WO2005077176A1 - Composes anthocyaniniques et procedes d'utilisation correspondants - Google Patents
Composes anthocyaniniques et procedes d'utilisation correspondants Download PDFInfo
- Publication number
- WO2005077176A1 WO2005077176A1 PCT/US2005/004553 US2005004553W WO2005077176A1 WO 2005077176 A1 WO2005077176 A1 WO 2005077176A1 US 2005004553 W US2005004553 W US 2005004553W WO 2005077176 A1 WO2005077176 A1 WO 2005077176A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- anthocyanin
- derivative
- glucoside
- galactoside
- anthocyanins
- Prior art date
Links
- 0 CC(C=C[C@]1C(*)=C(*)C(*)=C(*)C1*C=C)=O Chemical compound CC(C=C[C@]1C(*)=C(*)C(*)=C(*)C1*C=C)=O 0.000 description 1
- YHNUDLCUIKMNSN-UHFFFAOYSA-N O=C([n]1ncnc1)[n]1ncnc1 Chemical compound O=C([n]1ncnc1)[n]1ncnc1 YHNUDLCUIKMNSN-UHFFFAOYSA-N 0.000 description 1
- OIJUPWXNUKTEIW-OVOGRAIMSA-N O[C@H]([C@@H](COC(/C=C/c(cc1)cc(O)c1O)=O)C[C@H]([C@@H]1O)OC2=C(c(cc3O)cc(O)c3O)O[C](C=C(C=C3O)O)C3=C2)[C@@H]1O Chemical compound O[C@H]([C@@H](COC(/C=C/c(cc1)cc(O)c1O)=O)C[C@H]([C@@H]1O)OC2=C(c(cc3O)cc(O)c3O)O[C](C=C(C=C3O)O)C3=C2)[C@@H]1O OIJUPWXNUKTEIW-OVOGRAIMSA-N 0.000 description 1
Classifications
-
- 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
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
Definitions
- Anthocyanins are a class of polyphenolic compounds, and these water-soluble pigments account, in part, for the red, purple and blue colors of fruits and flowers.
- Anthocyanins belong to the flavonoid family of compounds and occur in nature as glycosides (anthocyanosides).
- a particularly rich source of anthocyanins is the bilberry plant. While anthocyanins have the structure of flavylium cations (2-phenyl-benzopyrylium salts), six different aglycones (the non-sugar portion of the anthocyanin) are known to occur naturally, all varying in degree of unsaturation and in the number and arrangement of hydroxyl and methyl groups.
- each aglycone can be glycosylated by different sugars, the most common being glucose, although galactose, arabinose, rhamnose and xylose residues are sometimes found.
- the present invention is directed to methods for treating or preventing eye diseases or disorders by administering one or more anthocyanins and/or Anthocyanin Derivatives (ADs). These methods are based in part on the present findings that anthocyanins can prevent and/or reduce cell death and damage induced by A2E. Further, the present invention provides methods for identifying particular anthocyanins and ADs that are effective for treating or preventing eye diseases or disorders, and compositions comprising ADs and specific mixtures of anthocyanins.
- ADs Anthocyanin Derivatives
- the present invention provides an anthocyanin derivative comprising a compound having the structure according to formula (IIA) or (IIB) (collectively referred to as "the Anthocyanin Derivatives”):
- R 1 , R 2 and R 3 are each independently -H, -OH, -O-td-Ce alkyl) or R 10 ;
- R 4 andR 5 are each independently -H, -OH or R 10 ;
- R° and R' are each independently -H, -OH, -OR 1 ⁇ 0 ⁇ or -CH 2 OR ( 1'0.
- R and R are each independently -OH or R ⁇ o. ; and [0010] R is a group derived from an antioxidant, wherein a compound of formula
- (IIA) can have only one R 10 group.
- R .1 1 , R2 z and R J are each independently -H, -OH, -O-(C ⁇ -C 6 alkyl) or R 1 ⁇ 0 ⁇ ;.
- R andR are each independently -H, -OH or R ⁇ o.
- R 6 is -H, -OH, -OR 10 or -CH 2 OR 10 ;
- R and R are each independently -OH or R . 10 ;.
- R 10 is a group derived from an antioxidant, wherein a compound of formula (IIB) can have only one R 10 group.
- the invention provides an anthocyanin derivative comprising a compound depicted by formula (IIA) wherein: R 1 is -OCH 3 ; R 2 , R 3 , R 8 and R 9 are -OH; R 4 is -H or -OH; R 5 is -H or -OH; R 6 is R 10 , -OR 10 , -H, -OH or -CH 2 OR 10 ; and R 7 is R 10 , -OR 10 , - H, -OH or -CH 2 OR 10 .
- formula (IIA) wherein: R 1 is -OCH 3 ; R 2 , R 3 , R 8 and R 9 are -OH; R 4 is -H or -OH; R 5 is -H or -OH; R 6 is R 10 , -OR 10 , -H, -OH or -CH 2 OR 10 ; and R 7 is R 10 , -OR 10 , - H, -OH or -CH 2 OR
- the invention provides an anthocyanin derivative comprising a compound depicted by formula (IIB) wherein: R 1 is -OCH 3 ; R 2 , R 3 , R 8 and R 9 are -OH; R 4 is -OH; R 5 is -OH; and R 6 is -CH 2 OR 10 .
- the invention provides an anthocyanin derivative comprising a compound depicted by formula (IIA) wherein: R 1 , R , R R and R 0 are -OH; R " is -OCH 3 ; R 4 is -H or -OH; R 5 is -H or -OH; R 6 is R 10 , -OR 10 , -H, -OH or -CH 2 OR 10 ; and R 7 is R 10 , -OR 10 , -H, -OH or -CH 2 OR 10 .
