archyophora dentula. Termites may infest the

trunk. In India, paper or plastic bags or other covers may be put over the fruits to

protect them from borers, birds, bats and squirrels.

Food Uses

For enjoying out-of-hand or at the table, the fruit is deeply scored several times

vertically and then broken apart; then the clusters of juice sacs can be lifted out of the

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rind and eaten. Italians and other pomegranate fanciers consider this not a laborious

handicap but a social, family or group activity, prolonging the pleasure of dining.

In some countries, such as Iran, the juice is a very popular beverage. Most simply, the

juice sacs are removed from the fruit and put through a basket press. Otherwise, the

fruits are quartered and crushed, or the whole fruits may be pressed and the juice

strained out. In Iran, the cut-open fruits may be stomped by a person wearing special

shoes in a clay tub and the juice runs through outlets into clay troughs. Hydraulic

extraction of juice should be at a pressure of less than 100 psi to avoid undue yield of

tannin. The juice from crushed whole fruits contains excess tannin from the rind (as

much as .175%) and this is precipitated out by a gelatin process. After filtering, the

juice may be preserved by adding sodium benzoate or it may be pasteurized for 30

minutes, allowed to settle for 2 days, then strained and bottled. For beverage

purposes, it is usually sweetened. Housewives in South Carolina make pomegranate

jelly by adding 7 1/2 cups of sugar and 1 bottle of liquid pectin for every 4 cups of

juice. In Saudi Arabia, the juice sacs may be frozen intact or the extracted juice may

be concentrated and frozen, for future use. Pomegranate juice is widely made into

grenadine for use in mixed drinks. In the Asiatic countries it may be made into a thick

sirup for use as a sauce. It is also often converted into wine.

In the home kitchen, the juice can be easily extracted by reaming the halved fruits on

an ordinary orange-juice squeezer.

In northern India, a major use of the wild fruits is for the preparation of "anardana"–

the juice sacs being dried in the sun for 10 to 15 days and then sold as a spice.

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INTRODUCTION TO HEALTH BENEFITS OF POMEGRANATE

The last decades have seen an increase of interest in active ingredients and natural

products with proven health benefits of plants. The “most medicinal fruit of all”, the

Punica granatim Linn (pomegranate) has been an important key to a rising interest of

scientist in researching alternatives (or additions) to conventional pharmaceutical

approaches.

The pomegranate tree - especially its fruit - possesses a vast ethnomedical history and

represents a phytochemical reservoir of heuristic medicinal value. The tree/fruit can

be divided into several anatomical compartments: (1) seed, (2) juice, (3) peel, (4) leaf,

(5) flower, (6) bark, and (7) roots, each of which has interesting pharmacologic

activity.

- Juice and peels have potent antioxidant properties;

- Juice, peel and oil are all weakly estrogenic and heuristically of interest for

the treatment of menopausal symptoms and sequellae. Juice, peel and oil

have also been shown to possess anticancer activities, including

interference with tumour cell proliferation, cell cycle, invasion and

angiogenesis.

The phytochemistry and pharmacological actions of all Punica granatum components

suggest a wide range of clinical applications for the treatment and prevention of

- 21 -

cancer, as well as other diseases where chronic inflammation is believed to play an

essential etiologic role.

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ACTIVE INGREDIENTS OF THE POMEGRANATE

Punicalagin

Ellacic Acid

ellagitannins punicalagin A and punicalagin B

potent tannins

anthocyanins (delphinidin, cyanidin, pelargonidin, idin, malvidin, petunidin)

caffeic acid

luteolin

punicic acid

gallic acid

Punicotannic Acid

Gallic Acid

Mannite

Pelletierine

N-Methylisopelletierine

Pelargonidin

Punicalin

Punicalagin

syringic acid

sinapic acid

protocatechuic acid

ferulic acid

3,4-dihydroxy-phenylacetic acid (PAA)

- 23 -

Quercetin

Kaempferol

Antioxidants

Any substance that reduces oxidative damage (damage due to oxygen) such as that

caused by free radicals is called an antioxidant. Free radicals are highly reactive

chemicals which attack molecules by capturing electrons and thus modifying

chemical structures. Oxygen damage (oxidation) to the cells is partly responsible for

the effects of aging and certain diseases.

As part of their normal function, cells produce free radicals, which are toxic

molecules. A free radical is a damaged molecule, because it is missing an electron.

Because the free-radical molecule needs its full complement of electrons, it reacts

with any molecule from which it can take an electron. By taking an electron from

certain key components in the cell, such as fat, protein or DNA molecules, free

radicals can damage cells. Antioxidants that occur naturally in the body and certain

foods may block this damage by donating electrons to stabilize and neutralize the

harmful effects of the free radicals.

Even though most free radical damage is repaired, a fraction may still remain. The

environment is also a source of free radicals caused by ultraviolet radiation or

airborne pollutants, such as cigarette smoke. Eventually, free radical damage may

- 18 -

overwhelm the body's natural defence. As cell damage accumulates, it may contribute

to aging and certain diseases like cardiovascular disease and some cancers. More

antioxidant vitamins from one's diet may help counter some of the damage.

Antioxidants are also commonly added to food products like vegetable oils and

prepared foods to prevent or delay their deterioration from the action of air.

Research has shown a relationship to a number of diseases. Scientists theorize that

low-density lipoprotein (LDL) cholesterol damages the lining of the arteries when it

becomes oxidized. Vitamin C, vitamin E and carotenoids may help protect against the

oxidation of LDL cholesterol by neutralizing free radicals. It is suspected that

cataracts develop partly as a result of oxidation of proteins in the lens of the eye, and

some studies have shown that antioxidants might be effective in reducing age-related

macular degeneration and the resulting vision loss.

Evidence from more than a hundred studies suggests that eating fruits and vegetables

rich in vitamin C or carotenoids is linked with a reduced risk of many cancers.

Despite the support for the health benefits of vitamin C, vitamin E and carotenoids,

there are also studies which suggest that one should not take large supplemental

doses.

Antioxidants may possibly reduce the risks of cancer and age-related macular

degeneration (AMD). Antioxidants clearly slow the progression of AMD.

The cellular protection against the deleterious effects of reactive oxidants generated in

aerobic metabolism, called oxidative stress, is organized at multiple levels. The

defense strategies include three levels of protection: prevention, interception, and

- 24 -

repair. Regulation of the antioxidant capacity includes the maintenance of adequate

levels of antioxidant and the localization of antioxidant compounds and enzymes.

Short-term and long-term adaptation and cell specialization in these functions are new

areas of interest. Control over the activity of pro-oxidant enzymes, such as NADPH

oxidase and NO synthases, is crucial. Synthetic antioxidants mimic biological

strategies.

Free radical production occurs continuously in all cells as part of normal cellular

function. However, excess free radical production originating from endogenous or

exogenous sources might play a role in many diseases. Antioxidants prevent free

radical induced tissue damage by preventing the formation of radicals, scavenging

them, or by promoting their decomposition.

In one research, the antioxidant activity of pomegranate juices was evaluated.

Four different methods (ABTS, DPPH, DMPD, and FRAP) were used.

ABTS (2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) is chemical compound

used to observe the reaction kinetics of specific enzymes. A common use for it is in

the enzyme-linked immunosorbent assay (ELISA) to detect for binding of molecules

to each other.

DPPH (1,1-diphenyl-2-picrylhydrazyl) is used for free radical scavenging assays.

DMPD/TMPD are effective electron-transfer mediators for an enzyme.

FRAP ferric reducing antioxidant power.

The antioxidant activity of the pomegranate juices were compared to those of red

wine and a green tea infusion. Commercial pomegranate juices showed an antioxidant

- 25 -

activity (18-20 TEAC) {Teac = trolox equivalent antioxidant capacity} three times

higher than those of red wine and green tea (6-8 TEAC). The activity was higher in

commercial juices extracted from whole pomegranates than in experimental juices

obtained from the arils only (12-14 TEAC). HPLC-DAD and HPLC-MS analyses of

the juices revealed that commercial juices contained the pomegranate tannin

punicalagin (1500-1900 mg/L) while only traces of this compound were detected in

the experimental juice obtained from arils in the laboratory. This shows that

pomegranate industrial processing extracts some of the hydrolyzable tannins present

in the fruit rind. This could account for the higher antioxidant activity of commercial

juices compared to the experimental ones. In addition, anthocyanins, ellagic acid

derivatives, and hydrolyzable tannins were detected and quantified in the

pomegranate juices.

Phenolics

Phenolics are plant compounds (structurally characterized by an alcohol group on an

aromatic ring) that impart a variety of functions to plants, including defence

mechanisms and interactions with other organisms. Phenolics can also determine

plant properties such as flavour and palatability. They naturally exist in grape stems,

skins, seeds, juice, and pulp.

Phenols, sometimes called phenolics, are a class of chemical compounds consisting of

a hydroxyl group (- O H) attached to an aromatic hydrocarbon group. The simplest of

- 26 -

the class is phenol (C6H5OH). Phenols are similar to alcohol, but they have unique

properties and relatively higher acidities due to the aromatic ring's tight coupling with

the oxygen and a relatively loose bond between the oxygen and hydrogen. Some

phenols are germicidal and are used in formulating disinfectants. Others possess

estrogenic or endocrine disrupting activity.

Phenolic compounds represent the most studied phytochemicals and have been widely

exploited as model systems in different areas of plant research. The research is still

active due to the complexity of the structures and the biosynthetic pathways.

Example: the nature and functions of enzymes involved in lignin synthesis have been

revisited several times, even in recent years. More recently, molecular biology and

genomics have provided additional understanding of the mechanisms underlying the

synthesis of these compounds with special emphasis on the regulation of gene

expression by environmental factors. The extensive characterization of genes

encoding the different enzymatic steps of flavonoid synthesis and cytochrome P450

genes have been among the most recent advances in this area. Metabolic engineering

of lignins and flavonoids has been deeply investigated. These studies have revealed a

substantial and sometimes unexpected network of regulatory interactions. In the

present time, the demand and an increasing interest for practical applications has

stimulated a wide range of biological and epidemiological studies aiming at

characterizing the health promoting properties of specific phenolic compounds with

antioxidant activities towards cancer, cardiovascular and neurodegenerative diseases

or for use in anti-aging or cosmetic products.

