WO2003074067A1 - Compositions a base de fraise et de framboise, et utilisations therapeutiques de ces compositions - Google Patents

Compositions a base de fraise et de framboise, et utilisations therapeutiques de ces compositions Download PDF

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Publication number
WO2003074067A1
WO2003074067A1 PCT/US2003/006279 US0306279W WO03074067A1 WO 2003074067 A1 WO2003074067 A1 WO 2003074067A1 US 0306279 W US0306279 W US 0306279W WO 03074067 A1 WO03074067 A1 WO 03074067A1
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Prior art keywords
extract
berry
disease
fraction
isolated
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PCT/US2003/006279
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English (en)
Inventor
Gary D. Stoner
John M. Cassady
James E. Klaunig
Dale Stokes
Original Assignee
Stoner Gary D
Cassady John M
Klaunig James E
Dale Stokes
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Application filed by Stoner Gary D, Cassady John M, Klaunig James E, Dale Stokes filed Critical Stoner Gary D
Priority to AU2003217848A priority Critical patent/AU2003217848A1/en
Publication of WO2003074067A1 publication Critical patent/WO2003074067A1/fr
Priority to US10/951,413 priority patent/US20050136141A1/en
Priority to US12/137,952 priority patent/US20130115322A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/07—Retinol compounds, e.g. vitamin A
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
    • A61K31/355—Tocopherols, e.g. vitamin E
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A61K31/375—Ascorbic acid, i.e. vitamin C; Salts thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • A61K31/525—Isoalloxazines, e.g. riboflavins, vitamin B2
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/73—Rosaceae (Rose family), e.g. strawberry, chokeberry, blackberry, pear or firethorn

Definitions

  • the present invention provides novel compounds and therapeutic compositions (e.g., formulations) derived from fruits, in particular berries, and more particularly strawberry and raspberry, as well as novel uses for the compounds and compositions.
  • the compounds are formulated as a pharmaceutical, a foodstuff (e.g., added to a foodstuff to enhance its nutritional and/or medical value), or a dietary supplement.
  • the compounds and compositions contain, or are enriched for, health promoting components (e.g., antioxidants, vitamin A, vitamin E (tocochromonals), vitamin C (ascorbic acid), folic acid, carotenoids, phenolic compounds, phytosterols, and minerals) that are useful in treating or preventing a variety of health-related disorders and diseases.
  • the invention provides methods of efficiently producing berry, e.g., strawberry and raspberry extracts (and fractions thereof) enriched for antioxidant activity (and other desirable components) such that the extracts (or fractions) can be added to foodstuffs or used as a dietary supplement or a pharmaceutical composition.
  • berry e.g., strawberry and raspberry extracts (and fractions thereof) enriched for antioxidant activity (and other desirable components)
  • the extracts (or fractions) can be added to foodstuffs or used as a dietary supplement or a pharmaceutical composition.
  • the present invention provides a method for treating or preventing a disease in a subject, particularly a malignancy (e.g., a cancer), by administering to the subject (e.g., orally or, when appropriate, by other routes) a therapeutically-effective amount of a compound or composition (e.g. , an extract or extract fraction) of the invention.
  • a malignancy e.g., a cancer
  • the malignancy can be, for example, metastatic, an aerodigestive tract cancer, or a metastatic aerodigestive tract cancer (e.g., an oral, pharyngeal, laryngeal, esophageal, stomach, or colon cancer).
  • the present invention provides a method for treating or preventing other diseases or disorders associated with oxidative damage such as skin cancer, cardiovascular disease (e.g., due to high cholesterol, i.e., hypercholesterolemia), neurodegenerative disease (e.g., stroke), immunological diseases or conditions, inflammatory diseases or conditions such as arthritis, dermatological conditions, and opthalmological conditions, in a subject, by administering to the subject a therapeutically-effective amount of a compound or composition of the invention (e.g., an extract or extract fraction of the invention).
  • a compound or composition of the invention e.g., an extract or extract fraction of the invention.
  • the compounds or compositions of the invention when administered to a subject, may also be used to retard aging.
  • compositions of the invention are derived (e.g., isolated) from, or contain components of strawberry and raspberry fruits, for example, strawberry and black raspberry, and combinations thereof.
  • Particular compositions identified by way of the present invention as having significant therapeutic value include and/or are derived from strawberry and/or raspberry (e.g., black raspberry) fruits which have been, for example, pureed, freeze-dried (referred to as a berry extract), organically extracted (e.g., by solvent extraction of a berry extract, thereby resulting in a berry extract fraction), and combinations thereof.
  • Such berry extracts and fractions thereof can then be formulated in a variety of manners, such as a dietary supplement, a pharmaceutical, or as an additive to a foodstuff. They may also contain additional desirable compounds such as carbohydrates, some proteins, fiber, and combinations thereof.
  • the present invention further provides therapeutic compositions containing novel combinations and/or ratios of health-promoting compounds derived (e.g. , isolated) from strawberry and raspberry (e.g. , black raspberry).
  • health-promoting compounds derived (e.g. , isolated) from strawberry and raspberry (e.g. , black raspberry).
  • Such compounds can be isolated from, for example, strawberry and raspberry (e.g., black raspberry), extracts and/or fractions.
  • such compounds can include antioxidants, vitamins (e.g., vitamin A, vitamin E (tocochromonals), vitamin C (ascorbic acid), folic acid, carotenoids, phenolic compounds, phytosterols, minerals, or combinations thereof.
  • the invention provides a method for isolating berry extracts, and optionally, fractions thereof, so that the extracts and/or fractions can be administered to a patient or to an animal as a therapeutic agent.
  • the method involves freeze drying the berries, followed by pulverization into a powder, then exposing the resultant extract to low temperature, and removing an amount of water content, e.g., under a vacuum (e.g., about half an atmosphere, e.g., 380 millitorr, e.g., by sublimation), thereby resulting in a freeze-dried extract enriched for antioxidant activity and other beneficial compounds.
  • a vacuum e.g., about half an atmosphere, e.g., 380 millitorr, e.g., by sublimation
  • the berry extract is then exposed to an organic solvent to produce an extract/solvent mixture, and the solvent portion of the extract/solvent mixture is then removed, thereby producing an isolated berry extract fraction substantially free of solvent, e.g., greater than 90% free of solvent, preferably, greater than 99% free of solvent.
  • solvent is well tolerated by an animal, e.g., ethanol
  • the solvent concentration can remain as high as appropriate to deliver the beneficial components to the animal (e.g., fractions in 50% ethanol).
  • solvents include dichloromethane, methanol, ethanol, acetone, and combinations thereof, with preferred combinations being about a 1:1 combination of dichloromethane and methanol, about a 1 :1 combination of dichloromethane and ethanol, about a 1:1 combination of acetone and methanol, or about a 1 : 1 combination of acetone and ethanol.
  • Fractions derived from an extract preferably represent at least about 50 to 55% of the starting extract material.
  • the berry extract or extract fraction of the above method is enriched, by about 1-5 fold, preferably 5-10 fold, more preferably by about 10 fold or greater, for antioxidant activity and the presence of, e.g. , one or more of the following: a vitamin (e.g., vitamin A, vitamin E, vitamin C, folic acid), carotenoid (e.g., ⁇ -carotene, ⁇ -carotene, zeaxanthin, and lutein), a phenolic compound (e.g., ellagic acid, ferulic acid, anthocyanin, cyanidin, quercetin, pelargonidin, kaempferol, and analogs thereof), phytosterol (e.g., ⁇ -sitosterol, campesterol, stigmasterol, and analogs thereof), and a mineral (e.g., calcium, magnesium, potassium, zinc, and selenium).
  • a vitamin e.g., vitamin A, vitamin E, vitamin C,
  • the extracts of the methods (or fractions thereof) enriched for, e.g., antioxidant activity are suitable for use in a foodstuff, a dietary supplement, or a pharmaceutical composition. Accordingly, the extracts of the invention (or fractions thereof) can be used in the treatment of a subject in need of an antioxidant therapy or having an antioxidant responsive disease or condition, such that treatment is achieved.
  • Figure 3 shows the inhibition of oral cancer in the hamster cheek pouch (HCP) in hamsters fed diets containing 5% and 10% black raspberry extracts as compared to controls.
  • Figure 4 shows the inhibition of BPDE induced activator protein- 1 ( AP- 1 ) activity in mouse epidermal cells (i.e., JB-6 clone 41) transfected with AP-1 (P + l-l cells) treated with black raspberry extract fractions as compared to controls.
  • Figure 5 shows the dose-dependent inhibition of BPDE induced AP-1 activity in cells (P + l-l cells) treated with the methanol extract fraction of black raspberries.
  • Figure 6 shows the inhibition of BPDE induced NFKB (b) activity in cells (massl cells) treated with black raspberry extract fraction (RU-ME).
  • Figure 7 shows the dose-dependent inhibition of BPDE induced NFKB activity in cells (massl cells) treated with the methanol extract fraction of black raspberries.
  • Figure 8 shows the inhibition of BPDE induced activation of MAPKs and I ⁇ B ⁇ phosphorylation and degradation in mouse epidermal cells (JB-6 clone 41) treated with black raspberry extract fraction (RU-ME) as compared to controls as determined by immunoblot.
  • Figure 9 shows that the inhibitory activity of the black raspberry extract fractions is independent of BPDE induced p53-dependent transcription activity in mouse epidermal cells (JB-6 clone 41) transfected with p53 (massl cells).