- formula (IIA) wherein: R 1 , R , R R and R 0 are -OH; R " is -OCH 3 ; R 4 is -H or -OH; R 5 is -H or -OH; R 6 is R 10 , -OR 10 , -H, -OH or -CH 2 OR 10 ; and R 7 is R 10 , -OR 10 , -H, -OH or -CH 2 OR
- the invention provides an anthocyanin derivative comprising a compound depicted by formula (IIA) wherein: R 1 and R 3 are OCH 3 ; R 2 , R 8 and R 9 are - OH; R 4 is -H or -OH; R 5 is -H or -OH; R 6 is R 10 , -OR 10 , -H, -OH or -CH 2 OR 10 ; and R 7 is R 10 , -OR 10 , -H, -OH or -CH 2 OR 10 .
- formula (IIA) wherein: R 1 and R 3 are OCH 3 ; R 2 , R 8 and R 9 are - OH; R 4 is -H or -OH; R 5 is -H or -OH; R 6 is R 10 , -OR 10 , -H, -OH or -CH 2 OR 10 ; and R 7 is R 10 , -OR 10 , -H, -OH or -CH 2 OR 10 .
- the invention provides an anthocyanin derivative comprising a compound depicted by formula (IIB) wherein: R 1 and R 3 are OCH 3 ; R 2 , R 8 and R 9 are -OH; R 4 is -OH; R 5 is -OH; and R 6 is -CH 2 OR 10 .
- the invention provides an anthocyanin derivative comprising a compound depicted by formula (IIA) wherein: R 1 , R 2 , R 3 , R 8 and R 9 are -OH; R 4 is -H or - OH; R 5 is -H or -OH; R 6 is R 10 , -OR 10 , -H, -OH or -CH 2 OR 10 ; and R 7 is R 10 , -OR 10 , -H, -OH or -CH 2 OR 10 .
- formula (IIA) wherein: R 1 , R 2 , R 3 , R 8 and R 9 are -OH; R 4 is -H or - OH; R 5 is -H or -OH; R 6 is R 10 , -OR 10 , -H, -OH or -CH 2 OR 10 ; and R 7 is R 10 , -OR 10 , -H, -OH or -CH 2 OR 10 .
- the invention provides an anthocyanin derivative comprising a compound depicted by formula (IIA) wherein: R 1 is -OCH 3 ; R 2 , R 8 and R 9 are -OH; R 3 is - H; R 4 is -H or -OH; R 5 is -H or -OH; R 6 is R 10 , -OR 10 , -H, -OH or -CH 2 OR 10 ; and R 7 is R 10 , - OR 10 , -H, -OH or -CH 2 OR 10 .
- formula (IIA) wherein: R 1 is -OCH 3 ; R 2 , R 8 and R 9 are -OH; R 3 is - H; R 4 is -H or -OH; R 5 is -H or -OH; R 6 is R 10 , -OR 10 , -H, -OH or -CH 2 OR 10 ; and R 7 is R 10 , - OR 10 , -H, -OH
- the invention provides an anthocyanin derivative comprising a compound depicted by formula (IIB) wherein: R 1 is -OCH 3 ; R 2 , R 8 and R 9 are -OH; R 3 is - H; R 4 is -OH; R 5 is -OH; and R 6 is -CH 2 OR 10 .
- the invention provides an anthocyanin derivative comprising a compound depicted by formula (IIA) wherein: R 1 , R 2 , R 8 and R 9 are -OH; R 3 is -H; R 4 is - H or -OH; R 5 is -H or -OH; R 6 is R 10 , -OR 10 , -H, -OH or -CH 2 OR 10 ; and R 7 is R 10 , -OR 10 , -H, -OH or -CH 2 OR 10 .
- formula (IIA) wherein: R 1 , R 2 , R 8 and R 9 are -OH; R 3 is -H; R 4 is - H or -OH; R 5 is -H or -OH; R 6 is R 10 , -OR 10 , -H, -OH or -CH 2 OR 10 ; and R 7 is R 10 , -OR 10 , -H, -OH or -CH 2 OR 10 .
- the invention provides an anthocyanin derivative comprising a compound depicted by formula (IIB) wherein: R 1 , R 2 , R 8 and R 9 are -OH; R 3 is -H; R 4 is - OH; R 5 is -OH; and R 6 is -CH 2 OR 10 .
- the invention provides an anthocyanin derivative comprising a compound depicted by formula (IIA) wherein: R 1 and R 3 are -H; R 2 , R 8 and R 9 are -OH; R 4 is -H or -OH; R 5 is -H or -OH; R 6 is R 10 , -OR 10 , -H, -OH or -CH 2 OR 10 ; and R 7 is R 10 , -OR 10 , - H, -OH or -CH 2 OR 10 .
- formula (IIA) wherein: R 1 and R 3 are -H; R 2 , R 8 and R 9 are -OH; R 4 is -H or -OH; R 5 is -H or -OH; R 6 is R 10 , -OR 10 , -H, -OH or -CH 2 OR 10 ; and R 7 is R 10 , -OR 10 , - H, -OH or -CH 2 OR 10 .
- the invention provides an anthocyanin derivative comprising a compound depicted by formula (IIB) wherein: R 1 and R 3 are -H; R 2 , R 8 and R 9 are -OH; R 4 is -OH; R 5 is -OH; and R 6 is R 10 .