- 27 -

TANNINS

Since ancient times it is known that certain organic substances have tanning properties

and are able to tan animal skins to form leather. Prehistoric tribes already knew about

the tanning of protective animal hides with brain material and the fat of the killed

animals. However, precisely what happens to the skin during the tanning process was

only elucidated during the twentieth century with the help of modern analytical

techniques. Real tanning is understood as the cross-linking of the skin’s collagen

chains, while false tanning entails the filling of hollow spaces between the skin’s

collagen chains.

In medicine, especially in Asian (Japanese and Chinese) natural healing, the tannin-

containing plant extracts are used as astringents, against diarrhoea, as diuretics,

against stomach and duodenal tumours, and as anti-inflammatory, antiseptic, and

haemostatic pharmaceuticals. As tannins can precipitate heavy metals and alkaloids

(except morphine), they can be used in poisonings with these substances. It is also

becoming clear that tannins often are the active principles of plant-based medicines.

Tannins are used in the dyestuff industry as caustics for cationic dyes (tannin dyes),

and also in the production of inks (iron gallate ink). In the food industry tannins are

used to clarify wine, beer, and fruit juices. Other industrial uses of tannins include

textile dyes, as antioxidants in the fruit juice, beer, and wine industries, and as

coagulants in rubber production.

Recently the tannins have attracted scientific interest, especially due to the increased

incidence of deadly illnesses such as AIDS and various cancers. The search for new

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compounds for the development of novel pharmaceuticals has become increasingly

important, especially as the biological action of tannin-containing plant extracts has

been well documented.

During the last twenty years many representatives of this class of compounds have

been isolated and characterized. Currently known tannins with unambiguously

determined structures already number far more than 1000 natural products. In

extensive biological tests many representatives of this class were found to have

antiviral, antibacterial, and, especially, anti-tumour activity. For example, certain

tannins can selectively inhibit HIV replication. Tannins are polyphenolic secondary

metabolites of higher plants. Corresponding polyphenolic natural products have not

yet been isolated from lower plants such as algae, or from the animal kingdom. The

polyphenolic structure of the secondary metabolites from higher plants is a necessary

but not sufficient requirement for membership of the tannin class.

(Gallotannins, Ellagitannins, Complex Tannins, Condensed Tannins).

- 29 -

Punicalagin

Punicalagin, the main ingredient of pomegranate (Punica granatum L.) husk, is a high

molecular weight polyphenolic compound. It has shown remarkable pharmacological

activities attributed in the presence of dissociable OH groups. To isolate punicalagin,

previous methods included labour intensive and expensive solid phase extractions by

column chromatography (C-18, polyamides, dellulose, Sephadex Lipophilic LH-20,

Diaion HP20). High-speed countercurrent chromatography (HSCCC) was used for

isolation and purification of punicalagin from pomegranate husk. Using preparative

HSCCC about a 350 mg amount of the crude extract was separated, yielding 105 mg

of punicalagin at a high-purity of over 92%. Eighty milligrams of gallic acid was

simultaneously separated as another product, at a purity of 75%. Punicalagin (PCG)

- 30 -

isolated from the fruit of Punica granatum was identified as a potent immune

suppressant, based on its inhibitory action on the activation of the nuclear factor of

activated T cells (NFAT). PCG downregulated the mRNA and soluble protein

expression of interleukin-2 from anti-CD3/anti-CD28-stimulated murine splenic

CD4+ T cells and suppressed mixed leukocytes reaction (MLR) without exhibiting

cytotoxicity to the cells.

The protective bioactivity of punicalagin, a high molecular weight polyphenol

isolated from pomegranate fruit pith and carpellary membrane, against oxidative

damages to lipids, amino acids constituting the proteins, and guanosine as a model for

DNA has been investigated.

Ellagic Acid

(Benzoaric acid, eleagic acid, elagostasine, gallogen)

Ellagic acid is a fused four-ring polyphenol. Pure ellagic acid is a cream to light

yellow crystalline solid.

It is present in many red fruits and berries, including raspberries, strawberries,

blackberries, cranberries, pomegranate and some nuts including pecans and walnuts.

Ellagic acid prevents the destruction of P53 gene by cancer cells. It can bind with

cancer causing molecules, thereby making them inactive. In their study “The effects

of dietary ellagic acid on rat hepatic and oesophageal mucosal cytochromes P450 and

- 31 -

phase II enzymes.” Ahn et al showed that ellagic acid causes a decrease in total

hepatic mucosal cytochromes and an increase in some hepatic phase II enzyme

activities, thereby enhancing the ability of the target tissues to detoxify the reactive

intermediates. Ellagic acid showed also a chemo-protective effect against various

chemically induced cancers.

Ellagic acid has also antiviral and antibacterial activities.

n 1996, a Nottingham, PA University research team learned that pomegranate extract

could destroy several viruses nearly on contact. The discovery of this anti-viral

activity instigated further experimentation and clinical trials. One study confirmed

that Ellagic acid effectively protects cells from damaging free radicals. Additional

phenolic compounds found in pomegranate known as anthocynadins (also well known

scavengers of free radicals) combine synergistically with Ellagic acid to greatly

augment pomegranate's potency as an antioxidant. Initial experimentation by Stoner

and Mukhtar showed that Ellagic acid decreased the number of chemically induced

lung tumours. Mukhtar further illustrated that topical application of Ellagic acid

provided protection against chemically induced skin tumours.

Ellagic acid has several mechanisms of actions by which it exhibits its chemo-

preventive properties. Research by Barch et al demonstrated that Ellagic acid could

actually bind to DNA, thus preventing its carcinogenic alteration. Additionally,

ellagic acid has been shown to induce the production of phase 11 detoxification

enzymes through its manipulation of gene expression. With an increased

concentration of these enzymes, various tissues ability to detoxify harmful

- 32 -

compounds is augmented. Finally, Ellagic acid was found to be a potent inhibitor of

tyrosine protein kinase, a molecule whose activity has been associated with the ability

of certain viruses to transform normal cells into cancerous cells.

Pelagonidin, Cyanidin

Pelargonidin and Cyanidin are both types of anthocyanidins. These are antioxidants

that have been found to help improve blood vessel function in humans and animals.

Anthocyanidins are found in blue, purple and red fruits and vegetables such as:

blueberries

blackberries

plums

cranberries

raspberries

strawberries

pomegranate

Anthocyanidins are found in the cell's cell sap (unlike chlorophyll and carotene that

are attached to cell membranes). The colour of the pigment is altered by the pH of the

cell sap. More acidic cell sap gives a red colour. Less acidic cell sap express a more

- 33 -

purple colour. Anthocyanins are used in processed foods (drinks and confectionery) to

avoid using synthetic additives.

Gallic Acid (3,4,5-Trihydroxybenzoic acid)

… is a free molecule or occurs a part of the tannin molecule. It has anti-fungal and

anti-viral properties and shows cytotoxicity against cancer cells without harming

healthy cells. Gallic acid acts as a antioxidant and helps to protect our cells against

oxidative damage.

It also inhibits histamine release and pro-inflammatory cytokine production in mast

cells and can therefore be of importance in future treatment for allergies.

Quercetin

… is also a powerful anti-histamine. It is a bioflavonoid that gives pigment to many

plants and herbs. The substance has been shown to exert anti-inflammatory,

antihistamine, antioxidant, and anticancer properties. In a Japanese study published in

the Journal of Allergy and Clinical Immunology, quercetin was effective against

symptom-causing histamine activity in mast cells. The study found that histamine

release was reduced 46 to 96 percent by the nutraceutical. Additional studies have

found high intakes quercetin (and of course other flavonoids) lower the risk of certain

respiratory diseases such as asthma, bronchitis, and emphysema.

- 34 -

POMEGRANATE AND CANCER

There is currently a shifting focus towards finding natural compounds that may

prevent or treat cancer, due to the problems that exist with current chemotherapeutic

regimens. The fruit of the pomegranate contains hundreds of phytochemicals and

pomegranate has been shown to exhibit antioxidant properties.

Cancer develops when the balance between cell proliferation and cell death is

disrupted, and the ensuing aberrant proliferation leads to tumour growth.

It is important to develop non-cytotoxic therapies for solid malignancies such as

prostate and breast cancer.

Flavonoids are a group of differentiation-inducing chemicals with a potentially lower

toxicology profile than retinoids. Flavonoid-rich polyphenol fractions from the

pomegranate fruit exert anti-proliferative, anti-invasive, anti-eicosanoid, and pro-

apoptotic actions in breast and prostate cancer cells and anti-angiogenic activities in

vitro and in vivo.

Pomegranate juice (PJ) and its ellagitannins inhibited proliferation and induced

apoptosis in HT-29 colon cancer cells. One study examined the effects of PJ on

inflammatory cell signaling proteins in the HT-29 human colon cancer cell line. At a

concentration of 50 mg/L PJ significantly suppressed TNFalpha-induced COX-2

protein expression by 79% (SE = 0.042), total pomegranate tannin extract (TPT) 55%

- 35 -

(SE = 0.049), and punicalagin 48% (SE = 0.022). Additionally, PJ reduced

phosphorylation of the p65 subunit and binding to the NFkappaB response element

6.4-fold. TPT suppressed NFkappaB binding 10-fold, punicalagin 3.6-fold, whereas

ellagic acid (EA) (another pomegranate polyphenol) was ineffective. PJ also

abolished TNFalpha-induced AKT activation, needed for NFkappaB activity.

Therefore, the polyphenolic phytochemicals in the pomegranate can play an important

role in the modulation of inflammatory cell signalling in colon cancer cells.