  • Figure 10 shows a chromatogram obtained upon HPLC analysis of the methanol fraction of ly ophilized black raspberry extracts .
  • Figure 11 shows the chemical structure of several active compounds identified in the ly ophilized black raspberry extracts of the invention, i.e., cyanidin, quercetin, pelargonidin, and kaempferol.
  • Figures 12-14 show LC-ESI-MS chromatograms obtained upon analysis of the methanol fraction of ly ophilized black raspberries for the presence of cyanidin, quercetin, pelargonidin, and kaempferol, and sugar conjugates thereof.
  • analog as in " a compound or analog thereof, is intended to include compounds that are structurally similar but not identical to the compound, but retain some or all of the anti-cancer properties of the compound.
  • anti-cancer activity refers to the inhibition (in part or in whole) or prevention of a cancer as defined herein.
  • Anti- cancer activity includes, e.g., the ability to reduce, prevent, or repair genetic damage, modulate undesired cell proliferation, modulate misregulated cell death, or modulate mechanisms of metastasis (e.g., ability to migrate).
  • anti-hypercholesterolemic activity and “cholesterol lowering activity” refers to the ability to regulate cholesterol metabolism or reduce serum cholesterol levels in a subject.
  • antioxidants includes chemical compounds that can absorb an oxygen radical, e.g. , ascorbic acid and phenolic compounds.
  • antioxidant activity refers to a measurable level of oxygen radical scavenging activity, e.g., the oxygen radical absorbance capacity (ORAC) of an extract, fraction, or compound.
  • ORAC oxygen radical absorbance capacity
  • antioxidant responsive condition includes any disease or condition that is associated with the presence of undesired oxidation, oxygen radicals, or other free radicals.
  • berry is intended to mean a fruit with external seeds such as a strawberry (e.g., strawberries of the genus Fragaria, e.g., Fragaria ananassa) and a fruit containing clustered berries (each with a seed) such as a raspberry (e.g., a red or black raspberry, e.g., raspberries of the genus Rubus, e.g., Rubus occidentalis).
  • a strawberry e.g., strawberries of the genus Fragaria, e.g., Fragaria ananassa
  • a fruit containing clustered berries each with a seed
  • a raspberry e.g., a red or black raspberry, e.g., raspberries of the genus Rubus, e.g., Rubus occidentalis.
  • berry extract includes a berry extract isolated from its natural context (i.e., the fruit), e.g., concentrated freeze-dried berries (e.g., lyophilized).
  • isolated berry extract of the invention is enriched for the presence of increased antioxidant activity, for example, has a high oxygen radical absorbance capacity (ORAC) (e.g.
  • ORAC oxygen radical absorbance capacity
  • vitamin A vitamin E (tocochromonal) content
  • vitamin C ascorbic acid
  • folic acid other desirable components
  • other desirable components e.g., carotenoids, phenolic compounds, phytosterols, and minerals
  • berry extract fraction includes a berry extract that has been fractionated with a solvent and is, preferably substantially free of solvent (e.g., at least 80-90%o, preferably 90-99%, more preferably greater than 99%, and most preferably greater than 99.7%) as determined by standard techniques (e.g., gas chromatography), and/or off-flavors (as determined by taste and smell).
  • solvent e.g., at least 80-90%o, preferably 90-99%, more preferably greater than 99%, and most preferably greater than 99.7%
  • standard techniques e.g., gas chromatography
  • off-flavors as determined by taste and smell
  • carcinoma i.e., usually derived from epithelial cells, e.g., aerodigestive tract cancer, such as an oral, esophageal, or colon cancer
  • sarcoma usually derived from connective tissue cells, e.g., a bone or muscle cancer
  • a cancer of the blood such as a erythroleukemia (a red blood cell cancer) or leukemia (a white blood cell cancer).
  • a "malignant” cancer i.e., a malignancy
  • cardiovascular disease includes, for example, hypercholesterolemia, thrombotic disease, and artherogenic disease.
  • dietary supplement includes a compound or composition used to supplement the diet of an animal or human.
  • exogenous means the component is derived or obtained from a source other than berries.
  • Exogenous compounds suitable for adding to a berry extract of the invention (or fractions thereof) include, for example, one or more pharmaceuticals, chemotherapeutic agents, and/or radiotherapy.
  • foodstuff includes any edible substance that can be used as or in food for an animal or human.
  • Foodstuffs also include substances that may be used in the preparation of foods such as cooking oils or food additives.
  • Foodstuffs also include dietary supplements designed to, e.g., supplement the diet of an animal or human.
  • health promoting refers to the prevention or treatment of a disease or condition in a human or other animal, or to the maintenance of good health in a human or other animal, resulting from the administration of a berry extract (or fraction thereof) of the invention, or a composition derived therefrom.
  • health benefits can include, for example, nutritional, physiological, mental, and neurological health benefits.
  • hypocholesterolemia refers to abnormally high serum levels of cholesterol, typically due to defective cholesterol metabolism in a subject or diet.
  • isolated refers to the removal or change of a composition or compound from its natural context, e.g., the berry.
  • mineral includes, e.g. , any mineral that is naturally present at some measurable level in the berry extracts (or fractions thereof) and includes, e.g., calcium, magnesium, potassium, zinc, and selenium.
  • composition refers to a composition formulated for therapeutic use and may further comprise, e.g. , a pharmaceutically acceptable carrier.
  • pharmaceutically effective amount refers to an amount effective to achieve a desired therapeutic effect, such as lowering tumor incidence, metastasis, undesired lipid levels in the blood, preventing thrombosis, preventing or treating inflammatory diseases, immunoregulatory diseases, fever, edema, cancer, or signs of aging.
  • phenolic compound includes a compound that has an aromatic acid having one or more hydroxyl groups on the benzene ring and is naturally present at some measurable level in the berry extract (or fraction thereof) and includes, for example, ellagic acid, ferulic acid, anthocyanin, cyanidin, quercetin, pelargonidin, kaempferol, and analogs thereof.
  • the term "physically disrupting” includes any appropriate physical manipulation (e.g., by mechanical means, e.g., using a masher, juicer, pulper, or, e.g., by sonication) that breaks (e.g., decharacterizes) the fruit into, e.g., skin, seeds and juice, e.g., into a puree.
  • the term "phytosterol” includes any sterol e.g., that is naturally present at some measurable level in the berry extracts (or fractions thereof) and includes, for example, ⁇ - sitosterol, campesterol, stigmasterol, and analogs thereof.
  • vitamin includes, for example, vitamin A, vitamin E, vitamin C, folic acid, but also any other art recognized vitamins.
  • vitamin A generally includes retinal, retinol, retinoic acid, or a combination thereof.
  • vitamin C generally refers to ascorbic acid.
  • vitamin E generally includes tocochromanol compounds such as tocopherol or tocotrienol compounds.
  • the present invention is based on the identification of therapeutic berry extracts, for example, strawberry and black raspberry extracts, and fractions or compounds isolated from the extracts (e.g., a berry extract fraction), having novel therapeutic and/or health promoting value.
  • therapeutic berry extracts of the invention (and fractions thereof) are shown herein to exhibit significant anti-cancer and anti- hypercholesterolemia activity when administered to a subject in vivo and when tested in vitro.
  • therapeutic methods of the invention employ physically disrupted berry fruit, preferably a puree free of cap stems, which is freeze- dried to produce a berry extract substantially free of water content and is enriched for a number of health promoting compounds and exhibits e.g., significant anti-cancer properties when administered to a subject.
  • berry fruit preferably a puree free of cap stems
  • a berry extract substantially free of water content and is enriched for a number of health promoting compounds and exhibits e.g., significant anti-cancer properties when administered to a subject.
  • health promoting compounds derived from prepared berry have been identified and are discussed below.
  • therapeutic methods of the invention employ berry products (e.g., extracts, or fractions thereof) which are novel sources of compounds having significant therapeutic value, in, for example, the prevention or treatment of cancer, particularly, aerodigestive cancers.
  • berry products e.g., extracts, or fractions thereof
  • a subset of these berry derivatives are also enriched with compounds suitable for treating cardiovascular disease related to, for example, high cholesterol (hypercholesterolemia).
  • therapeutic methods of the invention employ compounds derived from berry extracts which, as shown herein, have anticancer activity (e.g., reduced metastasis rates) e.g., when prepared in concentrated form and administered to a mammal in vivo.
  • the identification of particular beneficial compounds in berry extracts and derivatives thereof has allowed for the development of convenient methods and compositions (e.g. , formulations) for administering therapeutic compounds to treat or prevent particular diseases.
  • the therapeutic compounds and compositions described herein have the additional advantage of being readily manufactured into palatable forms (e.g., as foodstuffs such as juices and food bars or as dietary supplements) for convenient oral administration.
  • Methods for obtaining and preparing the berry extracts of the invention, identifying (e.g., characterizing) and obtaining therapeutic components of the products, evaluating biological activity in vitro and in vivo of the products and components, and methods of using the products and novel compositions containing the products or combinations of components isolated from the products are discussed in the following subsections.
  • Berry extracts of the invention may be isolated from whole berry, preferably freshly harvested berries, using any suitable art recognized method.
  • Preferred derivatives include berry extract, or fractions thereof, that optionally, have been freeze-dried.
  • the berries may be freeze-dried using any art recognized method.
  • the berries are freeze-dried by first physically disrupting the berries resulting in a puree which is then further processed to be substantially free of impurities or undesired solids, e.g., stems.