- the present invention provides Anthocyanin Derivatives according to formula (IIA) or (IIB), wherein group R 10 is derived from a caffeic acid, a carotenoid, a linoleic acid, curcumin, or vitamin E.
- R 10 is a group of formula (III):
- R 11 , R 12 , R 13 , R 14 and R 15 are each independently -H, -OH or -O-(C ⁇ -
- the present invention provides an anthocyanin derivative having the structure depicted below by formula VI, V, VI, VII, VIII, IX, X, XI, XII, XIII, TV, XV, XVI, XVII, XVIII, XIX, XX, XXI or XXII.
- the present invention provides a composition comprising an Anthocyanin Derivative and a physiologically acceptable carrier or vehicle.
- the present invention provides a method for treating an eye disease in a subject comprising administering to the subject an amount of an anthocyanin and/or an Anthocyanin Derivative of formula (IIA) or formula (IIB) effective to ameliorate the eye disease and thereby treat the disease.
- the Anthocyanin Derivatives can comprise, for example, one or more of delphinidin 3-O- galactoside, delphinidin 3-O-glucoside, cyanidin 3-O-galactoside, cyaniding 3-O-glucoside, petunidin 3-O-galactoside, cyanidin 3-O-arabinoside, petunidin 3-O-glucoside, peonidin 3-O- galactoside, peonidin 3-O-glucoside, malvidin 3-O-glycoside, or any combination thereof.
- the amount of the anthocyanin and/or the anthocyanin derivative can comprise from about 5 mg to about 50 mg, from about 50 mg to about 100 mg, or from about 100 mg to about 500 mg per kg of the body weight of the subject.
- the anthocyanin(s) and/or the Anthocyanin Derivatives can be administered to a subject by an ophthalmic delivery, an oral delivery, a nasal delivery, or by injection.
- the anthocyanins and the Anthocyanin Derivatives can be, for example, injected into the vitreous or subretinal compartments of the eye.
- anthocyanins and/or the Anthocyanin Derivatives can be administered in combination with isotretinoin (13-c ⁇ -retinoic acid), all-tr ⁇ ns-retinol- dehydrogenase, an anti-phosphatidylethanolamine moiety, vitamin E, zinc, an anti-vascular endothelial growth factor moiety, or any combination thereof.
- Derivatives are directed to treating or preventing eye diseases or disorders, such as age- related macular degeneration (AMD), juvenile macular degeneration, retinitis pigmentosa, choroidal neovascular membrane (CNVM), macular hole, pattern dystrophy, cone-rod dystrophy, bulls-eye maculopathy cataracts or glaucoma.
- AMD age-related macular degeneration
- CNVM choroidal neovascular membrane
- AMD can be a neovascular or a non-neovascular condition.
- the degeneration can be cone-rod dystrophy, corneal dystrophy, Fuch's dystrophy, Sorsby's macular dystrophy, Best disease, Stargadt's disease, or juvenile retinoschisis.
- pattern dystrophy can comprise adult-onset foveomacular vitelliform dystrophy, butterfly-shaped pigment dystrophy, reticular dystrophy of the retinal pigment epithelium, multifocal pattern dystrophy simulating fundus flavimaculatus or fundus pulverulentus.
- the present invention provides a method for conferring resistance to blue-light damage to retinal pigment epithelial (RPE) cells in a subject comprising administering to the subject an amount of one or more antl ⁇ ocyanin(s) and/or Anthocyanin Derivatives effective to quench singlet oxygen in the RPE cells, thereby increasing resistance to blue-light damage in the RPE cells.
- RPE retinal pigment epithelial
- the present invention provides a method for reducing
- RPE cell death in a subject comprising administering to the subject an amount of one or more anthocyanin(s) and/or anthocyanin derivative(s) effective to quench singlet oxygen in the RPE cells, thereby reducing RPE cell death in the subject.
- the present invention provides a method for reducing or preventing lipofuscin accumulation in a RPE cell in a subject comprising administering to the subject an amount of one or more anthocyanin(s) and or anthocyanin derivative(s) effective to prevent or reduce lipofuscin levels in the RPE cell.
- the lipofuscin can comprise A2E and/or A2E- PE.
- the present invention provides a method for a stabilizing an
- the cell membrane which comprises contacting the RPE cell with an amount of one or more anthocyanin(s) and/or anthocyanin derivative(s).
- the cell membrane can comprise a plasma membrane, a lysosomal membrane, a nuclear membrane or a mitochondrial membrane. Further, the method can stabilize cell membranes that are subject to damage by lipid peroxidation.
- a method for treating or preventing an eye disease in a subject comprises contacting a cell in vitro with one or more anthocyanin(s) and/or anthocyanin derivative(s); and implanting the cell into an eye of the subject, thereafter treating or preventing an eye disease in the subject.
- anthocyanin(s) and/or anthocyanin derivative(s) comprises contacting a cell in vitro with one or more anthocyanin(s) and/or anthocyanin derivative(s); and implanting the cell into an eye of the subject, thereafter treating or preventing an eye disease in the subject.
- a number of different cell types or cell lines are contemplated for implantion, such as a retinal progenitor cell, a stem cell or a retinal pigment epithelial cell.
- the cells for implantation can be obtained from the subject itself, or the cells can be obtained from tissue culture cell lines or from donors other than the subject.
- the cells Prior to implantation, the cells can be manipulated or modified by integrating an ⁇ bcr gene cassette or an all-tra «,s-retinol-dehydrogenase gene cassette into the genome of the cell in vitro.
- gene cassettes can be stably or transiently transfected into the cells.