Prostate Cancer (CaP) is one of the leading causes of cancer-related deaths among

males in most Western countries. CaP is an ideal candidate disease for

chemoprevention, because it is typically diagnosed in men over 50 years of age, and

thus even a modest delay in disease progression - achieved through pharmacological

or nutritional intervention - could significantly impact the quality of life of these

patients.

Dietary antioxidants are proven chemo-preventative agents for CaP patients.

Pomegranates possess strong antioxidant and anti-inflammatory agents. Pomegranate

fruit extracts (PFE) inhibits the cell-growth and induces apoptosis of the highly

aggressive human prostate carcinoma PC3 cells, which are cancer cells derived from a

bone marrow metastasis. This is done through modulations in the cyclin kinase

inhibitor/cyclin kinase dependent machinery.

The cyclin dependent kinase (cdk) is a family of kinases that - once activated by

cyclin - regulate the cell cycle by adding phosphate groups to a variety of protein

substrates that control processes in the cycle. CDKs are considered a potential target

- 36 -

for anti-cancer medication. If it is possible to selectively interrupt the cell cycle-

regulation in cancer cells by interfering with CDK action, the cell will die. Much

more research is necessary, because the disruption of the CDK pathways can also lead

to serious consequences/side-effects.

The answers maybe found in more intensified studies of active ingredients in natural

products. Shifting research towards the effects for the use of natural products like

active ingredients in pomegranate, which inhibit cell growth and induce apoptosis,

before developing new drugs, is one way forward.

The event of modulation of CDKs is associated with alterations of Bax and Bcl-2 –

shifting the Bax:Bcl-2 ratio in favour of apoptosis.

The biochemical mechanism of apoptosis, or programmed cell death (PCD), is an area

of extensive study because of the importance of maintaining the homeostatic balance

in response to pro- or anti-apoptotic stimuli. The balance between cell proliferation

and apoptosis is crucial for the healthy functioning of organisms. Dysregulation of

apoptosis is implicated in many degenerative and autoimmune diseases, including

cancer, acquired immune deficiency syndrome, neurodegenerative disorders, and viral

and bacterial infections. The mitochondrial apoptotic pathway is largely mediated

through Bcl-2 family proteins, which include both pro-apoptotic members such as

Bax, Bak, and BNIP3 that promote mitochondrial permeability, and anti-apoptotic

members such as Bcl-2 and Bcl-xL that inhibit their effects, or inhibit the

mitochondrial release of cyt c .

- 37 -

Another important component is the tumor suppressor protein p53, which

simultaneously suppresses Bcl-2 and activates Bax. Cyt c leakage supports the

formation of an apoptosome complex by binding to apoptotic protease activating

factor-1 (Apaf-1), which activates the caspase-9 molecules (upon cleavage of the

bound zymogen procaspases-9), which in turn activate caspase-3. Caspase-3 cleaves

the inhibitor of caspase activated DNase (ICAD), leading to DNA degradation or

fragmentation, whereas the inhibitor of apoptosis (IAP) inhibits both caspase-3 and

caspase-9 activities.

The cyclin-dependent kinase inhibitor p21 is induced by both p53-dependent and -

independent mechanisms following stress, and induction of p21 may cause cell cycle

arrest. As a proliferation inhibitor, p21 is poised to play an important role in

preventing tumour development. a number of recent studies have pointed out that in

addition to being an inhibitor of cell proliferation, p21 acts as an inhibitor of apoptosis

in a number of systems, and this may counteract its tumor-suppressive functions as a

growth inhibitor.

P21 is often responsible for stress-induced p53-dependent and p53-independent cell

cycle arrest. Cell cycle arrest permits cells to pause and to repair damage and then to

continue cell division. On one hand, the function of p21 to inhibit cell proliferation

may contribute to its ability to act as tumor suppressor. On the other hand, the

capacity of p21 to induce cell cycle arrest after stress can protect cells from stress-

induced apoptosis. Anti-apoptotic activity of p21 may contribute to its potential to act

as an oncogene.

- 38 -

Anticancer drugs kill cancer cells by inducing p53-dependent and p53-independent

apoptosis, and p21 protects cells from anticancer drug-induced apoptosis. Because

loss of p21 usually increases sensitivity of tumor cells to apoptosis induced by

different chemotherapeutic agents, small molecules that eliminate p21 expression may

improve the action of anticancer drugs. Therefore, functional p21 may suppress

tumour growth in the organism, but at the same time elimination of p21 may be

beneficial during chemotherapy.

The role of natural products/anti-oxidants of influencing/changing the activity of p53

and p21 should be studied intensively.

Here some examples of studies and the results: (detailed information regarding the

studies please see reference list)

A)

Four pure chemicals, ellagic acid (E), caffeic acid (C), luteolin (L) and punicic acid

(P), all important components of the aqueous compartments or oily compartment of

pomegranate fruit, and each belonging to different representative chemical classes and

showing known anticancer activities, were tested as potential inhibitors of in vitro

invasion of human PC-3 prostate cancer cells in an assay employing Matrigel artificial

membranes. All compounds significantly inhibited invasion when employed

individually. When C, P, and L were equally combined at the same gross dosage (4

microg/ml) as when the compounds were tested individually, a supradditive inhibition

- 39 -

of invasion was observed, measured by the Kruskal-Wallis non-parametric test. This

test compares three or more unpaired groups. To perform the Kruskal-Wallis test,

Prism first ranks all the values from low to high, disregarding which group each value

belongs. If two values are the same, then they both get the average of the two ranks

for which they tie. The smallest number gets a rank of 1. The largest number gets a

rank of N, where N is the total number of values in all the groups. Prism then sums

the ranks in each group, and reports the sums. If the sums of the ranks are very

different, the P value will be small.

The discrepancies among the rank sums are combined to create a single value called

the Kruskal-Wallis statistic (some books refer to this value as H). A larger Kruskal-

Wallis statistic corresponds to a larger discrepancy among rank sums.

The P value answers this question: If the populations really have the same median,

what is the chance that random sampling would result in sums of ranks as far apart (or

more so) as observed in this experiment? More precisely, if the null hypothesis is true

then what is the chance of obtaining a Kruskal-Wallis statistic as high (or higher) as

observed in this experiment.

If your samples are small and no two values are identical (no ties), Prism calculates an

exact P value. If your samples are large or if there are ties, it approximates the P value

from the chi-square distribution. The approximation is quite accurate with large

samples. With medium size samples, Prism can take a long time to calculate the exact

P value. While it does the calculations, Prism displays a progress dialog and you can

- 40 -

press Cancel to interrupt the calculations if an approximate P value is good enough for

your purposes.

The four chemicals - ellagic acid (E), caffeic acid (C), luteolin (L) and punicic acid

(P), - on its own showed important anti-cancer activities, but the combination – the

synergism of all four - increased the activity even more.

B)

Another study tested flavonoid-rich fractions from fresh (J) and fermented (W)

pomegranate juice and from an aqueous extraction of pomegranate pericarps (P) <the

fruit wall> as potential differentiation-promoting agents of human HL-60

promyelocytic leukaemia cells. Four assays were used to assess differentiation: nitro

blue tetrazolium reducing activity, nonspecific esterase activity, specific esterase

activity, and phagocytic activity. In addition, the effect of these extracts on HL-60

proliferation was evaluated. Extracts W and P were strong promoters of

differentiation in all settings, with extract J showing only a relatively mild

differentiation-promoting effect. The extracts had proportional inhibitory effects on

HL-60 cell proliferation. The results highlight an important, previously unknown,

mechanism of the cancer preventive and suppressive potential of pomegranate (here:

fermented juice and pericarp.

C)

Another study showed the inhibition of the proliferation of LNCaP, PC-3, and DU

145 human cancer cell lines. In this research scientists focussed on the quantities of

- 41 -

used active ingredients to induce apoptosis. Overall, this study demonstrates

significant anti-tumour activity of pomegranate-derived materials against human

prostate cancer.

D)

Developing novel mechanism-based chemo-preventive approaches for lung cancer

through the use of dietary substances which humans can accept has become an

important goal. In one study, employing normal human bronchial epithelial cells

(NHBE) and human lung carcinoma A549 cells, scientists first compared the growth

inhibitory effects of pomegranate fruit extract (PFE). Treatment of PFE (50-150

microg/ml) for 72 h was found to result in a decrease in the viability of A549 cells but

had only minimal effects on NHBE cells as assessed by the MTT and Trypan blue

assays. PFE treatment of A549 cells also resulted in dose-dependent arrest of cells in

G0-G1 phase of the cell cycle (as assessed by DNA cell cycle analysis). Researchers

further found that PFE treatment also resulted in (i) induction of WAF1/p21 and

KIP1/p27, (ii) decrease in the protein expressions of cyclins D1, D2 and E, and (iii)

decrease in cyclin-dependent kinase (cdk) 2, cdk4 and cdk6 expression. The treatment

of cells with PFE inhibited (i) phosphorylation of MAPK proteins, (ii) inhibition of

PI3K, (iii) phosphorylation of Akt at Thr308, (iv) NF-kappaB and IKKalpha, (v)

degradation and phosphorylation of IkappaBalpha, and (vi) Ki-67 and PCNA. The

scientists also found that PFE treatment to A549 cells resulted in inhibition of NF-

kappaB DNA-binding activity. Oral administration of PFE (0.1 and 0.2%, wt/vol) to

- 42 -

athymic nude mice implanted with A549 cells resulted in a significant inhibition in

tumour growth. The results provide a suggestion that PFE can be a useful

chemopreventive/chemotherapeutic agent against human lung cancer.

E)

One study investigated whether dissimilar biochemical fractions originating in

anatomically discrete sections of the pomegranate fruit might act synergistically

against proliferation, metastatic potential, and phosholipase A2 (PLA2) expression of

human prostate cancer cells. Proliferation of DU 145 human prostate cancer cells was

measured following treatment with a range of therapeutically active doses of

pomegranate, fermented pomegranate juice polyphenols (W) and sub-therapeutic

doses of either pomegranate pericarp (peel) polyphenols (P) or pomegranate seed oil

(Oil). Supradditive, complementary and synergistic effects were proven in all models

(again) by the Kruskal-Wallis non-parametric H test at p < 0.001 for the proliferation

tests, p < 0.01 for invasion, and p < 0.05 for PLA2 expression.