  • the puree is then poured into a shallow vessel and quickly exposed to low temperature, i. e. , flash frozen, for example at -20°C or lower, preferably under a vacuum for removal of water content (lyophilization).
  • the resultant berry extract as compared to the native fruit by weight, is typically enriched for, e.g., antioxidant activity, antioxidant compounds, and other compounds described herein, by a factor of at least about 1-5 (i.e., -100-500%), preferably, by a factor of at least about 5-10 (/. e. , -500-1000%), more preferably by a factor of at least about 10 or more (i.e., -1000% or more).
  • the resultant extract i.e., lyophilized
  • an isolated berry extract results that is suitable for use in a foodstuff, dietary supplement, or pharmaceutical composition.
  • the berry extracts or fractions are preferably obtained in a form suitable for use in a foodstuff, dietary supplement, or pharmaceutical composition.
  • any of the techniques for preparing a berry extract or derivative described herein it may also be desirable to avoid exposing the derivative, or component thereof, to oxygen by, e.g., protective blanketing of the derivative or component with an inert gas (e.g., carbon dioxide or nitrogen gas), or by, e.g., exposing the derivative or component, where appropriate, to low temperature, a stabilizer, or a combination of these conditions.
  • an inert gas e.g., carbon dioxide or nitrogen gas
  • berry extracts including strawberry and black raspberry (including fractions thereof) were analyzed for health promoting antioxidant activity and various compounds. These berry extracts were analyzed using both chemical analysis and bioactivity assays as described herein. In addition, a number of berry extract fractions were also studied for their in vitro and in vivo therapeutic activity and analyzed for health promoting compounds (see Tables 1-3).
  • berry extracts are novel sources of therapeutically beneficial antioxidant activity (e.g., as measured by the oxygen radical absorbance capacity (ORAC) of the extracts) as well as compounds such as a vitamin A, vitamin E (tocochromanols), vitamin C (ascorbic acid), folic acid, carotenoids, phenolic compounds, phytosterols, minerals, and combinations thereof.
  • ORAC oxygen radical absorbance capacity
  • the berry extracts prepared as described herein provide several advantages over currently known sources of such therapeutically beneficial compounds including, for example, remarkably high levels of antioxidant activity as well as the presence of many desirable components. Accordingly, the berry extracts of the invention, or components thereof, can be used in foodstuffs, dietary supplements, and pharmaceutical compositions.
  • the invention provides a berry extract, for example, a strawberry or black raspberry extract, or a composition comprising one or more components of such an extract, as listed, respectively, in Tables 2 and 3, which promotes health in a human or other animal.
  • the berry extracts or composition derived therefrom are also preferably substantially enriched for antioxidant activity, for example, possess a high value for oxygen radical absorbance capacity (ORAC) as shown in Table 1.
  • ORAC oxygen radical absorbance capacity
  • the berry extracts or composition derived therefrom also can contain one or more exogenous (i.e., externally added) compounds to further enhance the therapeutic value of the berry extracts or composition derived therefrom, for example, by acting in synergism with one or more native components of the berry extract.
  • the strawberry and black raspberry extracts of the invention can contain one or more of the following compounds: vitamins (e.g., vitamin A, vitamin E, vitamin C, and folic acid); carotenoids (e.g., ⁇ -carotene, ⁇ -carotene, zeaxanthin, lutein); phenolic compounds (e.g., ellagic acid, ferulic acid, anthocyanin, cyanidin, quercetin, pelargonidin, kaempferol, and analogs thereof); phytosterols (e.g., ⁇ -sitosterol, campesterol, and stigmasterol, and analogs thereof); and minerals (e.g., calcium, magnesium, potassium, zinc, and selenium).
  • exogenous compounds such as other vitamins (e.g., vitamins underrepresented) and /or chemotherapeutic agents, can be added to the berry extracts of the invention and compositions derived therefrom, to achieve a synergistic effect.
  • the berry extracts of the invention contain high levels of antioxidant activity as measured by the oxygen radical absorbance capacity (ORAC) of the extracts.
  • ORAC oxygen radical absorbance capacity
  • black raspberry extracts are especially enriched for such antioxidants. Accordingly, the berry extracts have a high antioxidant activity (in addition to other properties discussed herein).
  • an important activity found in the berry extracts of the invention is antioxidant activity, e.g., as determined by the oxygen radical absorbance capacity (ORAC) value found for each extract.
  • ORAC oxygen radical absorbance capacity
  • the berry extracts of the invention (and fractions thereof) have the advantage of being potent delivery systems for antioxidants.
  • Antioxidants include, e.g., vitamin E, vitamin C, and phenolic compounds.
  • the berry extracts of the invention also contain vitamin A which generally includes any member (or combination thereof) of a family of fat-soluble vitamins such as retinol, retinal, and retinoic acid. These compounds play an important role in vision, bone growth, reproduction, cell division and differentiation, immunoregulation, and lowering cancer risk.
  • vitamin A generally includes any member (or combination thereof) of a family of fat-soluble vitamins such as retinol, retinal, and retinoic acid.
  • Vitamin E Tocochromanols
  • the berry extracts of the invention also contain vitamin E which generally comprises tocochromanols (a class of compounds that includes tocopherols and tocotrienols).
  • vitamin E generally comprises tocochromanols (a class of compounds that includes tocopherols and tocotrienols).
  • the berry extracts of the invention and compositions derived therefrom can be used to treat respiratory, inflammatory, neurological, dermatological, opthalmological, and gastroenterological diseases.
  • the amount of vitamin E (tocochromanols) determined to be in the berry extracts of the invention is present at high levels in both strawberry and black raspberry extracts (respectively, 5-6 mg/lOOgm; -11 mg/lOOgm).
  • vitamin E as an anticarcinogenic agent has been recognized for a number of years (Haenszel et al., Int. J. Cancer, 36:43-48 (1985); Menkes et al, N. Engl. J. Med., 315:1250-1204 (1986); Stahelin et al. , Ann. NY Acad. Set, 570:391-399 (1989)).
  • in vitro and in vivo studies, including human studies have demonstrated that vitamin E interferes with the development of carcinogenesis that results from exposure to various environmental factors known to enhance oxidant stress (Borek et al.
  • the berry extracts of the invention also contain vitamin C (ascorbic acid) which can function as an antioxidant. Vitamin C is also useful for promoting healthy teeth and gums, absorption of iron, maintenance of connective tissue and the immune system.
  • vitamin C ascorbic acid
  • Berry extracts of the invention also contain measurable levels of folic acid which acts a coenzyme (with other vitamins (vitamins B-12 and vitamin C) in the metabolism of proteins and in the synthesis of new proteins) and is necessary for the production of red blood cells and the synthesis of DNA, tissue growth and cell function. Adequate levels of folic acid are required to prevent neural tube defects during human embryogenesis.
  • Carotenoids act a coenzyme (with other vitamins (vitamins B-12 and vitamin C) in the metabolism of proteins and in the synthesis of new proteins) and is necessary for the production of red blood cells and the synthesis of DNA, tissue growth and cell function. Adequate levels of folic acid are required to prevent neural tube defects during human embryogenesis.
  • Carotenoids are required to prevent neural tube defects during human embryogenesis.
  • Berry extracts of the invention also contain measurable levels of carotenoids.
  • Typical carotenoids found within the berry extracts of the invention include ⁇ -carotene, ⁇ -carotene, zeaxanthin, and lutein.
  • the health promoting effects of the carotenoids of the invention include reducing the risk of developing several kinds of cancer, including stomach, colorectal, esophagus, larynx, and lung cancer.
  • Berry extracts of the invention also can contain one or more phenolic compounds, such as ellagic acid, ferulic acid, and anthocyanins (but also, e.g., hydrobenzoic acid, hydroxy cinnamic acid, flavonoids (e.g., cyanidin, quercetin, pelargonidin, kaempferol, and analogs thereof), flavanols, flavan-3-ols, and/or tannins).
  • phenolic compounds can act as potent antioxidants and, therefore, can prevent or delay oxidation reactions which cause various diseases.
  • the berry extracts of the invention and compositions derived therefrom e.g., certain extract fractions
  • the berry extracts of the invention and compositions derived therefrom can be used to prevent cancer with few side effects.
  • the black raspberry and strawberry extracts of the invention contain significant quantities of various polyphenols including ellagic acid, ferulic acid, and multiple anthocyanins.
  • Ellagic acid alone has demonstrated inhibitory effects against skin, lung, liver, esophagus and colon cancer in animals.
  • ellagic acid activates Hageman factor (involved in blood clotting); inhibits replication of certain DNA viruses such as adenovirus and herpesvirus; inhibits enzymes involved in the synthesis of HTLV-3 (AIDs) virus; inhibits the bioactivation and stimulates the detoxification of certain chemical carcinogens; scavenges the ultimate carcinogenic metabolite of benzo(a) ⁇ yrene, a ubiquitous environmental carcinogen; exhibits antimutagenic activity in the AMES mutagenesis assay; and, has therapeutic effects against tumors in animals.
  • ellagic acid is a strong antioxidant. Ferulic acid also exhibits antimutagenic and antioxidant activity. The anthocyanins impart color to berries and many are polyphenols that exhibit antioxidant activity. Phytosterols
  • the berry extracts of the invention can contain one or more phytosterols (plant sterols), including, but not limited to, ⁇ -sitosterol, campesterol, and stigmasterol, and analogs thereof.
  • phytosterols have been shown to inhibit the absorption of cholesterol from the intestine, and decrease blood serum cholesterol. It has been proposed that, in the intestine, phytosterols act by reducing the solubility of cholesterol in the lipid and micellar phases with a consequential decrease in cholesterol absorption. Plant sterols are also reported to inhibit colon cancer and breast cancer development.