- the present invention is also directed to compositions comprising anthocyanins and Anthocyanin Derivatives.
- the present invention provides a therapeutic composition comprising one or more anthocyanin(s) and/or anthocyanin derivative(s) and a carbomer or a polymer.
- the polymer can comprise a biodegradable microsphere.
- the invention provides a composition comprising one or more anthocyanin(s) and/or anthocyanin derivative(s) encapsulated in liposomes.
- the invention provides an topical ophthalmic composition comprising one or more anthocyanin(s) and or anthocyanin derivative(s) solubilized in a pharmaceutically acceptable solution.
- the present invention also provides a particular anthocyanin mixture- composition, wherein the anthocyanin composition comprises delphinidin 3-O-galactoside, delphinidin 3-O-glucoside, cyanidin 3-O-galactoside, cyanidin 3-O-glucoside, petunidin 3-O- galactoside, cyanidin 3-O-arabinoside, petunidin 3-O-glucoside, peonidin 3-O-galactoside, peonidin 3-O-glucoside, and malvidin 3-O-glycoside.
- the anthocyanin composition comprises delphinidin 3-O-galactoside, delphinidin 3-O-glucoside, cyanidin 3-O-galactoside, cyanidin 3-O-glucoside, petunidin 3-O- galactoside, cyanidin 3-O-arabinoside, petunidin 3-O-glucoside, peonidin 3-O-galactoside, peonidin
- an anthocyanin composition that consists essentially of one or more of delphinidin 3-O- galactoside, delphinidin 3-O-glucoside, cyanidin 3-O-galactoside, cyanidin 3-O-glucoside, petunidin 3-O-galactoside, cyanidin 3-O-arabinoside, petunidin 3-O-glucoside, peonidin 3-O- galactoside, peonidin 3-O-glucoside, and malvidin 3-O-glycoside.
- an anthocyanin composition wherein delphinidin 3 -galactoside, delphinindin 3-glucoside, cyanidin 3-galactoside and cyanidin 3-glucoside comprise greater than about 50% of the anthocyanins in the anthocyanin composition.
- the present invention also provides a method for identifying an anthocyanin and/or anthocyanin derivative composition useful for treating or preventing macular degeneration comprising: administering one or more anthocyanin(s) and/or anthocyanin derivate(s) to a first abcr gene deficient animal; administering a negative control to a second abcr gene deficient animal; recovering retinal pigment epithelial cells from the first and second abcr gene deficient animals; and comparing the health of the recovered retinal pigment epithelial cells from the first and second abcr gene deficient animals, whereby a therapeutically effective composition of anthocyanin(s) is determined.
- the methods can comprise assaying A2E levels; assaying A2E subcellular localization; assaying plasma membrane integrity; assaying cell viability levels; DNA damage, lipid peroxidation; changes in gene expression; formation of advanced glycation endproducts (AGEs); or any combination thereof.
- the present invention provides a method for identifying an anthocyanin effective against A2E-mediated cell damage comprising: providing a plant extract comprising a plurality of anthocyanins; isolating anthocyanin fractions from the plant extract by reverse-phase high-pressure liquid chromatography, wherein each of the anthocyanin fractions comprise a distinct anthocyanin; and testing the anthocyanin fractions for activity against A2E-mediated cell damage.
- This method for identifying an anthocyanin effective against A2E-mediated damage can further comprise incubating a first population of cells with one of the anthocyanin fractions; incubating a second population of cells with a negative control; incubating the first population and second population of cells with A2E; and comparing the health of the first and second population of cells, wherein the comparing comprises assaying A2E levels; assaying A2E subcellular localization; assaying plasma membrane integrity; assaying cell viability levels; DNA damage, lipid peroxidation; changes in gene expression; formation of advanced glycation endproducts (AGEs); or any combination thereof.
- AGEs advanced glycation endproducts
- FIG. 1 Reverse phase HPLC profile of anthocyanins isolated from water- soluble extracts of bilberry.
- a lyophilized bilberry extract was first obtained from Pharmanex, Inc., and the organic phase of the bilberry extract was removed by EtOAc extraction prior to HPLC anthocyanin isolation.
- HPLC was conducted with a Vydac C18, 10mm, 22 X 250 mm, with a gradient mode A:B, 95:5 to75:25 (A: 5% formic acid, B: acetonitrile). Detection was performed at 520 nm.
- Figure 2 Structures of anthocyanins obtained from the HPLC bilberry aqueous fractions. The anthocyanin obtained from each fraction is identified by "Al” through “A10” as shown in Figure 1.
- FIG. 1 A2E (2- ⁇ 2-[2,6-Dimethyl-8-(2,6,6-trimethyl-cyclohex-l-enyl)-octa- l,3,5,7-tetraenyl]-4-[4-methyl-6-(2,6,6-trimethyl-cyclohex-l-enyl)-hexa-l,3,5-trienyl]- pyridin-l-yl ⁇ -ethanol) is pyridinium bisretinoid compound and fluorophore, which is a constitutent of RPE lipofuscin.
- A2E presents as a prominent peak along with its slightly less polar photoisomer iso-A2E and additional minor isomers.
- A2E has a rather unique structure - it consists of a positively charged pyridine ring with two side arms each derived from all-traws-retinal. NMR studies indicate that iso-A2E is a cis double bond isomer - cis at position C 13- 14 of one of the side arms of the molecule.
- the minor isomers are putative cis isomers at other positions.