The results suggest vertical as well as the usual horizontal strategies for discovering

pharmacological actives in plants.

F)

Another study focussed on the interaction/anti-proliferative activities of caffeic acid,

3,4-dihydroxyphenylacetic acid (PAA), syringic acid, sinapic acid, protocatechuic

acid and ferulic acid on the human breast cancer T47D cell line, at concentrations

more or less similar to those expected from normal consumption of foods. Again, the

- 43 -

results indicate that phenolic acids produce growth inhibition of cancer cells, in vitro,

indicating an additional protective effect on hormone-dependent breast tumours.

G)

In another study, three components of the pomegranate were used to study the effects

on breast cancer: fermented juice, aqueous pericarp extract and cold-pressed or

supercritical CO2-extracted seed oil.

The activities of these ingredients, and of the crude whole oil and crude fermented

and unfermented juice concentrate, were assessed in vitro for possible chemo-

preventive or adjuvant therapeutic potential in human breast cancer.

The ability to affect a blockade of endogenous active oestrogen biosynthesis was

shown by polyphenols from fermented juice, pericarp, and oil, which inhibited

aromatase activity by 60-80%. F (aromatase are a group of enzymes of the

cytochrome P450 superfamily, whose function is to aromatize androgens (that is, to

selectively increase their aromaticity), producing estrogens.) As such, it is an

important factor in sexual development.)

Fermented juice and pericarp polyphenols, and whole seed oil, inhibited 17-beta-

hydroxysteroid dehydrogenase Type 1 from 34 to 79%, at concentrations ranging

from 100 to 1,000 microg/ml according to seed oil.

- 44 -

In a yeast oestrogen screen (YES) lyophilized fresh pomegranate juice effected a 55%

inhibition of the estrogenic activity of 17-beta-estradiol; whereas the lyophilized juice

by itself displayed only minimal estrogenic action.

Inhibition of cell lines by fermented juice and pericarp polyphenols was according to

oestrogen-dependent (MCF-7) > estrogen-independent (MB-MDA-231) > normal

human breast epithelial cells (MCF-10A). In both MCF-7 and MB-MDA-231 cells,

fermented pomegranate juice polyphenols consistently showed about twice the anti-

proliferative effect as fresh pomegranate juice polyphenols. Pomegranate seed oil

effected 90% inhibition of proliferation of MCF-7 at 100 microg/ml medium, 75%

inhibition of invasion of MCF-7 across a Matrigel membrane at 10 microg/ml, and

54% apoptosis in MDA-MB-435 oestrogen receptor negative metastatic human breast

cancer cells at 50 microg/ml. In a murine mammary gland organ culture, fermented

juice polyphenols effected 47% inhibition of cancerous lesion formation induced by

the carcinogen 7,12-dimethylbenz[a]anthracene (DMBA). Although a higher

inhibition through seed oil and fermented juice was proven, the fresh juice activities

were also high enough to focus in further studies on the chemo-preventive and

therapeutic use of pomegranate juice in human breast cancer.

- 45 -

POMEGRANATE AND ATHEROSCLEROSIS

(… and implications re. stroke, heart attack, diabetes, high systolic/diastolic blood

pressure)

Atherosclerosis (or coronary artery disease) occurs, when the normal lining of the

arteries deteriorates the walls of the arteries thicken/harden, and fatty substances,

cholesterol, cellular waste products and other clotting materials like calcium and

fibrin is building up in the inner lining of an artery.

The antiatherogenic activity of pomegranate juice has been attributed to its

antioxidant polyphenols. The most potent in vitro antioxidant polyphenol from this

juice is the ellagitannin punicalagin. Its bioavailability and metabolism was studied to

assess the effect on several blood parameters (including cardiovascular risk disease

markers) and to compare the antioxidant activity of punicalagin with that of the in

vivo generated metabolites.

In one experiment, six healthy subjects (four men and two women) consumed 1 L of

pomegranate juice daily (5.58 g/L polyphenols, including 4.37 g/L punicalagin

isomers) for 5 days. The polyphenols and the in vivo generated metabolites were

measured by HPLC-DAD-MS-MS. Fourteen haematological and twenty

serobiochemical parameters including LDL, HDL and VLDL as well as cholesterol

and triglycerides in each lipoprotein were evaluated. In vitro antioxidant activity of

plasma (ABTS and FRAP assays) and urine (ABTS and DPPH) were determined.

- 46 -

RESULTS: Neither punicalagin nor ellagic acid present in the juice were detected in

both plasma and urine. Three microbial ellagitannin-derived metabolites were

detected: 3,8-dihydroxy-6H-dibenzo[b,d]pyran-6-one glucuronide, an unidentified

aglycone (tentatively, trihydroxy-6H-dibenzo[b,d]pyran-6-one) and hydroxy-6-H-

dibenzo[b,d]pyran-6-one glucuronide. These metabolites could reach up to 18.6

microM in plasma, although a large inter-individual variability was observed. In

urine, the same metabolites and their corresponding aglycones became evident after 1

day of juice consumption. Total urine excretion of metabolites ranged from 0.7 to

52.7% regarding the ingested punicalagin. No relevant effect was observed on any

blood parameter. The metabolites did not show significant antioxidant activity

compared to punicalagin from pomegranate juice.

Although other studies have clearly shown the anti-oxidant activity of the active

ingredients in pomegranate juice, this study shows that the potential systemic

biological effects of pomegranate juice ingestion should be attributed to the colonic

microflora metabolites rather than to the polyphenols present in the juice (more in

chapter ‘microbes’.)

Polyphenolic antioxidants are associated with the inhibition of low density

lipoproteins (LDL, the so called bad cholesterol), the macrophage foam cell formation

and attenuation of atherosclerosis development. One study investigated ‘the effects of

pomegranate juice (PJ, which contains potent tannins and anthocyanins) consumption

by atherosclerotic patients with carotid artery stenosis (CAS) on the progression of

- 47 -

carotid lesions and changes in oxidative stress and blood pressure. Ten patients were

supplemented with PJ for 1 year and five of them continued for up to 3 years. Blood

samples were collected before treatment and during PJ consumption. In the control

group that did not consume PJ, common carotid intima-media thickness (IMT)

increased by 9% during 1 year, whereas, PJ consumption resulted in a significant IMT

reduction, by up to 30%, after 1 year. The patients' serum paraoxonase 1 (PON 1)

activity was increased by 83%, whereas serum LDL basal oxidative state and LDL

susceptibility to copper ion-induced oxidation were both significantly reduced, by

90% and 59%, respectively, after 12 months of PJ consumption, compared to values

obtained before PJ consumption. Furthermore, serum levels of antibodies against

oxidized LDL were decreased by 19%, and in parallel serum total antioxidant status

(TAS) was increased by 130% after 1 year of PJ consumption. Systolic blood pressure

was reduced after 1 year of PJ consumption by 21% and was not further reduced

along 3 years of PJ consumption. For all studied parameters, the maximal effects were

observed after 1 year of PJ consumption. Further consumption of PJ, for up to 3 years,

had no additional beneficial effects on IMT and serum PON1 activity, whereas serum

lipid peroxidation was further reduced by up to 16% after 3 years of PJ consumption.

The results of the present study thus suggest that PJ consumption by patients with

CAS decreases carotid IMT and systolic blood pressure and these effects could be

related to the potent antioxidant characteristics of PJ polyphenols.

One study investigated whether daily consumption of pomegranate juice for 3 months

would affect myocardial perfusion in 45 patients who had Coronary Heart Disease

- 48 -

and myocardial ischemia in a randomized, placebo-controlled, double-blind study.

Patients were randomly assigned into 1 of 2 groups: a pomegranate juice group (240

ml/day) or a placebo group that drank a beverage of similar caloric content, amount,

flavour, and color. Participants underwent electrocardiographic-gated myocardial

perfusion single-photon emission computed tomographic technetium-99m tetrofosmin

scintigraphy at rest and during stress at baseline and 3 months. Visual scoring of

images using standardized segmentation and nomenclature (17 segments, scale 0 to 4)

was performed by a blinded independent nuclear cardiologist. To assess the amount of

inducible ischemia, the summed difference score (SDS) was calculated by subtracting

the summed score at rest from the summed stress score. The experimental and control

groups showed similar levels of stress-induced ischemia (SDS) at baseline (p >0.05).

After 3 months, the extent of stress-induced ischemia decreased in the pomegranate

group (SDS -0.8 +/- 2.7) but increased in the control group (SDS 1.2 +/- 3.1, p <0.05).

This benefit was observed without changes in cardiac medications, blood sugar,

hemoglobin A1c, weight, or blood pressure in either group. In conclusion, daily

consumption of pomegranate juice may improve stress-induced myocardial ischemia

in patients who have Coronary Heart Disease.

Diabetes is associated with increased oxidative stress and atherosclerosis

development. One study investigated the effects of pomegranate juice (PJ; which

- 49 -

contains sugars (!) and potent anti-oxidants) consumption by diabetic patients on

blood diabetic parameters, and on oxidative stress in their serum and macrophages.

Ten healthy test-persons (controls) and 10 non-insulin dependent diabetes mellitus

(NIDDM) patients who consumed PJ (50ml per day for 3 months) participated in the

study. In the patients versus controls serum levels of lipid peroxides and thiobarbituric

acid reactive substances (TBARS) were both increased, by 350% and 51%,

respectively, whereas serum SH groups content and paraoxonase 1 (PON1) activity,

were both decreased (by 23%). PJ consumption did not affect serum glucose,

cholesterol and triglyceride levels, but it resulted in a significant reduction in serum

lipid peroxides and TBARS levels by 56% and 28%, whereas serum SH groups and

PON1 activity significantly increased by 12% and 24%, respectively. In the patients

versus controls monocytes-derived macrophages (HMDM), the researchers observed

increased level of cellular peroxides (by 36%), and decreased glutathione content (by

64%). PJ consumption significantly reduced cellular peroxides (by 71%), and

increased glutathione levels (by 141%) in the patients' HMDM. The patients' versus

control HMDM took up oxidized LDL (Ox-LDL) at enhanced rate (by 37%) and PJ

consumption significantly decreased the extent of Ox-LDL cellular uptake (by 39%).