  • the berry extracts of the invention and compositions derived therefrom can be used, for example, in the treatment of patients with cardiovascular disease or as chemopreventative agents against colon cancer and breast cancer.
  • Berry extracts of the invention also contain high levels of minerals.
  • Typical minerals found within the berry extracts of the invention include calcium, magnesium, potassium, zinc, and selenium.
  • the health promoting effects of minerals found within the extracts of the invention include, for example, reducing osteoporosis and cancer risk (calcium), maintaining electrolyte balance (magnesium and potassium), maintaining immune system function (zinc), and reducing cancer risk (selenium).
  • phenolic compounds of the strawberry and raspberry extracts and derivatives of the invention can be analyzed and extracted using HPLC analysis and solvent extraction, respectively.
  • the isolated extracts can be dissolved in an organic solvent, for example, methanol (or ethanol, which can be administered to animals, e.g. , humans) and then extracted with a methanol/water solution (or ethanol/water) followed by centrifugation.
  • the extract can then be dried, and the residue can be resuspended in methanol/water for HPLC analysis.
  • extracts for example, carotenoids, phenolic compounds, phytosterols
  • TLC thin-layer chromatography
  • HPLC gas and high-performance liquid chromatography
  • Such isolated components which can be separated as "value added" fractions (e.g., fractions having therapeutic value), are typically rich in at least one beneficial component identified from the berry extracts or factions thereof described herein. These isolated components or fractions may be further combined to provide a composition rich in more than one component or, e.g., a desired combinations thereof.
  • a particular formulation intended for the treatment or prevention of a particular disease or condition may be formulated to be rich in those components having a therapeutic effect on the disease or condition (e.g. , associated with affecting a change in any of the mechanisms associated with that particular disease or condition).
  • a formulation suitable for administering to a subject with cancer is preferably rich in berry extract-derived components having antioxidant activity and other anticancer properties
  • a formulation for administering to a subject with cardiovascular disease e.g., hypercholesterolemia
  • a subject with a dietary need may be administered a formulation rich in, for example, beneficial vitamins or minerals.
  • the strawberry or raspberry extracts of the invention, and compositions derived therefrom can be tested for their in vivo therapeutic effect by administering (e.g., orally) the extracts or compositions in a suitable form (e.g., as a food stuff, dietary supplement, or pharmaceutical composition) to a human or other animal, and then observing the physiological effect (e.g. , compared to a control).
  • a suitable form e.g., as a food stuff, dietary supplement, or pharmaceutical composition
  • the human or animal can be, for example, suffering from a disease or condition, such as those described herein (e.g. , cancer or hypercholesterolemia).
  • a reduction in the physical symptoms of the disease can be measured as an indication of the therapeutic efficacy of the strawberry or raspberry extracts or compositions derived therefrom.
  • strawberry or raspberry extracts of the invention or compositions derived therefrom can be used in a controlled animal study where tumors are induced in the animal via diet (or by other appropriate routes such as injection, e.g., by intraperitoneal, subcutaneous, or intravenous injection), by applying a chemical tumor promoter to the skin, or by the implantation of tumor cells in the presence or absence of the test agent.
  • Various assays such as those described below, can then be used to examine the progression of carcinogenesis in the presence or absence of the administration of the extracts or compositions of the invention.
  • the health promoting properties of berry extracts of the invention and compositions derived therefrom also can be evaluated using a variety of art-recognized cell-based assays.
  • the antioxidant effects on cells caused by exposure to a berry extract of the invention or a composition derived therefrom can be determined by an oxygen radical absorbance capacity (ORAC) assay or electron spin resonance technology as described herein.
  • ORAC oxygen radical absorbance capacity
  • the extracts of the invention have enriched antioxidant activity as measured by either of these technologies.
  • the extracts of invention were also found to be non-toxic even at high doses.
  • a berry extract of the invention and compositions derived therefrom can be administered to a human or other animal to treat or prevent a variety of cancers.
  • the extracts of the invention are especially well-suited for inhibiting the development of cancers of the aerodigestive tract in animals and humans such as oral, laryngeal, pharyngeal, esophageal (squamous cell carcinoma and adenocarcinoma), stomach, and colon cancer.
  • Other disease indications include preneoplastic lesions in humans such as epithelial dysplasia of the esophagus, development of Barrett's esophagus, oral leukoplakia and erythroplakia, and colonic polyps.
  • the extract and compositions derived therefrom also can be administered in combination with other anti-cancer agents.
  • the berry extracts of the invention and compositions derived therefrom can be administered with other nutrients, chemotherapy, and/or radiotherapy for the treatment of, for example, an aerodigestive cancer.
  • chemopreventive agents suitable for coadministration for inhibiting development of tumors include glutathione, beta-carotene, limonin, retinyl acetate, Ocimum sanctum, diallyl sulfide, vitamin E, protease inhibitors from soybeans, ibuprofen, green coffee beans, green tea polyphenols, curcumin, quercetin, and mint.
  • beta-carotene retinyl acetate, Ocimum sanctum, diallyl sulfide, retinoids, protease inhibitors, green tea, curcumin, and similar synthetic compounds are suitable for preventing tumor formation when given post-initiation, i.e., after exposure to a chemical carcinogen.
  • berry extracts of the invention and compositions derived therefrom can be used to treat or prevent heart disease.
  • vitamin E tocochromonals
  • the berry extracts of the invention can be used in the treatment of high cholesterol (cholesterolemia) and other associated conditions such as heart disease.
  • high cholesterol cholesterol
  • other associated conditions such as heart disease.
  • the berry extracts of the invention and compositions derived therefrom can be used in the treatment or prevention of a wide range of other diseases and disorders that include, respiratory, inflammatory, neurological, dermatological (e.g., actinic keratosis and dysplastic nevi of the skin, skin cancer), cardiovascular disease, stroke, inflammatory diseases (e.g., arthritis), as well as inhibiting aging.
  • other diseases and disorders include, respiratory, inflammatory, neurological, dermatological (e.g., actinic keratosis and dysplastic nevi of the skin, skin cancer), cardiovascular disease, stroke, inflammatory diseases (e.g., arthritis), as well as inhibiting aging.
  • a large volume of reported research provides evidence that antioxidants (and other compounds, e.g., vitamin E) play a critical role in the above-mentioned conditions.
  • the berry extracts of the invention and compositions derived therefrom having both of these properties are especially well suited for the prevention and/or treatment of a broad spectrum of biological conditions.
  • such extracts and compositions of the invention also are well suited to the treatment of any yet to be characterized biological disorders or diseases that, at some level, are affected by or controlled by a mechanism associated with these properties.
  • Hypercholesterolemic diseases and conditions that can be treated using the berry extracts of the invention and compositions derived therefrom include, but are not limited to, atherosclerosis, arteriosclerosis, xanthomatosis, hyperlipoproteinemias, and familial and hypercholesterolemia.
  • Thrombotic diseases and conditions that may be treated using berry extracts of the invention and compositions derived therefrom include, but are not limited to, pulmonary disease (for example, involving reduced conductance, compliance, or constriction), excessive fluid accumulation or pulmonary edema, respiratory distress, asthma, pulmonary vascular permeability, pulmonary vasoconstriction, pulmonary hypertension, pulmonary embolism, cardiac ischemia, myocardial infarction, cardiopulmonary bypass associated dysfunction, vasoconstriction, organ dysfunction, platelet dysfunction, cardiac disease, chronic obstructive arterial disease caused by arteriosclerosis, vasoconstriction, renal artery stenosis, myocardial infarction, stroke, deep vein thrombosis, peripheral arterial occlusion, and other blood system thromboses.
  • pulmonary disease for example, involving reduced conductance, compliance, or constriction
  • excessive fluid accumulation or pulmonary edema for example, involving reduced conductance, compliance, or constriction
  • respiratory distress asthma
  • Antiatherogenic diseases and conditions that can be treated using berry extracts of the invention and compositions derived therefrom include, but are not limited to, atherosclerosis, arteriosclerosis, myocardial infarction, ischemia (i.e., myocardial ischemia, brain ischemia, and renal ischemia) and strokes.
  • Inflammatory diseases and conditions that can be treated using berry extracts of the invention and compositions derived therefrom include, but are not limited to, essential hypertension, hypertension of congestive heart failure, renal dysfunction caused by reduced myocardia output, endotoxemia, chronic liver disease or hypertension, pulmonary inflammation in asthma, lung injury (bronchitis, pneumonia, or acute); rheumatic diseases (for example, rheumatoid arthritis or systemic lupus erythematosus), inflammatory bowel disease (for example, ulcerative colitis), irritable bowel disease (such as villous adenoma), gastrointestinal disorders caused by excess acids, pepsin or bile salts, skin diseases or trauma (such as burns or acid or caustic injury), rheumatoid diseases.
  • rheumatic diseases for example, rheumatoid arthritis or systemic lupus erythematosus
  • inflammatory bowel disease for example, ulcerative colitis
  • Immunoregulatory diseases and diseases that can be treated using berry extracts of the invention and compositions derived therefrom include, but are not limited to, autoimmune diseases, for example, AIDS, chronic fatigue syndrome, graft rejections, and other viral diseases that impair the immune system.
  • the extracts of the invention are capable of inhibiting any of the diseases or conditions described herein through the modulation, for example, via its antioxidant. activity, of one or more mechanisms.
