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 The Claims Scientific Classification Nutritional Value Compact History Cultivars Pollination Climate Soil Propagation Culture Harvesting and Yield Keeping Quality & Storages Pest and Disease Food Uses Introduction to Health Benefits of Pomegranate Active Ingredients of the Pomegranate Antioxidants Phenolics TANNINS Tannin Table I Punicalagin Ellagic Acid Pelagonidin, Cyanidin Gallic Acid Quercetin Pomegranate and Cancer Pomegranate and Atherosclerosis page page page page page page page page page page page page page page page page page page page page page page page page page page page page page 01 02 04 04 05 08 12 14 14 15 15 15 12 16 18 18 20 22 23 25 27 29 29 30 32 33 33 34 45

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Chronic Obstructive Pulmonary Disease Osteoarthritis Increase in Sperm Quality Erectile Dysfunction Alzheimers Disease Menopausal Symptoms Cosmeceutical Antimicrobial

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54 56 58 59 61 62 63 67

Reference List List of Research Centers

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77 84

Correspondence Address:

MSML c/o Hale Clinic, 7 Park Crescent, London W1B 1PF Senior biomedical Scientist: Dirk Budka Senior Practitioner: Dr. Peter Gruenewald, MD dirk.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 whats wrong in using natural products, inexpensive and available in the next shop, to treat ailments/diseases?

Lets 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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Alzheimers 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 Division Class Subclass Order Family Genus Species

Plantae Magnoliophyta Magnoliopsida Rosidae Myrtales Lythraceae Punica P. granatum

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The Nutritional Value per 100 g of the Pomegranate (aril only)

Carbohydrates -sugars - dietary fibre Fat Protein Thiamin (B1) Riboflavin (B2) Niacin (B3) Pantothenic acid (B5) Vitamin B6 Folate (B9) Vitamin C Calcium Iron Magnesium Phosphorus Potassium Zinc 16.57 g 0.60 g

17.17 g

0.30 g 0.95 g 0.030 mg 0.063 mg 0.300 mg 0.596 mg 0.105 mg 6 g 3 mg 3 mg 0.30 mg 3 mg 8 mg 259 mg 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, LDLcholesterol. 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 childs sensitization.

-8HISTORY

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 Solomons 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: Dont 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 thirstquenching 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, mediumsweet 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 mediumhard seeds.

'Muscat White'large, creamy-white tinged with pink; thin rind; fleshy, creamcolored, 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 12 F (-11.11 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 airlayered 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 sunscaldbrown, 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 32 to 41 F (0-5 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 41 F (5 C); for longer periods at 50 F (10 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 35.6 F (2 C). Subsequent transfer to 68 F (20 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

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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)

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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

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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

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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

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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

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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.

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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 skins collagen chains, while false tanning entails the filling of hollow spaces between the skins collagen chains. In medicine, especially in Asian (Japanese and Chinese) natural healing, the tannincontaining 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).

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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)

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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

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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 chemopreventive 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

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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

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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.

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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 proapoptotic 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%

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(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

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

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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 stressinduced apoptosis. Anti-apoptotic activity of p21 may contribute to its potential to act as an oncogene.

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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

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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 KruskalWallis 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

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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

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

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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

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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 chemopreventive 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-betahydroxysteroid dehydrogenase Type 1 from 34 to 79%, at concentrations ranging from 100 to 1,000 microg/ml according to seed oil.

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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 antiproliferative 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.

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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.

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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-Hdibenzo[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

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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

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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

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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 Nacyl-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 polyphenolinduced 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 dosedependent 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

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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 lipoproteincholesterol (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 antiinflammatory 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)-1 - 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 antiinflammatory 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-1 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-1induced proteoglycan breakdown in cartilage explants in vitro. At the cellular level, PFE (6.2525 mg/L) inhibited the IL-1induced 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-1 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-1induced 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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ALZHEIMERS 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 Alzheimers 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 womans 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 womans 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 womens 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. UVAmediated 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 signalregulated 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, chocolateflavoured 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

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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 methicillinresistant 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 XAD16 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), XADEtOAc, 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 methicillinresistant 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.