It was concluded that PJ consumption by diabetic patients did not worsen the diabetic

parameters, but rather resulted in anti-oxidative effects on serum and macrophages,

which could contribute to attenuation of atherosclerosis development in these patients.

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Paraoxonase 2 (PON2), a member of the paraoxonase gene family, was shown to

protect macrophages against oxidative stress. . Pomegranate juice (PJ), which

contains potent polyphenol anti-oxidants, exhibits similar effects.

{(Additionally to PON2, PON1 and PON3 also hydrolyze and thereby inactivate N-

acyl-homoserine lactones, which are quorum-sensing signals of pathogenic bacteria

(more in chapter Microbes)}.

One study showed the association of pomegranate juice polyphenolics, macrophage

oxidative stress, and cellular PON2 expression, in relation to the activation of specific

PON2 transcription factors. Incubation of J774A.1 macrophages with PJ (0-50

microM of total polyphenols) dose-dependently increased expression (mRNA,

protein) and activity and reduced macrophage oxidative status. These effects could be

attributed to the PJ unique polyphenols, punicalagin and gallic acid. PJ polyphenol-

induced up-regulation of PON2 was inhibited by 40% upon using the PPAR gamma

inhibitor GW9662 (50 microM). Accordingly, the PPAR gamma ligand, rosiglitazone,

dose-dependently stimulated macrophage PON2 expression, by up to 80%. Inhibition

of AP-1 activation with SP600125, attenuated PJ-induced up-regulation of PON2 by

40%. Similarly, incubation of macrophages with PJ polyphenols in the presence of

GW9662 or SP600125, significantly reduced their capacity to protect the cells against

oxidative stress. It can be concluded that the anti-oxidative characteristics of PJ

unique phenolics punicalagin and gallic acid could be related, at least in part, to their

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stimulatory effect on macrophage PON2 expression, a phenomenon which was shown

to be associated with activation of the transcription factors PAPR gamma and AP-1.

The anti-atherosclerotic properties in pomegranate juice showed in one study a clear

reduction in blood pressure. The effects of pomegranate juice consumption (50 ml,

1.5mmol of total polyphenols per day, for 2 weeks) by hypertensive patients on their

blood pressure and on serum angiotensin converting enzyme (ACE) activity were

tested in this research. A 36% decrement in serum ACE activity and a 5% reduction in

systolic blood pressure were noted. Similar dose-dependent inhibitory effect (31%) of

pomegranate juice on serum ACE activity was observed also in vitro. As reduction in

serum ACE activity, even with no decrement in blood pressure, was previously shown

to attenuate atherosclerosis, pomegranate juice can offer a wide protection against

cardiovascular diseases which could be related to its inhibitory effect on oxidative

stress and on serum ACE activity.

Pomegranate juice (PJ) can also revert the potent downregulation of the expression of

endothelial nitric-oxide synthase (NOSIII) induced by oxidized low-density liporotein

(oxLDL) in human coronary endothelial cells. analyses showed a significant decrease

of NOSIII expression after a 24-h treatment with oxLDL. A significant dose-

dependent reduction in nitric oxide bioactivity represented by both basal and

bradykinin-stimulated cellular cGMP accumulation was observed. These phenomena

were corrected significantly by the concomitant treatment with PJ. The data of this

study, done by the Department of General Pathology and Excellence Research Center

- 52 -

on Cardiovascular Disease, University of Naples, Italy, suggest that PJ can exert

beneficial effects on the evolution of clinical vascular complications, coronary heart

disease, and atherogenesis in humans by enhancing the NOSIII bioactivity.

A study done by Department of Human Nutrition, National Nutrition and Food

Technology Research Institute, Tehran, Iran was undertaken to assess the effect of

concentrated pomegranate juice consumption on lipid profiles of type II diabetic

patients with hyperlipidemia (total cholesterol or triglycerides > or = 200 mg/dL). In

this pilot study 22 diabetic patients were recruited from the Iranian Diabetes Society.

They were free of any other chronic diseases. The patients were followed for eight

weeks to obtain more detailed data about their diet before concentrated pomegranate

juice (CPJ) consumption period began. In this pre-study period a 24-hour food recall

and a food record (containing flavonoid-rich foodstuffs) were completed every ten

days. At the end of the eighth week, anthropometric and biochemical assessments

were done. Thereafter the patients consumed 40 g CPJ for eight weeks. During this

period, dietary assessment was continued. After completion of the study

anthropometric and blood indices were evaluated again. The Wilcoxon signed-rank

test was used for statistical analysis. P-value was considered significant at p < 0.05.

There were 14 women (63.6%) and 8 men (36.4%) in this survey. Mean (+/- SD) of

age, weight, and duration of diabetes were 52.5 (+/- 5.2) years, 71.5 (+/- 10.3) kg, and

7.9 (+/- 6.6) years, respectively. After consumption of concentrated pomegranate juice

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significant reductions were seen in total cholesterol (p < 0.006), low-density

lipoprotein-cholesterol (LDL-c) (p < 0.006), LDL-c/high-density lipoprotein-

cholesterol (HDL-c) (p < 0.001), and total cholesterol/HDL-c (p < 0.001). However

there were no significant changes in serum triacylglycerol and HDL-c concentrations.

Anthropometric indices, physical activity level, types and doses of oral hypoglycemic

agents, and the intake of nutrients and flavonoid-rich foodstuffs did not change during

the CPJ consumption period. It is concluded that CPJ consumption could modify heart

disease risk factors in these hyperlipidemic patients. Therefore, its inclusion in their

diets may be beneficial.

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CHRONIC OBSTRUCTIVE PULMONARY DISEASE (COPD)

… also known as Chronic Obstructive Airway Disease is a pathological limitation of

airflow in the airway, which is irreversible. It includes chronic bronchitis,

emphysema, and different other lung disorders. It can include ‘classical’ asthma, but

more often it is an asthma-related breathing problem. The decrease in the expiratory

flow rates leads to an increase in the total lung capacity. Patients with this condition –

triggered by smoking or other airborne irritants such as asbestos, solvents, coal-dust

or congenital conditions (e.g. alpha-1-antitrypsin deficiency) – are very prone to acute

respiratory failure from infections.

The Department of Food Science and Technology, Research Group on Quality, Safety

and Bioactivity of Plant Foods, CEBAS-CSIC, Murcia, Spain, did a double blind,

placebo-controlled, 5-week randomized study with the use of pomegranate juice in

COPD-patients.

This – and other research looking into interstitial lung diseases, like lung fibrosis,

have shown, that antioxidants can be effective in attenuating fibroproliferative

responses in the lung of animals and humans. In the Spanish study it was concluded

that Pomegranate Juice (PJ) supplementation does not add benefits to the current

standard therapy in patients with stable COPD. The high TEAC of PJ cannot be

extrapolated in vivo probably due to the metabolism of its polyphenols by the colonic

microflora. The understanding of the different bioavailability of dietary polyphenols

is critical before claiming any antioxidant-related health benefit.

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It it/was proven that polyphenols in PJ can be very beneficial, but the colonic

microflora of each individual plays a major role in bioavailability. More research is

necessary.

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OSTEROARTHRITIS

The pomegranate has antioxidant AND anti-inflammatory properties. We will discuss

the latter also in the ‘Microbe/Bacteria’ section.

Arthritis is one of the foremost diseases for which patients seek herbal or traditional

medicine treatments.

Using tissue samples of human cartilage affected by osteoarthritis, researchers added

a water extract of pomegranate fruit to the culture using a well-established in vitro

model. The findings showed a new activity for pomegranate fruit extract -- namely

cartilage protection -- in addition to its previously discovered antioxidant and anti-

inflammatory properties.

A case study done by the Western Reserve University School of Medicine showed the

significant effects of Pomegranate fruit extract (PFE). Here is a study extract:

Interleukin (IL)-1ß - a pro-inflammatory protein molecule that plays a key role in

cartilage degradation in osteoarthritis - induces the expression of matrix

metalloproteinases (MMPs) implicated in cartilage resorption and joint degradation in

osteoarthritis (OA). Pomegranate fruit extract (PFE) was recently shown to exert anti-

inflammatory effects in different disease models. However, no studies have been

undertaken to investigate whether PFE constituents protect articular cartilage. In the

present studies, OA chondrocytes or cartilage explants were pretreated with PFE and

then stimulated with IL-1ß at different time points in vitro. The amounts of

proteoglycan released were measured by a colorimetric assay. The expression of

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MMPs, phosphorylation of the inhibitor of B (I B ) and mitogen-activated protein

kinases (MAPKs) was determined by Western immuno-blotting. Expression of

mRNA was quantified by real-time PCR. MAPK enzyme activity was assayed by in

vitro kinase assay. Activation of nuclear factor- B (NF- B) was determined by

electrophoretic mobility shift assay. PFE inhibited the IL-1ß–induced proteoglycan

breakdown in cartilage explants in vitro. At the cellular level, PFE (6.25–25 mg/L)

inhibited the IL-1ß–induced expression of MMP-1, -3, and -13 protein in the medium

(P < 0.05) and this was associated with the inhibition of mRNA expression. IL-1ß–

induced phosphorylation of p38-MAPK, but not that of c-Jun-N-terminal kinase or

extracellular regulated kinase, was most susceptible to inhibition by low doses of PFE,

and the addition of PFE blocked the activity of p38-MAPK in a kinase activity assay.