  • Such mechanisms include, modulation of a chemical carcinogen prior to its metabolism or contact with a cell; modulation of the metabolism of a carcinogen, modulation of a carcinogen metabolite (e.g., by scavenging or binding to the metabolite before it can cause oxidative damage of a lipid, protein, or genetic material); and/or modulation of a cellular pathway (e.g., signal transduction or gene transcription).
  • berry extracts of the invention are administered to a subject with an additional (exogenous) compound, e.g., an anti-cancer agent such as a chemotherapeutic compound and/or in combination with, for example, radiotherapy for the treatment of cancer.
  • an additional (exogenous) compound e.g., an anti-cancer agent such as a chemotherapeutic compound and/or in combination with, for example, radiotherapy for the treatment of cancer.
  • an additional (exogenous) compound e.g., an anti-cancer agent such as a chemotherapeutic compound and/or in combination with, for example, radiotherapy for the treatment of cancer.
  • an additional (exogenous) compound e.g., an anti-cancer agent such as a chemotherapeutic compound and/or in combination with, for example, radiotherapy for the treatment of cancer.
  • Administration of berries or their fractions along with chemotherapeutic drugs can permit more long-term, low-dose treatment of cancer patients with chemotherapy.
  • the berry extracts of the invention and compositions derived therefrom can be administered to a subject in any suitable form.
  • the extracts and compositions of the invention are sufficiently stable such that they can be readily prepared in a form suitable for adding to various foodstuffs including, for example, juice, fruit drinks, carbonated beverages, milk, nutritional drinks (e.g., EnsureTM, MetracalTM), ice cream, breakfast cereals, biscuits, cakes, muffins, cookies, toppings, bread, bagels, fiber bars, soups, crackers, baby formulae (e.g., SimilacTM), teas, salad dressings, cooking oils, and meat extenders.
  • the berry extracts of the invention may also be delivered in the form of jellies, jams, or preserves.
  • berry extracts of the invention and compositions derived therefrom can be formulated as a pharmaceutical composition (e.g., a medicinal drug) for the treatment of specific disorders.
  • a pharmaceutical composition e.g., a medicinal drug
  • berry extracts of the invention and compositions derived therefrom can be formulated as a dietary supplement.
  • compositions may take the form of tablets, capsules, emulsions, suspensions and powders for oral administration, sterile solutions or emulsions for parenteral administration, sterile solutions for intravenous administration and gels, lotions and cremes for topical application.
  • the pharmaceutical compositions may be administered to humans and animals in a safe and pharmaceutically effective amount to elicit any of the desired results indicated for the compounds and mixtures described herein.
  • the extracts of the invention may be used in cosmetics.
  • compositions of this invention typically comprise a pharmaceutically effective amount of a berry extract or fraction thereof containing, for example, a berry extract with antioxidant activity, and, if suitable, a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier may be solid or liquid, such as, for example, cornstarch, lactose, sucrose, olive oil, or sesame oil. If a solid carrier is used, the dosage forms may be tablets, capsules or lozenges. Liquid dosage forms include soft gelatin capsules, syrup or liquid suspension.
  • Therapeutic and prophylactic methods of this invention comprise the step of treating patients or animals in a pharmaceutically acceptable manner with the compositions and mixtures described herein.
  • compositions of this invention maybe employed in a conventional manner for the treatment and prevention of any of the aforementioned diseases and conditions. Such methods of treatment and prophylaxis are well- recognized in the art and may be chosen by those of ordinary skill in the art from the available methods and techniques. Generally, dosage ranges may be from about 1 to about 1000 mg/day. However, lower or higher dosages maybe employed. The specific dosage and treatment regimens selected will depend upon factors such as the patient's or animal's health, and the severity and course of the patient's (or animal's) condition and the judgment of the treating physician.
  • a diet is formulated to include the freeze dried berry powders of the invention in a concentration from about 1% to about 25% by weight. In a preferred embodiment, the concentration is about 5% by weight. In another preferred embodiment, the concentration is about 10%. In yet another preferred embodiment, the concentration is about 15%. In still another embodiment, the concentration is about 20%.
  • the berry extracts of the invention and compositions derived therefrom also can be used in combination with conventional therapeutics used in the treatment or prophylaxis of any of the aforementioned diseases.
  • Such combination therapies advantageously utilize lower dosages of those conventional therapeutics, thus avoiding possible toxicity incurred when those agents are used alone.
  • other nutrients or medications for example, cholesterol lowering drugs, chemotherapeutic agents, and/or radiotherapy.
  • the berry extracts of the invention and compositions derived therefrom can be used with any suitable carrier or edible additive.
  • the berry extracts of the invention may be used in foodstuffs, such as baked goods (for example, breads, muffins, and pastries), and cereals.
  • the berry extracts of the invention and compositions derived therefrom also can be emulsified and used in a variety of water-based foodstuffs, such as drinks, for example, juice drinks, sports drinks, and drink mixes.
  • the above-mentioned foodstuffs may be included in low fat, low cholesterol, or otherwise restricted dietary regimens.
  • compositions, dietary supplements, and foodstuffs of the present invention can be administered to humans and animals such as, for example, livestock and poultry.
  • the practice of the present invention employs, unless otherwise indicated, conventional techniques of chemistry, e.g., food chemistry.
  • Other techniques for carrying out the invention for example, for preparing fruit extracts (and fractions thereof) and performing animal or cell-based assays for determining the anti-cancer properties of an extract (or fractions thereof), can be found, for example, in: Carlton et al, Carcinogenesis, 22:441-446 (2001); Stoner et al, Tox. Sciences Supp. 95-100 (1999); Carlton et al, Cancer Letter, 159:113-117 (2000); Xue et al, Carcinogenesis, 22:351-356 (2001); Harris et al, Proc. Amer. Assoc. Can.
  • Extract fractions were carried out as described in the examples (e.g., Example 3) and as diagrammed in the figures of the application (Fig.
  • the alcohol fraction isolated using methanol (ME) is isolated using ethanol (Et) in some studies.
  • Ethanol is a preferred extraction vehicle for extracts intended for human administration.
  • Antioxidant activity was typically measured as a function of the oxygen radical absorbance capacity (ORAC) of the sample which was determined using standard techniques. Briefly, in the ORAC assay mixture, B-PE was used as a target of free radical change, AAPH as a peroxyl radical generator, and Trolox as a control standard, and fluorescence (at, e.g., the following wavelengths of 540 nm (excitation) and 565 nm
  • ESR electron spin resonance
  • compounds from the berry extracts of the invention are analyzed using art-recognized techniques such as solvent extraction and HPLC analysis.
  • Components of the extracts for example, carotenoids, phenolic compounds, phytosterols can be extracted and analyzed using, for example, thin layer chromatography and high- performance liquid chromatography.
  • the material can be fractionated on thin-layer chromatography (TLC) plates where the individual bands that are subsequently resolved can be scraped and extracted with a chloroform/methanol solvent. These resultant samples can then be analyzed using, e.g., gas and high-performance liquid chromatography (HPLC).
  • chromatographic techniques may be used for isolating components of the berry extracts of the invention, in sufficient and pure quantities, such that the component may be administered alone or as part of a composition or product described herein (e.g., foodstuffs, dietary supplements, pharmaceuticals, etc.).
  • gas liquid cliromatography gas solid chromatography
  • HPLC high pressure or high performance liquid chromatography
  • ion exchange chromatography or size exclusion chromatography
  • size exclusion chromatography can be employed as described, for example, in Advances in Chromatography, Brown, Eds., Marcel Dekker, Pub. (1998); Basic Gas Chromatography, Harold et al, John Wiley & Sons, Pub. (1997); Column Handbook for Size Exclusion Chromatography, Wu, Ed., Academic Press, Pub. (1999); Fundamentals of Preparative and Nonlinear Chromatography, Guichon et al, Eds., Academic Press, Pub.
  • the agent 7,12-dimethylbenz(a)anthracene (DMBA) was obtained from Sigma- Aldrich (Milwaukee,WI) and dissolved at an 0.2% concentration in dimethylsulfoxide (DMSO) obtained from Fisher Scientific, Pittsburgh, PA 4-(methylnitrosamino)-l-(3- pyridyl)-l-butanone (NNK) was obtained from Toronto Research Chemicals, Ontario, Canada and benzo(a)pyrene (BaP) from Sigma Chemical Co., St. Louis, MO. The BaP/NNK mixture was prepared at a 1% concentration in DMSO.
  • the agent NMBA obtained from Ash Stevens Corp., Detroit, Michigan, and azoxymethane, obtained from Sigma Chemical Co., were purified by HPLC to greater than 98% purity.
  • black raspberries (Jewel variety) were supplied by the Dale Stokes Berry Farm (Wilmington, OH) and shipped frozen to Van Drunen Farms (Momence IL) for freeze-drying.
  • the composition of the berry powder was determined by Covance Laboratories (Madison, WI) and is presented in Table 1 along with the composition of two other ly ophilized black raspberry (LBR) extracts used for inhibition of rat esophageal tumors.
  • the LBR powder was mixed into a modified AIN- 76A diet at 5% and 10% concentrations with the concentration of cornstarch adjusted to maintain an isocaloric diet among all experimental groups.
  • Berry-containing and control diets were prepared every two weeks, 140-170 grams measured into pint rat feeding jars, and stored at 4°C. Two jars were placed into each cage, feeding jars were rotated with each jar being replaced every 5-8 days with fresh feed, and the before and after weights of the jars recorded. Diets comprising 5% and 10% lyophilized black raspberries (LBR) were prepared as described above and determined to comprise the following components as indicated in Table 1. Concentration of components is expressed as mg/100g of LBR: selenium is expressed as ⁇ g/100g. b Lot used for inhibition of oral tumors in HCP.