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REFERENCE LIST

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Siriporn Stonsaovapak, Pornthip Chareonthamawat and Malai Boonyaratanakornkit. Inhibitory Effects of Selected Thai Spices and Medicinal Plants on Escherichia coli O157 : H 7 and Yersinia enterocolitica. Sumner MD, Elliott-Eller M, Weidner G, Daubenmier JJ, Chew MH, Marlin R, Raisin CJ, Ornish D. Effects of pomegranate juice consumption on myocardial perfusion in patients with coronary heart disease. 2005. Am J Cardiol, 96. Syed DN, Malik A, Hadi N, Sarfaraz S, Afaq F, Mukhtar H. Photochemopreventive effect of pomegranate fruit extract on UVA-mediated activation of cellular pathways in normal human epidermal keratinocytes. 2006 Photochem Photobiol 82 (2) Toi M, Bando H, Ramachandran C, Melnick SJ, Imai A, Fife RS, Carr RE, Oikawa T, Lansky EP. Preliminary studies on the anti-angiogenic potential of pomegranate fractions in vitro and in vivo. 2003. Angiogenesis 6 (2) Tuerk G et al. Effects of pomegranate juice consumption on sperm quality, spermatogenic cell density, antioxidant activity and testosterone level in male rats. 2008. Clin Nutr. 27 (2) West T, Atzeva, M, Holtzman, DM. Pomegranate Polyphenols and Resveratrol Protect the Neonatal Brain against Hypoxic-Ischemic Injury. Hope Center for Neurological Disorders and Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Mo., USA

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http://edis.ifas.ufl.edu/MG056 http://pomegranateinformation.com/history-of-pomegranate.html http://www.pomegranate-benefits.com/pomegranate-history.html http://www.healthscout.com/ency/68/252/main.html http://pbi-ibp.nrc-cnrc.gc.ca http://en.wikipedia.org http://www.ozgrenade.com.au http://www.phytochemicals/info http://www.lef.org/magazine/mag2006/apr2006_cover_menopause_01.htm

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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, 97 Lisburn Road, Belfast, BT9 7BL, UK. Department of Dermatology, University of Wisconsin, Madison 53706, USA. Laboratory of Molecular Modeling & Drug Design, Lindsley F. Kimball Research Institute, 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. Box 14115-336, Tehran, Iran Biochemical Virology Laboratory, Lindsley F. Kimball Research Institute, New York Blood Center, New York, USA Center for Human Nutrition, David Geffen School of Medicine, University of California, Los Angeles, CA 90095, USA Istituto di Farmacologia e Farmacognosia, Universit 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, Jamia Hamdard, Hamdard Nagar, New Delhi 110062, India. The Lipid Research Laboratory, Technion Faculty of Medicine, The Rappaport Family Institute for Research in the Medical Sciences, 31096 Haifa, Israel Research Group on Quality, Safety and Bioactivity of Plant Foods, Department of Food Science and Technology, CEBAS-CSIC, P.O. Box 4195, 30080 Murcia, Spain. Departamento de Biologia Geral, Instituto de Cincias Biolgicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil Walter Reed Army Institute of Research, Division of Pathology, Department of Molecular Pathology, 503 Robert Grant Avenue, Silver Spring, MD 20910, USA Departamento de Qumica, Instituto de Cincias Exatas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil Laboratrio de Enterotoxinas, Fundao Ezequiel Dias, Brazil Faculdade de Cincias da Sade, Universidade Metodista de Piracicaba, Piracicaba, SP, Brasil. Faculdade de Cincias Farmacuticas, Universidade de So Paulo, So Paulo, SP, Brasil Department of Microbiology, Muthayammal College of Arts and Science, Rasipuram 637 408, Tamil Nadu, India. Ncleo de Pesquisas de Produtos Naturais, Bloco H, Centro de Cincias da Sade, Ilha do Fundo, 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, Aristotle University of Thessaloniki, Greece

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Laboratrio de Qumica de Produtos Naturais e Central Analtica, FarManguinhos - 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 York Blood Center, New York, USA Laboratory of Molecular Modeling & Drug Design, Lindsley F. Kimball Research Institute, New York Blood Center, New York, USA Department of Food Science and Technology, Research Group on Quality, Safety and Bioactivity 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 Arrixaca University 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 School of Medicine, St. Louis, MO, USA David Geffen School of Medicine, University of California School of Medicine, Los Angeles, CA, USA Department of General Pathology and Excellence Research Center on Cardiovascular Disease, 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 Qumica, Instituto de Cincias Exatas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil Laboratrio de Enterotoxinas, Fundao Ezequiel Dias, Brazil

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Departamento de Biologia Geral, Instituto de Cincias Biolgicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil Walter Reed Army Institute of Research, Division of Pathology, Department of Molecular Pathology, 503 Robert Grant Avenue, Silver Spring, MD 20910, USA Fachbereich Chemie und Chemietechnik der Universitt Paderborn, Germany. Department of Chemistry, University of Venda,Republic of South Africa Faculty of Health Sciences, Soroka Hospital Medical Center, Ben-Gurion University of the Negev, Israel. Department of Human Nutrition, National Nutrition and Food Technology Research Institute, Tehran, Iran