PFE also inhibited the IL-1ß–induced phosphorylation of I B and the DNA binding

activity of the transcription factor NF- B in OA chondrocytes. Taken together, these

novel results indicate that PFE or compounds derived from it may inhibit cartilage

degradation in OA and may also be a useful nutritive supplement for maintaining joint

integrity and function.

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INCREASE IN SPERM-QUALITY

Regarding sperm-quality, only one study was done so far… with rats. Pomegranate

fruit is inescapably linked with fertility, birth and eternal life because of its many

seeds. The aim of this study was to investigate the effects of pomegranate juice (PJ)

consumption on sperm quality, spermatogenic cell density, antioxidant activity and

testosterone level of male healthy rats. METHODS: Twenty-eight healthy adult male

Wistar rats were divided into four groups; each group containing seven rats. One

milliliter distilled water, 0.25 mL PJ plus 0.75 mL distilled water, 0.50 mL PJ plus

0.50 mL distilled water and 1 mL PJ were given daily for seven weeks by gavage to

rats in the first, second, third and fourth groups, respectively. Body and reproductive

organ weights, spermatogenic cell density, sperm characteristics, levels of antioxidant

vitamins, testosterone, and lipid peroxidation and, antioxidant enzyme activities were

investigated. All analyses were done only once at the end of the seven week study

period. Data were compared by analysis of variance (ANOVA) and the degree of

significance was set at P<0.05. RESULTS: A significant decrease in malondialdehyde

(MDA) level and marked increases in glutathione (GSH), glutathione peroxidase

(GSH-Px) and catalase (CAT) activities, and vitamin C level were observed in rats

treated with different doses of PJ. PJ consumption provided an increase in epididymal

sperm concentration, sperm motility, spermatogenic cell density and diameter of

seminiferous tubules and germinal cell layer thickness, and it decreased abnormal

sperm rate when compared to the control group. CONCLUSION: The results suggest

that PJ consumption improves sperm quality and antioxidant activity of rats.

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ERECTILE DYSFUNCTION

A randomized, placebo-controlled, double-blind, crossover pilot study, published by

the International Journal of Impotence Research (Nature Group) examined the

efficacy of pomegranate juice versus placebo in improving erections in 61 male

subjects. To qualify, participants had to experience mild to moderate ED for at least 3

months; be in a stable, monogamous relationship with a consenting female partner;

and be willing to attempt sexual intercourse on at least one occasion per week during

each study period.

For the first four weeks of the study, the subjects were assigned to drink either 8 oz.

of Pomegranate Juice (used juice: POM Wonderful) daily with their evening meal or

shortly after. After a two-week washout period during which the subjects did not

consume any study beverage nor utilize any erectile dysfunction treatment, they were

assigned to drink 8 oz. of the opposite study beverage every evening for another four

weeks. At the end of each four week period, efficacy was assessed using the

International Index of Erectile Function (IIEF) and Global Assessment Questionnaires

(GAQ). The IIEF is a validated questionnaire that has been demonstrated to correlate

with ED intensity. The GAQ elicits the patient's self-evaluation of the study

beverages' effect on erectile activity.

47% percent of the subjects reported that their erections improved with the

pomegranate juice, while only 32% reported improved erections with the placebo

(p=0.058). These results compare favourably to a recent 24-week study using a PDE5

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inhibitor (such as Cialis), in which roughly 73% of subjects reported a benefit from

the PDE5 inhibitor and 26% reported a "placebo effect" (i.e. experiencing

improvement while on the placebo).

Although the study did not achieve overall statistical significance, the authors

conclude that additional studies with more patients and longer treatment periods may

in fact reach statistical significance. Looking at several causes of ED (diabetes, high

blood pressure, arterial plague, heart disease) the anti-atherosclerosis activity of the

active pomegranate ingredients can also help with ED conditions.

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ALZHEIMER’S DISEASE

It is proven, that polyphenols are neuro-protective. Although there are no proven ways

to delay onset or slow progression of Alzheimer's disease (AD), studies suggest that

diet can affect the risk. A study with mice done by the Department of Psychology,

Loma Linda University, California, the Department of Neurology, Washington

University School of Medicine, the Hope Center for Neurological Disorders, the

Department of Molecular Biology and Pharmacology, (all: Washington University

School of Medicine, St. Louis, MO, USA) and the David Geffen School of Medicine,

University of California School of Medicine, California, showed, that that further

studies to validate and determine the mechanism of polyphenolic effects, as well as

whether substances in PJ may be useful in Alzheimer’s Diseases, should be

considered.

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MENOPAUSAL SYMPTOMS

Active ingredients in pomegranate are helpful once we look at the complete picture of

menopause.

Menopause is a complex health condition. Addressing menopause requires a diverse

approach that both restores normal hormone balance and protects against the

multitude of diseases that can arise during this period in a woman’s life. The onset of

menopause triggers profound changes in cardiac health, mental states, bone strength,

and cell proliferation, all of which combine to greatly elevate a woman’s risk for

contracting heart disease, osteoporosis, and certain cancers.

These health issues are not easy to correct, especially when they occur imultaneously.

The ideal approach to addressing menopause would be comprehensive and holistic,

providing fast-acting, short-term symptomatic relief as well as longer-term benefits

that support women’s health as their body chemistry changes. While declining levels

of oestrogen are what gives rise to the difficulties of menopause, both nutritional and

hormonal support are required to address and ameliorate the physiological symptoms

and other changes that accompany menopause.

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COSMECEUTICAL

Pomegranate seed oil is commonly used in cosmetic products to revitalize dull or

mature skin, assist with wrinkles, and to soothe minor skin irritations. Without

moisture, wrinkles become more abundant and pronounced, the skin looks tight and

lacks luster. Pomegranate seed oil adds moisture, has natural estrogenic properties,

anti-oxidants, is anti-inflammatory, anti-microbial, improves skin elasticity, and

protects the skin. It provides relief from minor skin irritations and inflammation,

including dry skin, eczema, psoriasis and sunburned skin. The conjugated fatty acids

give it strong anti-inflammatory properties, which help to reduce swelling and ease

muscular aches and pains.

Pomegranate seed oil, but not aqueous extracts of fermented juice, peel or seed cake,

was shown to stimulate keratinocyte proliferation in monolayer culture. In parallel, a

mild thickening of the epidermis (without the loss of ordered differentiation) was

observed in skin organ culture. The same pomegranate seed oil that stimulated

keratinocyte proliferation was without effect on fibroblast function. In contrast,

pomegranate peel extract (and to a lesser extent, both the fermented juice and seed

cake extracts) stimulated type I procollagen synthesis and inhibited matrix

metalloproteinase-1 (MMP-1; interstitial collagenase) production by dermal

fibroblasts, but had no growth-supporting effect on keratinocytes. These results

suggest heuristic potential of pomegranate fractions for facilitating skin repair in a

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polar manner, namely aqueous extracts (especially of pomegranate peel) promoting

regeneration of dermis, and pomegranate seed oil promoting regeneration of

epidermis.

A double-blind, placebo-controlled trial was performed in Japan to clinically evaluate

the protective and ameliorative effects of ellagic acid-rich pomegranate extract on

pigmentation in the skin after ultraviolet ray (UV) irradiation, using female subjects in

their 20s to 40s. Thirteen healthy volunteers per group were randomly assigned to

three groups; namely, high dose (200 mg/d ellagic acid), low dose (100 mg/d ellagic

acid) and control (0 mg/d ellagic acid: placebo). Each group received the respective

test foods for 4 wk. Each subject received a 1.5 MED (minimum erythema dose) of

UV irradiation on an inside region of the right upper arm, based on the MED value

measured on the previous day. Luminance (L), melanin and erythema values were

measured before the start of the test food intake, and after 1, 2, 3 and 4 wk following

the start of the test food intake. Further, questionnaires were conducted regarding the

condition of the skin before the start of the test food intake and at the termination of

the test food intake. As a result, decreasing rates of L values from the baseline in the

low- and high-dose groups were inhibited by 1.35% and 1.73% respectively, as

compared to the control group. Further, a stratified analysis using subjects with a

slight sunburn revealed an inhibited decrease of L values compared with the control

group at 1, 2 (p<0.01, respectively) and 4 wk (p<0.05) after the start of the test food

intake in the low-dose group, and at 2 and 3 wk (p<0.05) in the high-dose group.

Furthermore, the results of questionnaires showed ameliorating tendencies due to the

test food, in some items such as "brightness of the face" and "stains and freckles."

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Based on the above-mentioned results, it is suggested that ellagic acid-rich

pomegranate extract, ingested orally, has an inhibitory effect on a slight pigmentation

in the human skin caused by UV irradiation.

Cosmeceutical/Skin Cancer

UVA is the major portion (90-99%) of solar radiation reaching the surface of the earth

and has been described to lead to formation of benign and malignant tumors. UVA-

mediated cellular damage occurs primarily through the release of reactive oxygen

species and is responsible for immuno-suppression, photo-dermatoses, photo-aging

and photo-carcinogenesis. Pomegranate fruit extract (PFE) possesses strong

antioxidant and anti-inflammatory properties. One studiy has shown that PFE

treatment of normal human epidermal keratinocytes (NHEK) inhibits UVB-mediated

activation of MAPK and NF-kappaB pathways. Signal transducers and activators of

transcription 3 (STAT3), Protein Kinase B/AKT and Map Kinases (MAPKs), which

are activated by a variety of factors, modulate cell proliferation, apoptosis and other

biological activities. The goal of this study was to determine whether PFE affords

protection against UVA-mediated activation of STAT3, AKT and extracellular signal-

regulated kinase (ERK1/2). Immunoblot analysis demonstrated that 4 J/cm2 of UVA

exposure to NHEK led to an increase in phosphorylation of STAT3 at Tyr705, AKT

at Ser473 and ERK1/2. Pretreatment of NHEK with PFE (60-100 microg/mL) for 24

h before exposure to UVA resulted in a dose-dependent inhibition of UVA-mediated

phosphorylation of STAT3 at Tyr705, AKT at Ser473 and ERK1/2. mTOR,

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structurally related to PI3K, is involved in the regulation of p70S6K, which in turn

phosphorylates the S6 protein of the 40S ribosomal subunit. The study found out that

UVA radiation of NHEK resulted in the phosphorylation of mTOR at Thr2448 and

p70S6K at Thr421/Ser424. PFE pre-treatment resulted in a dose-dependent inhibition

in the phosphorylation of mTOR at Thr2448 and p70S6K at Thr421/Ser424. The data

further demonstrate that PFE pre-treatment of NHEK resulted in significant inhibition

of UVA exposure-mediated increases in Ki-67 and PCNA. PFE pre-treatment of

NHEK was found to increase the cell-cycle arrest induced by UVA in the G1 phase of

the cell cycle and the expression of Bax and Bad (proapoptotic proteins), with

downregulation of Bcl-X(L) expression (antiapoptotic protein). The data suggest that

PFE is an effective agent for ameliorating UVA-mediated damages by modulating

cellular pathways and merits further evaluation as a photochemopreventive agent.