- A2E accumulates in RPE cells, its synthesis actually begins in the photoreceptor cell outer segments and it is generated as a byproduct of the retinoid cycle. A2E forms from reactions with first one all-tr ⁇ s-retinal (ATR) molecule and then another ATR molecule to form first the precursor A2-PE (Hexadecanoic acid 2-[(2- ⁇ 2- [2,6-dimethyl-8-(2,6,6-trimethyl-cyclohex-l-enyl)-octa-l,3,5,7-tetraenyl]-4-[4-methyl-6- (2,6,6-trimethyl-cyclohex-l-enyl)-hexa-l,3,5-trienyl]-pyridin-l-yl ⁇ -ethoxy)-hydroxy- phosphoryloxy]-l-hexadecanoyloxy-ethyl ester)), and after phosphate hydrolysis A2E is liberated.
- ATR all-tr ⁇ s-
- the source of all-tr ⁇ ws-retinal for A2E formation is the visual cycle, with A2E formation being a byproduct of the visual cycle. Not all of the all-tr ⁇ r ⁇ -retinal forms A2E - only the fraction that escapes reduction to all-trans-retinol. But there is enough inefficiency in the system that over time, enough all-tr ⁇ Ms-retinal escapes to lead to a progressive accumulation of the non-degradable A2E. Since the availability of ATR for A2E formation is light driven, the implication is that light exposure determined the rate of A2E formation - (light of all wavelengths in the visible spectrum).
- A2E structure on the left
- A2E has the tendency to perturb cell membranes because it has an amphiphilic structure - with a polar head and two hydrophobic side-arms.
- Factors that are important in terms of A2E interactions with membranes are its amphiphilic structure, its bulky wedge-shape and the behaviour of its counter-ion (putatively chloride).
- the photoisomer iso-A2E structure on the right
- A2E becomes sequested within the lysosomal compartment of the cell, and A2E may traverse the plasma membrane by endovesiculation.
- FIG. 6 A cell culture model to study A2E cell toxicity.
- A2E is incubated with cells, for example the human RPE cell line - ARPE-19.
- A2E as depicted by the black ovals, accumulates in the cells from the culture medium.
- ARPE-19 cells do not have endogenous A2E, ARPE-19 cells that are not incubated with A2E provide a negative control.
- FIG. 7 Detection of internalized A2E by confocal microscopy. Confocal microscopy images were taken of RPE cells incubated with A2E (left and middle image) and without A2E (right image) in the cell culture system depicted in Figure 6. A2E is shown by the green fluorescence in a punctate perinuclear pattern. The perinuclear pattern is typical of the distribution of lysosomes, which is where A2E localizes in RPE cells in vivo.
- lysotracker that targets to lysosomes, indicating that A2E accumulates in the lysosomal compartment of the cultured cells, just as A2E does in vivo.
- A2E green fluorescence
- red fluorescence red fluorescence
- Figure 9A and 9B The plasma membrane as a model for the effects of A2E on membrane integrity.
- Figure 9A When A2E is introduced to the cells, it perturbs membrane integrity as indicated by the leakage of the cytoplasmic enzyme lactate dehydrogenase (LDH) out of the cells.
- Figure 9B Using the plasma membrane as a general model for the effects of A2E on membrane integrity (including lysosomal or mitochondrial membranes, for example), A2E was incubated (6 hours incubation) at various concentrations, and at concentrations greater than 10 ⁇ M, membrane disruption occurred as manifested by LDH levels in the cell culture medium after 24 hours.
- LDH lactate dehydrogenase
- FIG. 10 A2E induces membrane blebbing.
- a lentivirus vector CMV promotor and VSV-G envelope protein
- VSV-G envelope protein a lentivirus vector
- 105-107 infectious units were added to the medium for 24 hours (70% confluent).
- Wild-type GFP (of small size - 238 aa) localizes to the cytoplasm and can diffuse throughout.
- A2E-induced blebbing can be a result of loss of adhesion between the membrane and the cytoskeleton.
- FIG. 11 To aid in the understanding how the structure of A2E determines the manner in which it interacts with membranes, a single side arm counterpart to A2E, a compound called A1E (MW 352.7), has been synthesized.
- A1E is a non-physiological single side armed pyridinium molecule that retains both hydrophobic and hydrophilic elements (ends).
- ends The comparison of A2E and A1E is useful because A2E has the same positively charged hydrophilic head group - pyridine ring - but only one hydrophobic side arm.
- A1E and A2E have the same hydrophilic head group (pyridinium ring) yet the structure of A1E is more typical of a detergent.
- A1E penetrates and disrupts the cell membrane (as indicated by loss of viability) faster than A2E could be an effect of shape rather than just size. That A2E enters more sluggishly is consistent with the view that A2E may traverse the plasma membrane by endovesiculation. Since A2E becomes sequestered within the lysosomal compartment of the cell, these findings are important to understanding whether and how A2E may interact with lysosomal membranes.
- FIG. 12A and 12B A1E accumulates in RPE cells at a faster rate than A2E and causes more pronounced cell death.
- A1E and A2E were incubated with RPE cells at 20 ⁇ M for 2 days. Nuclei were stained with DAPI (microscopy filter set for DAPI, max 325, emission above 400).
- Figure 12A shows that A1E fluorescence is readily apparent in the cells (A IE presents as yellow intracellular fluorescence). A2E did not yet accumulate to detectable levels.
- Figure 12B shows that A1E induced more rapid membrane permeabilization, as indicated by the ability of a membrane impermeable dye to penetrate into the cells and stain the nuclei.
- the bar graph in Figure 12B presents percent nonviable cells in A1E and A2E-treated cultures after 24 hours of incubation. Nonviable nuclei were labeled with Dead Red and all nuclei with DAPI. Percent nonviable cells (mean +/- SEM; 6 fields/experiment, 3-4 experiments).