  • LBR lyophilized black raspberries
  • the three groups of animals were treated by painting both surfaces of each pouch 3 times weekly for 8 weeks with an 0.2% solution of DMBA dissolved in DMSO using a No.4 camel hair brush (Dachi et al, Cancer Res. 27:1183-1185 (1967)).
  • Group 1 animals received a diet with 5%> LBR
  • Group 2 received a diet containing 10% LBR
  • Group 3 received the ALN-76A diet alone. Tumors of sufficient mass in the control group (3-10 mm in greatest length) suitable for final analyses appeared in 70-77 days (10 to 11 weeks) after beginning DMBA treatment.
  • tumors were harvested, processed for evaluation, and final histologic examination performed after fixing and staining. Hematoxylin and eosin stained hamster cheek pouches were evaluated and histologically characterized by a board- certified oral pathologist in the College of Dentistry at The Ohio State University.
  • Assays for determining DMBA induced adduct formation were typically carried out as follows. Two groups of six hamsters each were treated with a 5%> concentration of black raspberry extract in a modified AIN-76A diet for two weeks. One day after cessation of berry treatment, both cheek pouches of each group were painted with an 0.2%) solution of DMBA in DMSO. Six control animals without berry treatment were painted with 0.2% DMBA+DMSO in the right cheek pouch or DMSO alone in the left pouch. Twenty-four and 48 hrs after DMBA or DMSO treatment, the animals were sacrificed by CO 2 euthanasia and the pouches quick frozen in liquid nitrogen.
  • DNA was isolated from the left and right cheek pouch tissue of each animal using a direct salt-precipitation method (Miller et al, Nucleic Acid Res. 16:1215 (1988); Schut et al, Cancer Lett. 67:117124 (1992)).
  • 32 P-postlabeling assays for DMBA-DNA adducts were run under intensification conditions (Randerath et al. , Carcinogenesis 6: . 1117-1126 (1985)). The assay conditions were identical to those used before (50), except for the D3 solvent that was used for the initial separation of adducts (3.5 M lithium formate, 7.0 M urea, pH 3.5) and the D5 solvent (1.0 M magnesium chloride).
  • DNA adduct levels were expressed as relative adduct labeling (RAL) values, after correction of the ⁇ RAL> values obtained under intensification conditions.
  • RAL relative adduct labeling
  • Cell-based assays were carried out using art-recognized techniques as described in the examples, and for example, as described in Xue et al, Carcinogenesis, 22:351- 356 (2001).
  • mouse epidermal cells i.e., JB-6 clone 41
  • AP-1-luciferase reporter gene construct P + l-l cells
  • NF ⁇ B-luciferase reporter gene construct Cl 41 NFKB massl cells
  • a p53-luciferase reporter gene construct Cl 41 PG13 massl cells
  • the cultures were dissociated with trypsin and transferred to new 75 cm culture flasks (Fisher, Pittsburgh, PA) from one to three times per week.
  • the substrate for the luciferase assay was obtained from Promega (Madison, WI); BPDE was obtained from Sigma (St. Louis, MO); and the phospho-specific antibodies against various phosphorylated sites of ERKs, p38 kinase, JNKs, and I ⁇ B ⁇ were obtained from New England Biolaboratories (Beverly, MA).
  • radiolabel ( ⁇ )-r-7, t-8-dihydroxy-t-9,10- epoxy-7,8,9,10-tetrahydro[l,3- H]benzo[a]pyrene [ H]-BPDE, specific activity, 2210 mCi/mmol) was obtained from ChemSyn Science Laboratories (NCI Chemical Carcinogen Repository, Kansas City, MO).
  • the AP-1 activity assay was typically performed using confluent monolayers of P + l-l cells cultured under standard conditions and subsequently incubated with different fractions of black raspberry extract dissolved in DMSO for 30 min at concentrations ranging from 1-100 ⁇ g/ml. Cells were then exposed to BPDE at a final concentration of 2 ⁇ M. The cells were extracted with lysis buffer (Promega, Madison, WI) at various periods of time (6-48 h) after BPDE exposure, and the luciferase activity was determined by the Luciferase assay using a luminometer (Wallac 1420 Victor 2 multilable counter system) after the addition of lysis buffer. The results are expressed as AP-1 activity relative to control medium containing DMSO (0.1% v/v) only (Relative AP-1 activity).
  • Immunoblots were performed with either phospho-specific antibodies or non- phosphorylated antibodies against various kinases, including ERKs, JNKs and p38 kinase, and also against IKBCC.
  • the protein band specifically bound to the primary antibody was detected using an anti-rabbit IgG-AP-linked and an ECF immunoblotting system (Amersham Biosciences, Piscataway, NJ).
  • freeze-dried (lyophilized) black raspberries (Rubus occidentalis) and strawberries (Fragaria ananassa) were made as follows. Several hundred pounds of fresh, ripe black raspberries and strawberries were picked, washed, and stored frozen at - 20°C. Berry puree, free of cap stems and seeds, was prepared by passing the whole berries through a pulper-finisher fitted with a screen having 0.020-inch perforations. The waste fraction was returned to the pulper three times to assure complete juicing of the harder white shoulders of the berries. The seed was pulverized and added to the puree. The puree containing pulverized seed was poured to a depth of approximately 1 inch into freeze-dryer trays lined with polyethylene film, and then frozen in a blast freezer. The frozen plates of puree were removed and stored at -20°C for subsequent freeze drying.
  • Freeze-drying was accomplished by means of a Virtis model 50-SRC-5 Sublimator. The shelf temperature was 40°C and the vacuum was 380 millitorr. One defrost cycle was required for each batch containing about 70 pounds of puree. Approximately three days are required to dry each batch of puree. When dry, the thickest portion of each plate of dried material was visually checked for remaining ice. If ice was found, freeze-drying was continued. When the product was found to be dry, it was packaged in doubled polyethylene bags, placed in carton boxes, and stored at -20°C. The berry extracts were then used as the source material for further analysis and fractionation described below. T US03/06279
  • the berry extracts isolated using the methods of the invention described above, were subjected to a detailed analysis of its beneficial components.
  • samples of freeze-dried strawberries and black raspberries prepared as described above where analyzed for their overall antioxidant activity as well as the presence of selected vitamins, carotenoids, phenolic compounds, phytosterols and minerals.
  • the overall antioxidant activity for each extract was determined using techniques described herein and results are shown in Table 2.
  • beneficial compounds such as vitamins (e.g., Vitamin E, Vitamin C, and folic acid); carotenoids (e.g., ⁇ -carotene, ⁇ -carotene, zeaxanthin, lutein); phenolic compounds (e.g., ellagic acid, ferulic acid, and anthocyanins); phytosterols (e.g., ⁇ -sitosterol, campesterol, and stigmasterol, and analogs thereof); and minerals (e.g., calcium, magnesium, potassium, zinc, and selenium) were detected.
  • vitamins e.g., Vitamin E, Vitamin C, and folic acid
  • carotenoids e.g., ⁇ -carotene, ⁇ -carotene, zeaxanthin, lutein
  • phenolic compounds e.g., ellagic acid, ferulic acid, and anthocyanins
  • phytosterols e.g., ⁇ -sito
  • LC-ESI-MS Liquid chromatography-electrospray ionization-negative ion-mass spectrometry
  • glucoside the following known sugar conjugates of cyanidin, quercetin, pelargonidin, and kaempferol: glucoside, galactoside, glucuronide, sophoroside, and xylosylglucuronide were selected for ion monitoring analysis as shown in Figures 12-14.
  • the top panel of Figure 12 shows the UV trace
  • the second panel shows the chromatogram obtained when monitoring total ion current.
  • the third panel shows the chromatogram obtained by monitoring m/z 447, M-l of kaempferol glucoside and galactoside.
  • the peaks marked K-glu or gal (UV) also had UV spectra and MS consistent with kaempferol glucoside.
  • the fourth panel shows the results of selected ion monitoring for m/z 461, which is M-l of kaempferol glucuronide.
  • the peak marked K-gluc (UV) had UV and MS consistent with kaempferol-glucuronide.
  • the fifth panel shows selected ion monitoring for m/z 463, M-l of quercetin glucoside or galactoside.
  • the peak marked Q glu or gal (UV) had UV and MS consistent with queretin glucoside or galactoside.
  • Figures 13 and 14 show similar data, where P refers to pelargonidin and C to cyanidin. Collectively, these data demonstrate the presence of desirable flavonoids in the active fraction of freeze-dried black raspberries.
  • the RU-001 or FA-001 fractions were chromatographed on a silica gel column using dichloromethane:methanol (1 : 1) and methanol eluates, respectively.
  • the fraction eluted with dichloromethane methanol is termed the DM fraction ( . e. , RU-DM or FA-DM), and that eluted with methanol is termed the ME fraction (i.e., RU-ME or FA-ME).
  • the RU-001 or FA-001 fractions were chromatographed on a silica gel column using acetone:methanol (1:1) and methanol eluates, respectively.
  • the fraction eluted with methanol is termed the ME fraction (i.e., RU-ME or FA-ME), and that eluted with acetone:methanol was termed the AC fraction (i.e., RU-AC or FA- AC).