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ANTIMICROBIAL

Introduction: The necessity of natural antimicrobials.

The pharmacological industries have produced a number of new antibiotics in the last

three decades, but resistance to these drugs by micro-organisms has increased

dramatically.

In general, bacteria have the genetic ability to transmit and acquire resistance to

drugs, which are utilized as therapeutic agents. This causes of course concern,

because of the number of patients in hospitals who have suppressed immunity, and

due to new bacterial strains, which are multi-resistant. Consequently, new infections

can occur in hospitals resulting in high mortality.

From 1980 to 1990, Montelli and Levy documented a high incidence of resistant

micro-organisms in clinical microbiology in Brazil. This fact has also been verified in

other clinics around all over world.

The problem of microbial resistance is growing and the outlook for the use of

antimicrobial drugs in the future is uncertain. Therefore, actions must be taken to

reduce this problem.

This means: A stricter control in the use of antibiotics and increased research for a

better understanding of the genetic mechanisms of microbial drug-resistance. Of

course, the development of new drugs is very important… but not only synthetic, but

also natural drugs.

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For a long period of time, plants have been a valuable source of natural products for

maintaining human health, especially in the last decade, with more intensive studies

for natural therapies.

The use of plant compounds for pharmaceutical purposes has gradually increased.

According to the World Health Organization (WHO) medicinal plants would be the

best source to obtain a variety of drugs. About 80% of individuals from developed

countries use traditional medicine, which has compounds derived from medicinal

plants. Therefore, such plants should be investigated to better understand their

properties, safety and efficiency.

The use of plant extracts and phytochemicals, both with known antimicrobial

properties, can be of great significance in therapeutic treatments. In the last few years,

a number of studies have been conducted in different countries to prove such

efficiency. Many plants have been used because of their antimicrobial activity, which

are due to compounds synthesized in the secondary metabolism of the plant. These

products are known by their active substances, for example, the phenolic compounds.

The antimicrobial properties of plants have been investigated by a number of

researchers world wide, especially in Latin America. In Argentina, a research tested

122 known plant species used for therapeutic treatments. Inhibition of bacterial

growth was observed with the following bacteria:

Staphylococus aureus

Escherichia coli

Aspergillus niger

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Candida albicans

Klebsiella pneumoniae

Pseudomonas aeruginosa

Bacillus subtilis

Styaphylococcus aureus

Staphylococcus epidermidis

Bacillus cereus

Escherichia coli

Salmonella typhi

Salmonella paratyphi

Some research suggest that the potential systemic biological effects of pomegranate

juice ingestion should be attributed to the colonic microflora metabolites rather than

to the polyphenols present in the juice. A very important theory, which is interesting

enough to focus more on antimicrobial research and the promicrobial use of

pomegranate.

In recent years there has been a dramatic increase in the number of reported cases of

foodborne illness. Escherichia coli O157 : H 7 is a highly virulent bacterium. Its

transmission to human has recently been epidemiologically associated with the

consumption of contaminated foods. This pathogen is an etiological agent of

haemorrhagic colitis and haemolytic uremic syndrome in humans. Its reported

mortality rates was as high

as 31% . Although outbreaks of food-associated E. coli O157 : H 7 illness have been

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associated with the consumption of undercooked ground beef, this bacterium has been

isolated from drinking water, apple cider, mayonnaise and mayonnaise-based sauces.

Yersinia enterocolitica is an important foodborne pathogen because of its ability to

grow

at refrigeration temperatures. Yersinia readily withstand freezing and can survive in

frozen foods for extended periods even after repeated freezing and thawing.

Foodborne outbreaks due to Y. enterocolitica have been related to raw milk, powdered

milk, chocolate–flavoured

milk, tofu, bean sprouts, pork, cheese and pork.

Due to negative consumer perceptions of artificial preservatives, attention is shifting

towards alternatives that the consumers perceive as natural, an in particular, spices

and medicinal plants or herbs.

Of very high interest is the In searching for natural antimicrobial products, the present

study provides data on antibacterial activity of various spices and medicinal plants

that are available in Thailand against Y. enterolitica.

Cultures of E. coli O157 : H 7 ATCC 43889 and Y. enterocolitica ATCC 23715 were

btained

from the National Institute of Health, Ministry of Public Health, Bangkok, Thailand.

Cultures were grown in Tryptic Soy Agar, TSA (Merck, Darmstadt) at 37C and

maintained on TSA at 4C. Before preparing inoculum for the test media, cultures

were activated by two successive transfers in Tryptic Soy Broth (TSB) at 37C.

Inoculum population was determined by serially diluting the suspension with

- 71 -

phosphate buffer, pour plating with TSA and incubating at 37C for 48 hr before

counting colonies.

Spices and medicinal plants used in this experiment are shown in Table 1. They were

dried

and ground before extracting with 95% ethyl alcohol for 48 hr. The volumes were

adjusted to obtain the concentration of 100 mg/ml. The aqueous extract was sterilized

by millipore membrane. Sensitivity testing was done by well assay technique. One ml

of an overnight culture in TSB was inoculated into 100 ml of a TSA maintained at

45C. Approximately

25 ml was poured into a petri dish and allowed to solidify. Wells (4 mm in diameter)

were cut into the agar and filled with 40 l of filtered aqueous extracted of spices and

medicinal plants. All plates were incubated at 37C for 48 hr and the diameters of the

inhibition zones were measured.

MICs were determined according to the method of Richards et al. Briefly, 9.9 ml

volumes of TSB containing a series of dilutions of cloves, cinnamon, roselle,

leadwort, pomegranate,

sappan and betle leaf were inoculated with 0.1 ml from an 18 hr culture to give

approximately 3.0104 or 3.0106 cfu/ml E. coli O157 : H 7 and 6.0104 or

6.0106 cfu/ml Y. enterocolitica. The inoculated tubes were incubated for 24 hr at

37C and the lowest concentrations showing no growth in tubes were recorded as the

MICs.

Clove showed the highest inhibitory effect for E. coli, while pomegranate had the

highest inhibitory effects on Yersinia enterocolitica.

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One study evaluated the interaction between Punica granatum (pomegranate)

methanolic extract (PGME) and antibiotics against 30 clinical isolates of methicillin-

resistant Staphylococcus aureus (MRSA) and methicillin-sensitive Staphylococcus

aureus (MSSA). Susceptibility testing of the isolates to PGME and antibiotics was

performed by the broth dilution method. Synergic activity was detected between

PGME and the 5 antibiotics tested, chloramphenicol, gentamicin, ampicillin,

tetracycline, and oxacillin, ranging from 38% to 73%. For some isolates, PGME did

not interfere with the action of any of the antibiotics tested. The bactericidal activity

of PGME (0.1 × MIC) in combination with ampicillin (0.5 × MIC) was assessed using

chosen isolates by time-kill assays, and they confirmed the synergic activity. Using

this combination, cell viability was reduced by 99.9% and 72.5% in MSSA and

MRSA populations, respectively. PGME increased the post-antibiotic effect (PAE) of

ampicillin from 3 to 7 h. In addition, PGME demonstrated the potential to either

inhibit the efflux pump NorA or to enhance the influx of the drug. The detection of in

vitro variant colonies of S. aureus resistant to PGME was low and they did not

survive. In conclusion, PGME dramatically enhanced the activity of all antibiotics

tested, and thus, offers an alternative for the extension of the useful lifetime of these

antibiotics.

In Brazil, pomegranate (Punica granatum L. (Punicaceae)) is widely used as a

phytotherapeutic agent. Studies evaluated the effect of pomegranate extract on

Staphylococcus aureus FRI 722 growth and subsequent enterotoxin production.

Bacterial susceptibility was determined by tube dilution method and production of

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enterotoxin was assessed using membrane-over-agar (MOA) plates. At a low extract

concentration (0.01% v/v) bacterial growth was delayed, while a higher concentration

(1% v/v) eliminated bacterial growth. Most interestingly, a 0.05% (v/v) concentration

of extract was found to inhibit Staphylococcal enterotoxin (SE) A production. These

data further implicate pomegranate extracts as potential antibacterial therapeutics with

the added ability to inhibit enterotoxin production.

The Punica granatum L. (pomegranate) by-product POMx was partitioned between

water, EtOAc and n-BuOH. The EtOAc and n-BuOH extracts were purified by XAD-

16 and Sephadex LH-20 column chromatography to afford ellagic acid (1), gallagic

acid (2), punicalins (3), and punicalagins (4). Compounds 1 - 4 and the mixture of

tannin fractions (XAD-16 eluates) were evaluated for antioxidant, antiplasmodial, and

antimicrobial activities in cell-based assays. The mixture of tannins (TPT), XAD-

EtOAc, XAD-H2O, XAD-PJ and XAD-BuOH, exhibited IC50 values against reactive

oxygen species (ROS) generation at 0.8 - 19 microg/mL. Compounds 1 - 4 showed

IC50 values of 1.1, 3.2, 2.3 and 1.4 microM, respectively, against ROS generation and

no toxicity up to 31.25 microg/mL against HL-60 cells. Gallagic acid (2) and

punicalagins (4) exhibited antiplasmodial activity against Plasmodium falciparum D6

and W2 clones with IC50 values of 10.9, 10.6, 7.5 and 8.8 microM, respectively.