- Figure 13 depicts confocal microscopic images of ARPE-19 cells incubated with A1E. The images show that A1E does accumulate in RPE cells, and A1E is fairly evenly distributed within the cytoplasm. Cell borders were labeled by immunostaining with antibody to ZO-1 (red) and nuclei were staining with propidium iodide (red). Internalized A1E presents as a green autofluorescence (imaging with FITC-appropriate filters). Each image is a single optical section (1 ⁇ M).
- Figure 14A-C. A1E does not localize to the lysosomal compartment.
- FIG. 14A shows A1E autofluorescence (green signal) that was imaged using FITC-appropriate filters.
- Figure 14B shows lysosomes that were labeled with Lysotracker Red DND, a membrane diffusible acidophilic fluorophore (red signal).
- Figure 14C shows the merging of red and green fluorescence of Figures 14A and 14B, and reveals that A1E autofluorescence does not co-localize with Lysotracker Red.
- FIG. 15 Protective effects of anthocyanin pre-treatment on blue light- induced A2E epoxidation.
- Data are the mean +/- SEM of 3 experiments, p ⁇ 0.05 as compared to 430 nm irradiated A2E in the absence of anthocyanins (A2E 430 nm) (ANOVA).
- FIG. 16 Anthocyanins inhibit A2E-epoxidation by quenching singlet oxygen.
- the symbols (+) and (-) indicate the presence or absence, respectively, of a compound; p ⁇ 0.001 as compared to a mixture of A2E and endoperoxide in the absence of anthocyanin (ANOVA).
- Figure 17A and 17B Anthocyanins protect A2E-laden RPE from blue light- induced death.
- Figure 17A depicts the incorporation of delphinidin-3 -galactoside (del-gal) and cyanidin-galactoside (cya-gal) by ARPE-19 cells. Phase-contrast images. Scale bar, 10 mM.
- Figure 17B shows that anthocyanins decrease the amount of A2E mediated blue light induced cell death.
- RPE cells that had accumulated A2E were incubated with delphinidin galactoside and cyanidin galactoside for 3 days and were then irradiated at 430 nm. Values are mean +/- SEM of 3 experiments, * p ⁇ 0.001 as compared to the absence of anthocyanin.
- Figure 18 In cultured RPE that have incorporated anthocyanins, the accumulation of A2E is retarded. The accumulation of A2E and iso-A2E was quantified by HPLC and normalized to cellular protein levels. Values are mean +/- SEM of 3 experiments, p ⁇ 0.001.
- FIG. 19 Anthocyanins protect A2E-laden RPE from blue light-induced death.
- Figure 19 depicts that different types of anthocyanins can have varying degrees of protection against A2E mediated cell death, as discussed in Example 5.
- Figure 20 depicts the basic structure of anthocyanins.
- R] is -OCH 3
- R 2 is -OH and R 3 is -OH
- the aglycone component of the anthocyanin is petunidin.
- R ! is -OH
- R 2 is -OH and R 3 is -OCH
- the aglycone is petunidin.
- Ri is - OCH 3
- R 2 is -OH and R 3 is -OCH
- the aglycone is malvidin.
- R is -OH
- R 2 is - OH and R 3 is -OH
- the aglycone is delphinidin.
- R] When R] is -OCH 3 , R 2 is -OH and R 3 is -H, then the aglycone is peeonidin.
- R] When R] is -OH, R 2 is -OH and R 3 is -H, then the aglycone is cyanidin.
- i can be unsubstituted, or it can be a sugar, for example, galactose, glucose, arabinose, rhamnose and xylose. Where the source of anthocyanins is bilberry, then R 4 is galactose, glucose or arabinose.
- Figure 21 depicts the basic structures of Anthocyanin Derivatives of the present invention.
- Figure 22 depicts one possible anthocyanin derivative (formula V) of the present invention.
- Figure 23 shows the synthesis of an anthocyanin derivative (2) (formula (V)) where the reaction is described in Example 8.
- Figure 24 shows that an anthocyanin derivative (formula (V)), i.e., an acylated anthocyanin by caffeic acid, provides more than 2-times protection against A2E degradation than unmodified anthocyanin.
- concentration of anthocyanin (“Cy”, which stands for cyanidin-3-O-glucose) and anthocyanin derivative (“AcylCy" used was 50 ⁇ M.
- the present invention relates to compositions for and methods of treating or preventing eye disorders and diseases through the use of anthocyanins and Anthocyanin Derivatives.
- the present invention also provides anthocyanin and/or anthocyanin derivative- based therapeutic compositions and methods that involve specific effects against A2E- mediated cellular damage. Methods are also provided which identify the anthocyanins and Anthocyanin Derivatives that can prevent or reduce A2E-mediated cellular damage, and the dose amount and formulations that provide such therapeutic effects.
- -d-C ⁇ alkyl refers to a straight chain or branched non-cyclic hydrocarbon having from 1 to 6 carbon atoms, wherein one of the hydrocarbon's hydrogen atoms has been replaced with a single bond.
- Representative straight chain -C ⁇ -C 6 alkyls include -methyl, -ethyl, -zz-propyl, -ra-butyl, - «-pentyl, and -n-hexyl.