  • ME fraction i.e., RU-ME or FA-ME
  • AC fraction i.e., RU-AC or FA- AC
  • the various fractions derived from either freeze-dried black raspberries or strawberries were determined to have desirable health promoting activities as described below.
  • strawberry extracts (freeze-dried strawberries) prepared as described above were evaluated for their ability to inhibit chemically-induced tumors in rodents.
  • strawberry extracts added at 5% and 10% of the diet 2 weeks before, during, and after subcutaneous administration of the chemical carcinogen, N-nitrosomethylbenzylamine (NMBA), caused significant reductions in the development of both preneoplastic lesions (simple hyperplasia, low- and high-grade dysplasia) and the number of esophageal tumors per rat by 24 and 56%, respectively.
  • NMBA N-nitrosomethylbenzylamine
  • strawberry extracts added at 5% and 10%) of the diet were shown to influence the metabolism of NMBA to DNA damaging species as indicated by the observation that they reduced the formation of O - methylguanine adducts in esophageal DNA by 59 and 64%, respectively.
  • the strawberry extracts When added at 5% and 10% of the diet following subcutaneous treatment of rats with NMBA (i. e. , post-carcinogen treatment), the strawberry extracts also significantly reduced tumor multiplicity by 38 and 31%>, respectively.
  • the strawberry extracts were capable of suppressing the conversion of premalignant esophageal cells to malignant cells.
  • freeze-dried strawberries added at 10% of the diet one week before, during and after the administration of the tobacco carcinogens, benzo(a)pyrene or 4-(methylnitrosamino)-l-(3-pyridyl)-l-butanone (NNK) failed to inhibit lung tumor development in strain A/J mice.
  • the lung tumor assay in A/J mice is a model for adenocarcinoma of the human lung.
  • freeze-dried black raspberries prepared as described above were evaluated for their ability to inhibit chemically-induced tumors in rodents.
  • freeze-dried black raspberries added at 5% and 10%> of the diet 2 weeks before, during, and after subcutaneous administration of the carcinogen, N-nitrosomethylbenzylamine (NMBA) caused significant reductions in esophageal tumor multiplicity of 39 and 49%>, respectively.
  • NMBA N-nitrosomethylbenzylamine
  • black raspberry extracts added at 5% and 10%) of the diet were shown to influence the metabolism of NMBA to DNA damaging species as indicated by the observation that they reduced the formation of O - methylguanine adducts in esophageal DNA by 73 and 80%, respectively.
  • the black raspberries significantly reduced the formation of both premalignant lesions (i.e., low- and high-grade dysplasia) and the number of esophageal tumors per rat by 62 and 43%o, respectively.
  • HCP hamster cheek pouch
  • a cancer inducing agent /. e. , 0.2%> 7,12-dimethylbenz(a)anthracene in dimethylsulfoxide; hereafter DMBA
  • DMBA in DMSO solvent was administered via cheek pouch painting for 8 weeks (3x/week) beginning 2 weeks after LBR diets were started.
  • Hamsters were given 5% and 10% LBR diets during the 8 weeks of DMBA treatment and for two weeks following treatment.
  • animals in the DMBA treated and control groups (Groups 1,2 and 3) were sacrificed and cheek pouches analyzed for tumors, tumor size, and histopathologic changes.
  • organs were collected at necropsy from rats that had been fed 5%> or 10%> strawberry or black raspberry diets in the bioassays and evaluated for any gross pathology.
  • the organs collected e.g., esophagus, stomach, small intestine, colon, liver, kidney, bladder, spleen, heart and lungs
  • the organs collected were also fixed in 10%> neutral buffered formalin for subsequent histopathological analysis. Histopathological examination of these tissues revealed no abnormal changes that could be associated with consumption of either strawberry or black raspberry extracts.
  • F005, RU-DM, RU-ME) and strawberry extract fractions (FA-F001, FA-F003, FA- F004, FA-F005, FA-DM, FA-ME) isolated as described above were analyzed for anti- transformation activity in the Syrian hamster embryo (SHE) cell transformation model using benzo(a)pyrene (B[a]P) as the chemical carcinogen. None of the extract fractions by themselves produced an increase in morphological transformation.
  • SHE cells were treated with each extract fraction at doses ranging from 2-100 microgram per milliliter and B[a]P (10 microgram per milliliter) for seven days.
  • the RU-ME and FA-ME extract fractions isolated as described above produced a dose-dependent decrease in transformation as compared to B[a]P treatment only.
  • the raspberry extract fraction (RU-ME) and strawberry extract fraction (FAME) were further examined using a 24 hour co-treatment with B[a]P or a 6 day treatment following a 24 hour treatment with B[a]P. Both extract fractions significantly reduced B[a]P-induced transformation when co-treated with B[a]P for 24 hours. These results indicate that the methanol fractions from black raspberry extracts and strawberry extracts inhibit cell transformation through interference of the uptake, activation and/or detoxification of B[a]P and/or intervention of DNA binding and DNA repair.
  • the oxygen radical absorbance capacity (ORAC) assay was performed to test for the presence of antioxidant activity.
  • ORAC oxygen radical absorbance capacity
  • Both freeze-dried black raspberry and strawberry extracts were tested for antioxidant activity using the ORAC assay and as indicated in Table 3.
  • the ORAC values for both berry types was elevated.
  • electron spin resonance technology was used. Each fraction was evaluated for its ability to quench singlet oxygen and hydroxide ion (electron spin resonance (ESR)). The fractions exhibit varying abilities to quench these free radicals, and overall, are highly active when compared to control compounds with high levels of antioxidant activity.
  • a black raspberry extract was investigated for its ability to modulate transactivation of AP-1 and NFKB induced by benzo(a)pyrene diol-epoxide (BPDE), the resultant carcinogen of B(a)P, in mouse epidermal cells (i.e., JB-6 clone 41 cells).
  • BPDE benzo(a)pyrene diol-epoxide
  • JB-6 clone 41 cells mouse epidermal cells
  • RU-F004, RU-DM, and RU-ME see, e.g., Figs. 1-2) on BPDE-induced AP-1 activation on mouse P + l-l cells were examined. Specifically, P+l-1 cells were pretreated with each of four fractions (RU-F003, RU-F004, RU-DM and RU-ME) at 25 ⁇ g/ml for 30 min, and then exposed to 2 ⁇ M BPDE to induce AP-1.
  • Pretreatment of P + l-l cells with either the RU-F003, RU-DM or RU-ME fractions resulted in a significant inhibition (P ⁇ 0.05) of BPDE-induced AP-1 activity, while the RU-F004 extract had no effect (Fig. 4).
  • the RU-ME fraction was the most potent inhibitor of AP-1 activity among the extracts tested (Fig. 4), which is consistent with its potency as an inhibitor of B(a)P- induced cell transformation.
  • the RU-ME fraction was inl ibitory when added to the medium at only 1 ⁇ g/ml (Fig. 5).
  • BPDE-induced p53 -dependent activation was not affected by any of the fractions tested on massl cells (Fig. 9).
  • the most active fraction, RU-ME was added to cultured C141 cells at different times before or after exposure of the cells to 2 ⁇ M BPDE.
  • the inhibitory effect of the RU-ME fraction on both AP-1 and NFKB occurred only when RU-ME was added either before or along with the BPDE.
  • RU-ME was not effective when added to the cells 3 hours after treatment with BPDE.
  • I ⁇ B ⁇ phosphorylation in cells exposed to BPDE and RU-ME using phospho-specific antibody was determined. Results obtained indicate that pretreatment of cells with RU-ME inhibited BPDE- induced increase in phosphorylation of I ⁇ B at 90 min, and degradation of I ⁇ B ⁇ protein at 270 min, after BPDE treatment.
  • the RU-ME fraction was determined to be the most potent inhibitor of BPDE-induced AP-1 and NFKB activities among the fractions tested, which is consistent with its potency as an inhibitor of B(a)P-induced cell transformation.
  • the inhibitory effects of RU-ME on BPDE-induced activation of AP-1 and NFKB can be mediated via inhibition of MAP kinase activity and I ⁇ B ⁇ phosphorylation, respectively.
  • the hamster cheek pouch (HCP) animal model as described above was used to evaluate the ability of black raspberries to inhibit the formation of DNA adducts in the check pouches of animals treated with the cancer inducing agent, DMBA.
  • intensification conditions and using the 32 P-postlabeling technique a total of four DNA adducts could be detected in the cheek pouches of animals treated with DMBA.
  • intensification factors for adducts 1, 3, and 4 were found to be 37.7, 8.1, and 10.5, respectively.
  • a minor adduct (#2) was not detectable under standard assay conditions as it amounted to only 1.2 - 4.3 % of the total intensified adducts ( ⁇ RAL> values), except for four separate samples where it constituted 7.1- 9.8% of the total.
  • adducts 1, 3, and 4 constituted 38.8 - 59.0 %>, 21.3 - 35.9 %>, and 17.8 - 29.0%>, respectively, of the adduct burden.
  • total RAL's sum of adducts 1, 3, and 4
  • sum of specific adducts were used.
  • the cheek pouch of a model animal i.e., hamsters, hamster cheek pouch (HCP); 2-3 animals per group
  • DMBA polycyclic aromatic hydrocarbons
  • HCP hamster cheek pouch
  • HCPs Twenty-four hours after the final DMBA treatment or at 4, 7, and 10 wk of BaP/NNK treatment, hamsters were sacrificed and the HCPs were divided longitudinally into two sections, one for quick freezing and the second for histological examination.