Fractions XAD-EtOAc, XAD-BuOH, XAD-H2O and XAD-PJ compounds 1 - 4

revealed antimicrobial activity when assayed against Escherichia coli, Pseudomonas

aeruginosa, Candida albicans, Cryptococcus neoformans, methicillin-resistant

Staphylococcus aureus (MRSA), Aspergillus fumigatus and Mycobacterium

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intracellulare. Compounds 2 and 4 showed activity against P. aeruginosa, C.

neoformans, and MRSA. This is the first report on the antioxidant, antiplasmodial and

antimicrobial activities of POMx isolates, including structure-activity relationships

(SAR) of the free radical inhibition activity of compounds 1 - 4. Our results suggest a

beneficial effect from the daily intake of POMx and pomegranate juice (PJ) as dietary

supplements to augment the human immune system's antioxidant, antimalarial and

antimicrobial capacities.

In another study, researcher determined the effects of combinations of antibiotics and

plant polyphenols against 20 clinical isolates of MRSA. The in vitro activities of 10

antibiotics and 15 natural polyphenols against the isolates were evaluated by

determining minimum inhibitory concentrations (MICs). All isolates were susceptible

to vancomycin and resistant to rifampicin, while susceptibilities to ciprofloxacin

varied. Among the 15 natural polyphenols, kaempferol (3,4',5,7-tetrahydroxyflavone)

and quercetin (3,3',4',5,7-pentahydroxyflavone) showed the lowest MICs. In

checkerboard assays, combinations of rifampicin and either kaempferol or quercetin

acted synergistically or partially synergistically against the clinical MRSA isolates.

Rifampicin combined with kaempferol or quercetin exhibited good beta-lactamase

inhibitory effects (57.8 % and 75.8 %, respectively) against a representative isolate

according to nitrocefin analysis. The study results and ready availability and low

toxicity of plant polyphenols warrant further investigations on the therapeutic

potential of combination therapies for MRSA infections. FIC:fractional inhibitory

concentration MIC:minimum inhibitory concentration MRSA:methicillin-resistant

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New therapies are needed to address the public health problem posed by methicillin-

resistant Staphylococcus aureus.

HIV

In the absence of vaccines, topical microbicides, expected to block virus transmission,

offer hope for controlling the pandemic. Antiretroviral chemotherapeutics have

decreased AIDS mortality in industrialized countries, but only minimally in

developing countries. To prevent an analogous dichotomy, microbicides should be:

acceptable; accessible; affordable; and accelerative in transition from development to

marketing. Already marketed pharmaceutical excipients or foods, with established

safety records and adequate anti-HIV-1 activity, may provide this option.

Methods

Fruit juices were screened for inhibitory activity against HIV-1 IIIB using CD4 and

CXCR4 as cell receptors. The best juice was tested for inhibition of: (1) infection by

HIV-1 BaL, utilizing CCR5 as the cellular coreceptor; and (2) binding of gp120 IIIB

and gp120 BaL, respectively, to CXCR4 and CCR5. To remove most colored juice

components, the adsorption of the effective ingredient(s) to dispersible excipients and

other foods was investigated. A selected complex was assayed for inhibition of

infection by primary HIV-1 isolates.

Results: HIV-1 entry inhibitors from pomegranate juice adsorb onto corn starch. The

resulting complex blocks virus binding to CD4 and CXCR4/CCR5 and inhibits

infection by primary virus clades A to G and group O.

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Conclusion: These results suggest the possibility of producing an anti-HIV-1

microbicide from inexpensive, widely available sources, whose safety has been

established throughout centuries, provided that its quality is adequately standardized

and monitored.

- 77 -

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RESEARCH CENTERS:

Department of Pathology, The University of Michigan Medical School, MI 48109,USA.

Laboratory of Bio-Organic Chemistry, Tokyo Denki University, Saitama, Japan.

Department of Pharmacy, Pusan National University, Korea.

Department of Surgery, Tokyo Metropolitan Komagome Hospital, Tokyo, Japan.

Rimonest Ltd., P.O.B. 9945, Haifa, Israel.

School of Pharmacy, Medical Biology Centre, Queens University of Belfast, 97Lisburn Road, Belfast, BT9 7BL, UK.

Department of Dermatology, University of Wisconsin, Madison 53706, USA.

Laboratory of Molecular Modeling & Drug Design, Lindsley F. Kimball ResearchInstitute, New York Blood Center, New York, USA

Division of Rheumatic Diseases, Department of Medicine, Case Western University,Cleveland, OH 44106, USA.

Polifenoles Naturales SL, Poligono Industrial Las Majoreras Ingenio, Las Palmas,Canary Islands, Spain.

Pharmaceutics Department, University of Florida, Gainesville, Florida 32610, USA.

Rambam Medical Center in Haifa, Israel

Department of Food Science and Technology, Tarbiat Modares University, P.O. Box14115-336, Tehran, Iran

Biochemical Virology Laboratory, Lindsley F. Kimball Research Institute, New YorkBlood Center, New York, USA

Center for Human Nutrition, David Geffen School of Medicine, University ofCalifornia, Los Angeles, CA 90095, USA

Istituto di Farmacologia e Farmacognosia, Università degli Studi di Urbino Carlo Bo,Urbino, Italy

Punisyn Pharmaceuticals Ltd, Haifa, Israel.

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Department of Pharmacognosy, School of Pharmacy, The University of Mississippi,USA.

Department of Medical Elementology and Toxicology, Faculty of Science, JamiaHamdard, Hamdard Nagar, New Delhi 110062, India.

The Lipid Research Laboratory, Technion Faculty of Medicine, The RappaportFamily Institute for Research in the Medical Sciences, 31096 Haifa, Israel

Research Group on Quality, Safety and Bioactivity of Plant Foods, Department ofFood Science and Technology, CEBAS-CSIC, P.O. Box 4195, 30080 Murcia, Spain.

Departamento de Biologia Geral, Instituto de Ciências Biológicas, UniversidadeFederal de Minas Gerais, Belo Horizonte, Brazil

Walter Reed Army Institute of Research, Division of Pathology, Department ofMolecular Pathology, 503 Robert Grant Avenue, Silver Spring, MD 20910, USA

Departamento de Química, Instituto de Ciências Exatas, Universidade Federal deMinas Gerais, Belo Horizonte, Brazil

Laboratório de Enterotoxinas, Fundação Ezequiel Dias, Brazil

Faculdade de Ciências da Saúde, Universidade Metodista de Piracicaba, Piracicaba,SP, Brasil.

Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, São Paulo, SP,Brasil

Department of Microbiology, Muthayammal College of Arts and Science, Rasipuram637 408, Tamil Nadu, India.

Núcleo de Pesquisas de Produtos Naturais, Bloco H, Centro de Ciências da Saúde,Ilha do Fundão, Universidade Federal do Rio de Janeiro, 21921-590 Rio de Janeiro -RJ, Brazil

Laboratory of Experimental Endocrinology, University of Crete, Heraklion, Greece

Laboratory of Gastroenterology, University of Crete, Heraklion, Greece

Laboratory of Pharmacology, University of Crete, Heraklion, Greece

Laboratory of Food Chemistry and Technology, Department of Chemistry, AristotleUniversity of Thessaloniki, Greece

- 86 -

Laboratório de Química de Produtos Naturais e Central Analítica, Far-Manguinhos - FIOCRUZ, Rio de Janeiro - RJ, Brazil

Department of Molecular Genetics, University of Illinois College of Medicine,Chicago, Illinois 60607

Biochemical Virology Laboratory, Lindsley F. Kimball Research Institute, New YorkBlood Center, New York, USA

Laboratory of Molecular Modeling & Drug Design, Lindsley F. Kimball ResearchInstitute, New York Blood Center, New York, USA

Department of Food Science and Technology, Research Group on Quality, Safety andBioactivity of Plant Foods, CEBAS-CSIC, Murcia, Spain

Neumology Service, Virgen de La Arrixaca University Hospital, Murcia, Spain

Clinical Analysis Service, Laboratory of Biochemistry, Virgen de La ArrixacaUniversity Hospital, Murcia, Spain

Department of Psychology, Loma Linda University, Loma Linda, CA, USA

Department of Neurology, Washington University School of Medicine, St. Louis,MO, USA

Hope Center for Neurological Disorders, Washington University School of Medicine,St. Louis, MO, USA

Department of Molecular Biology and Pharmacology, Washington University Schoolof Medicine, St. Louis, MO, USA

David Geffen School of Medicine, University of California School of Medicine, LosAngeles, CA, USA

Department of General Pathology and Excellence Research Center on CardiovascularDisease, University of Naples, Italy

Anesthesiology Division, University of California at Los Angeles, Los Angeles, USA

Pharmacology Division, University of California at Los Angeles, Los Angeles, USA

Departamento de Química, Instituto de Ciências Exatas, Universidade Federal deMinas Gerais, Belo Horizonte, Brazil

Laboratório de Enterotoxinas, Fundação Ezequiel Dias, Brazil

- 87 -

Departamento de Biologia Geral, Instituto de Ciências Biológicas, UniversidadeFederal de Minas Gerais, Belo Horizonte, Brazil

Walter Reed Army Institute of Research, Division of Pathology, Department ofMolecular Pathology, 503 Robert Grant Avenue, Silver Spring, MD 20910, USA

Fachbereich Chemie und Chemietechnik der Universität Paderborn, Germany.

Department of Chemistry, University of Venda,Republic of South Africa

Faculty of Health Sciences, Soroka Hospital Medical Center, Ben-Gurion Universityof the Negev, Israel.

Department of Human Nutrition, National Nutrition and Food Technology ResearchInstitute, Tehran, Iran