- Representative branched -C ⁇ -C 6 alkyls include -isopropyl, -sec-butyl, -isobutyl, -tert-butyl, -isopentyl, -neopentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 1-ethylbutyl, 2-ethylbutyl, 3-ethylbutyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl, -isopropyl, -sec-butyl, -isobutyl, -neohexyl, -iso
- salts include, e.g., water-soluble and water-insoluble salts, such as the acetate, amsonate (4,4-diaminostilbene-2, 2 - disulfonate), benzenesulfonate, benzonate, bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate, calcium edetate, camphorsulfonate, camsylate, carbonate, chloride, citrate, clavulariate, dihydrochloride, edetate, edisylate, estolate, esylate, fiunarate, fumarate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexafluorophosphate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isothionate, lactate, lactate, lactate, lactate
- subject includes, but is not limited to, a non-human animal, such as a cow, monkey, horse, sheep, pig, chicken, turkey, quail, cat, dog, mouse, rat, rabbit, or guinea pig; and a human.
- a subject is a human.
- Anthocyanins are water-soluble pigments that impart colors ranging from violet and blue to most shades of red to flowers and other plant parts. Chemically, anthocyanins can be classified as both flavonoid and phenolic. In terms of their function within plants, anthocyanins absorb ultraviolet (UV) radiation thereby protecting plant DNA from sunlight damage.
- UV ultraviolet
- the present invention contemplates the use, isolation, identification and formulation of therapeutic anthocyanins from a wide range of plants (or from non-natural sources, such as plant cells grown in tissue culture).
- Most colored fruits and vegetables can be a source of anthocyanins.
- the fruits and vegetables include, but are not limited to, bilberries, blueberries, blackberries, strawberries, currants, cranberries, cherries, raspberries, grapes, elderberries, hibiscus flowers, bell peppers, red cabbage, pears, plums, purple com, legumes and violet sweet potatoes.
- Anthocyanins are comprised of an aglycone portion and a sugar portion.
- the structural formula (I) shown below (and Figure 20) presents a basic anthocyanin structure, which comprises an aglycone structure (which is also called an anthocyanidin, i.e., an anthocyanin without a sugar component) and a sugar (R ) that is attached to the aglycone at position 3.
- R 1 , R 2 and R 3 are each independently -H, -OH or -OCH 3 ; and and R 4 is a sugar, for example, R 4 can comprise galactose, glucose, arabinose, xylose or rhamnose.
- R 4 is either a galactose, glucose or arabinose.
- anthocyanins isolated from nature six different aglycones are known to comprise the anthocyanin compound: petunidin, malvidin, delphinidin, peonidin, cyanidin and pelargonidin.
- Aglycones can vary in their degree of unsaturation and in the number and arrangement of hydroxyl and methyl groups. For example, in respect to Formula (I) above (and Figure 20), when R 1 is -OCH 3 , R 2 is -OH and R 3 is -OH, then the aglycone is petunidin.
- R 1 is -OH
- R 2 is -OH and R 3 is -OCH 3
- the aglycone is petunidin.
- R 1 is -OCH 3
- R 2 is -OH and R 3 is -OCH 3
- the aglycone is malvidin.
- R 1 is -OH
- R 2 is -OH and R 3 is -OH
- the aglycone is delphinidin.
- R 1 is -OCH 3
- R 2 is -OH and R 3 is -H
- the aglycone is peonidin.
- R 4 can be substituted with soluble antioxidants for embodiments that relate to anthocyanidin derivatives.
- R 4 is a sugar, for example, galactose, glucose, arabinose, rhamnose or xylose.
- Anthocyanins can be purified or isolated from extracts derived from plant sources by a variety of means, such as by chromatography, (J.-P. Goiffon, M. Brun and M.-J. Bourrier, High-performance liquid chromatography of red fruit anthocyanins. Journal of Chromatography, 537, 101-121 (1991); see also PCT Publication No. WO 02/17945).
- chromatography J.-P. Goiffon, M. Brun and M.-J. Bourrier, High-performance liquid chromatography of red fruit anthocyanins. Journal of Chromatography, 537, 101-121 (1991); see also PCT Publication No. WO 02/17945.
- HPLC reverse- phase high pressure liquid chromatography
- the anthocyanins identified from the HPLC fractions were delphinidin-3-O-galactoside (del-gal), delphinidin-3-O-glucoside (del-glc), cyanidin-3-O-galactoside (cya-gal), cyanidin-3-O- glucoside (cya-glc), petunidin-3-O-galactoside (pet-gal), cyanidin-3-O-arabinoside (cya-ara), petunidin-3-O-glucoside (pet-glc), peonidin-3-O-galactoside (peo-gal), peonidin-3-O- glucoside (peo-glc), and malvidin-3-O-glucoside (mal-glc).
- the predominant compounds were delphinidin-3-O-galactoside, delphinidin-3-O-glucoside, cyanidin-3-O-galactoside and cyanidin-3-O-glucoside, where the predominant compounds constituted greater than about 50 % of the mixture.
- compositions where the composition comprises greater than about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% anthocyanin.
- the anthocyanin component of the composition can comprise a mixture of specific anthocyanins.
- the mixture of specific anthocyanins in an anthocyanin composition can comprise delphinidin-3-O-galactoside (del- gal), delphinidin-3-O-glucoside (del-glc), cyanidin-3-O-galactoside (cya-gal), cyanidin-3-O- glucoside (cya-glc), petunidin-3-O-galactoside (pet-gal), cyanidin-3-O-arabinoside (cya-ara), petunidin-3-O-glucoside (pet-glc), peonidin-3-O-galactoside (peo-gal), peonidin-3-O- glucoside (peo-glc), and malvidin-3-O-glucoside (mal-glc).