  • Control tissues that were treated only with DMSO solvent, had a normal histologic appearance with a normal orthokeratin pattern and no evidence of a hyperproliferative or inflammatory response upon histological examination. Histopathologic examination of sections taken 24 hrs after the last DMBA treatment showed morphologic changes ranging from a mild inflammatory response to areas of focal dysplasia, as evidenced by abnormal cell maturation, increased mitotic figures, and cellular pleomorphism.
  • Control hamster cheek pouch epithelium treated for 10 weeks with the DMSO vehicle showed a uniform histology characterized by a 3-4 epithelial cell thickness, lack of defined epithelial rete ridges, hyperorthokeratosis, and un-inflamed connective tissue upon histological examination.
  • the surface epithelium showed a slight basilar hyperplasia, increased thickness of the spinous layer (acanthoid), and a mild chronic inflammatory cell infiltrate in the superficial connective tissue upon histological examination.
  • the above animal model is suitable for determining the cancer inhibiting properties of the extracts described herein.
  • the above animal model is suitable for determining the cancer inhibiting properties of the extracts described herein.
  • Male Syrian Golden hamsters, 3-4 weeks of age can be fed 5% and 10%> lyophilized black raspberries (LBR) in the diet for two weeks prior to treatment (and/or during or after treatment) with a cancer inducing agent as described above.
  • LBR lyophilized black raspberries
  • the cancer agent can be applied to the oral cavities of the animals for eight weeks after which the animals were sacrificed 12-13 weeks from the beginning of treatment and the number and volume of tumors (mm 3 ) can be determined and/or histological examination is conducted on tissue samples of the oral cavity. Significant differences in the number, volume, or incidence of tumors or the degree of tissue dysplasia determined by histological examination are evaluated in animals fed a berry extract as compared to control animals
  • the chemoprevention studies above using a mixture of the tobacco- associated carcinogens, BaP and NNK, provide the ability to evaluate the anti-cancer properties of the berry extracts of the invention in a well-defined animal system that mimics the pathologic condition of former tobacco users.
  • anthocyanin-rich black raspberry extracts were investigated for their inhibitory properties on the proliferation of normal colon cell lines and cancerous colon cell lines (HT-29). All extracts (i.e., fractions DM, FOOl, F003, F004, and ET) inhibited the proliferation of the human colon cancer cell line, HT-29, within 24h of administration of the extract.
  • colon cancer cells were more susceptible to growth inhibition by anthocyanin-rich extracts at concentrations of 5 to 50 ⁇ g/ml than normal human colon cells.
  • Cell cycle analyses indicated that progression through the cell cycle was altered in extract-treated cells as compared to untreated controls.
  • the cellular (growth inhibiting and cytotoxic) effects of phytochemicals found in black raspberry fractions was determined (i.e., FOOl, F003, DM, and ME/Et, see Figs. 1 and 2).
  • the berry extract representing -55% of the total berry components (FOOl) did not affect the growth or induce cytotoxicity in the oral cell lines.
  • partitioning and chromatography of the FOO 1 extract yielded three fractions which exhibited varying degrees of growth inhibition in the oral cell lines.
  • the water soluble F003 fraction exhibited no growth inhibiting effects to any the cell lines.
  • the FOOl berry fraction portioned into chloroform (F003) was selectively growth inhibitory to the premalignant oral cell line.
  • the fractions eluting with dichloromethane (DM) and methanol/ethanol (Me/Et) were selectively growth inhibitory to the premalignant and malignant cell lines.
  • the extracts or fractions were not observed to be cytotoxic to the cells, indicating that phytochemicals in the DM, ethanol and methanol extracts are growth inhibitory without eliciting cytotoxicity.

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Abstract

L'invention concerne des extraits de fraise isolés et leurs constituants, présentés sous une forme appropriée pour être utilisés comme denrées alimentaires, compléments alimentaires ou compositions pharmaceutiques. Ces extraits de fraise isolés ou compositions comprennent un constituant ou une combinaison de constituants dérivés des extraits de fraise, peuvent être utilisés comme agents destinés à inhiber un grand nombre de maladies ou troubles tels que, par exemple, le cancer ou une maladie cardiovasculaire. L'invention concerne en outre de nouvelles méthodes de préparation d'extraits de fraise sous une forme appropriée permettant de les ajouter à des denrées alimentaires, compléments alimentaires, ou produits pharmaceutiques.
PCT/US2003/006279 2002-03-01 2003-02-28 Compositions a base de fraise et de framboise, et utilisations therapeutiques de ces compositions WO2003074067A1 (fr)

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US12/137,952 US20130115322A1 (en) 2003-02-28 2008-06-12 Therapeutic Compositions and Methods for Treating Cell Dysplasia Using Extracts From Raspberry and Strawberry

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WO2004026325A2 (fr) * 2002-09-18 2004-04-01 Interhealth Nutraceuticals Incorporated Methode et composition d'extraits de baie riche en anthocyanine inhibant l'angiogenese et helicobacter pilori et jouant le role d'un antioxydant puissant benefique pour la sante
EP1617837A1 (fr) * 2003-04-04 2006-01-25 Cellmics Co. Ltd. Composition permettant la prevention et le traitement des maladies allergiques, comprenant un extrait de riz noir et utilisation therapeutique de cette composition
WO2007039452A1 (fr) * 2005-09-20 2007-04-12 Nestec S.A. Composition dispersible dans l'eau et procédé servant à préparer celle-ci
AU2005301315B2 (en) * 2004-07-29 2008-04-10 Michigan State University Insulin secretion by anthocyanins and anthocyanidins
US7964223B2 (en) 2005-09-27 2011-06-21 University Of Kentucky Research Foundation Berry preparations and extracts
AU2009220028B2 (en) * 2003-08-26 2012-06-14 Mannatech, Inc. Antioxidant sensor, methods and compositions
CN102697893A (zh) * 2012-05-22 2012-10-03 辽宁大学 黑树莓提取物在制备治疗胃癌药物中的应用
US8367126B2 (en) 2005-09-27 2013-02-05 University Of Kentucky Research Foundation Berry preparations and extracts
CN103142720A (zh) * 2013-02-27 2013-06-12 辽宁大学 黑树莓提取物在制备抗糖尿病药物中的应用
WO2018115381A1 (fr) * 2016-12-23 2018-06-28 Naturex Sa Compositions

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CN110452276B (zh) * 2018-05-07 2023-05-12 新加坡力汇有限公司 草莓植株萃取物、萃取方法及其用于免疫调节及抗发炎的用途

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Cited By (18)

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Publication number Priority date Publication date Assignee Title
WO2004026325A3 (fr) * 2002-09-18 2004-07-15 Interhealth Nutraceuticals Inc Methode et composition d'extraits de baie riche en anthocyanine inhibant l'angiogenese et helicobacter pilori et jouant le role d'un antioxydant puissant benefique pour la sante
AU2003272586B2 (en) * 2002-09-18 2009-05-28 Interhealth Nutraceuticals Incorporated Composition of berry extracts that prevents or inhibits angiogenesis and helicobacter pylori
WO2004026325A2 (fr) * 2002-09-18 2004-04-01 Interhealth Nutraceuticals Incorporated Methode et composition d'extraits de baie riche en anthocyanine inhibant l'angiogenese et helicobacter pilori et jouant le role d'un antioxydant puissant benefique pour la sante
EP1617837A1 (fr) * 2003-04-04 2006-01-25 Cellmics Co. Ltd. Composition permettant la prevention et le traitement des maladies allergiques, comprenant un extrait de riz noir et utilisation therapeutique de cette composition
EP1617837A4 (fr) * 2003-04-04 2008-08-06 Tg Biotech Corp Composition permettant la prevention et le traitement des maladies allergiques, comprenant un extrait de riz noir et utilisation therapeutique de cette composition
AU2009220028B2 (en) * 2003-08-26 2012-06-14 Mannatech, Inc. Antioxidant sensor, methods and compositions
AU2005301315B2 (en) * 2004-07-29 2008-04-10 Michigan State University Insulin secretion by anthocyanins and anthocyanidins
WO2007039452A1 (fr) * 2005-09-20 2007-04-12 Nestec S.A. Composition dispersible dans l'eau et procédé servant à préparer celle-ci
JP2009508508A (ja) * 2005-09-20 2009-03-05 ネステク ソシエテ アノニム 水分散性組成物及びその調製方法
JP4891325B2 (ja) * 2005-09-20 2012-03-07 ネステク ソシエテ アノニム 水分散性組成物及びその調製方法
AU2006298842B2 (en) * 2005-09-20 2012-05-10 Nestec S.A. Water dispersible composition and method for preparing same
US7964223B2 (en) 2005-09-27 2011-06-21 University Of Kentucky Research Foundation Berry preparations and extracts
US8367126B2 (en) 2005-09-27 2013-02-05 University Of Kentucky Research Foundation Berry preparations and extracts
CN102697893A (zh) * 2012-05-22 2012-10-03 辽宁大学 黑树莓提取物在制备治疗胃癌药物中的应用
CN103142720A (zh) * 2013-02-27 2013-06-12 辽宁大学 黑树莓提取物在制备抗糖尿病药物中的应用
CN103142720B (zh) * 2013-02-27 2015-09-09 辽宁大学 黑树莓提取物在制备抗糖尿病药物中的应用
WO2018115381A1 (fr) * 2016-12-23 2018-06-28 Naturex Sa Compositions
US11219657B2 (en) 2016-12-23 2022-01-11 Naturex Sa Method for treating or preventing inflammation

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