- delphinidin-3-O-galactoside delphinidin-3-O-glucoside
- del-glc cyanidin-3-O-galact
- delphinidin-3-O-galactoside, delphinidin-3-O-glucoside, cyanidin-3-O- galactoside and cyanidin-3-O-glucoside constitute greater than about 50 % of the anthocyanin mixture.
- the present invention contemplates isolating essentially any anthocyanin by methods such as HPLC in order to subsequently test the anthocyanins and specific mixtures of anthocyanins for therapeutic uses against eye disorders and diseases.
- additional anthocyanins include, but are not limited to, pelargonidin-3-O- glucoside, cyanidin-3-O-(6-malonyl-glucoside), pelargonidin-3-O-(6-malonyl-glucoside), peonidin-3-O-(6-malonyl glucoside), delphinidin-3-O-arabinoside, petunidin-3-O- arabinoside, malvidin-3-O-galactoside, peonidin-3-O-arabinose and malvidin-3-O-arabinose.
- an anthocyanin derivative can be a compound represented by formula (IIA) or formula (IIB):
- R 1 , R 2 , R 3 , R 4 , R 5 ' R 6 , R 7 , R 8 , R 9 and R 10 are defined above for the compounds of formulas (IIA) and (IIB).
- an anthocyanin derivative comprises an anthocyanin that is derivatized by the conjugation of an antioxidant compound to a free hydroxyl group of the anthocyanin.
- the antioxidant compound is an organic molecule with antioxidant properties.
- the conjugation of an antioxidant compound to a free hydroxyl group can occur via a dehydrogenation or a condensation reaction, such that the oxygen of the free hydroxyl group is released as part of a water molecule as the byproduct of the reaction and that the antioxidant compound is then covalently attached to the anthocyanin molecule.
- any antioxidant compound can be attached to an anthocyanin to form an anthocyanin derivative.
- the present invention provides Anthocyanin Derivatives having an antioxidant conjugated to a free hydroxyl group of the anthocyanin, wherein the antioxidant is caffeic acid, a carotenoid, linoleic acid, curcumin, or vitamin E.
- the invention provides an anthocyanin derivative comprising an antioxidant as the R 1 , R 2 or R 3 group of formula (IIA) or (IIB), such that the antioxidant is conjugated to the "B Ring" phenolic portion of the aglycone portion of the anthocyanin.
- the invention provides an anthocyanin derivative comprising an antioxidant as the R 4 , R 5 , R 6 or R 7 group of formula (IIA) or (IIB), such that the antioxidant is conjugated to the sugar portion of the anthocyanin.
- an anthocyanin derivative comprises an antioxidant as the R 8 or R 9 group of formula (IIA) or (IIB), such that the antioxidant is conjugated to the "A Ring" phenolic portion of the agyclone portion of the anthocyanin.
- the invention provides an anthocyanin derivative as a petunidin comprising derivative, where the anthocyanin derivative comprises a compound according to formula (IIA), wherein: R 1 is -OCH 3 ; R 2 , R 3 , R 8 and R 9 are -OH; R 4 is -H or - OH; R 5 is -H or -OH; R 6 is R 10 , -OR 10 , -H, -OH or -CH 2 OR 10 ; and R 7 is R 10 , -OR 10 , -H, -OH or -CH 2 OR 10 .
- formula (IIA) wherein: R 1 is -OCH 3 ; R 2 , R 3 , R 8 and R 9 are -OH; R 4 is -H or - OH; R 5 is -H or -OH; R 6 is R 10 , -OR 10 , -H, -OH or -CH 2 OR 10 ; and R 7 is R 10 ,
- the invention provides an anthocyanin derivative as a petunidin comprising derivative, where the anthocyanin derivative comprises a compound according to formula (IIB), wherein: R 1 is -OCH 3 ; R 2 , R 3 , R 8 and R 9 are -OH; R 4 is -OH; R 5 is -OH; and R 6 is -CH 2 OR 10 .
- formula (IIB) wherein: R 1 is -OCH 3 ; R 2 , R 3 , R 8 and R 9 are -OH; R 4 is -OH; R 5 is -OH; and R 6 is -CH 2 OR 10 .
- the invention provides an anthocyanin derivative as a malvidin comprising derivative, where the anthocyanin derivative comprises a compound according to formula (IIA), wherein: R 1 , R 2 , R 8 and R 9 are -OH; R 3 is -OCH 3 ; R 4 is -H or - OH; R 5 is -H or -OH; R 6 is R 10 , -OR 10 , -H, -OH or -CH 2 OR 10 ; and R 7 is R 10 , -OR 10 , -H, -OH or -CH 2 OR 10 .
- formula (IIA) wherein: R 1 , R 2 , R 8 and R 9 are -OH; R 3 is -OCH 3 ; R 4 is -H or - OH; R 5 is -H or -OH; R 6 is R 10 , -OR 10 , -H, -OH or -CH 2 OR 10 ; and R 7 is R 10 , -
- the invention provides an anthocyanin derivative as a malvidin comprising derivative, where the anthocyanin derivative comprises a compound according to formula (IIA), wherein: R 1 and R 3 are OCH 3 ; R 2 , R 8 and R 9 are -OH; R 4 is -H or -OH; R 5 is -H or -OH; R 6 is R 10 , -OR 10 , -H, -OH or -CH 2 OR 10 ; and R 7 is R 10 , -OR 10 , -H, -OH or -CH 2 OR 10 .
- formula (IIA) wherein: R 1 and R 3 are OCH 3 ; R 2 , R 8 and R 9 are -OH; R 4 is -H or -OH; R 5 is -H or -OH; R 6 is R 10 , -OR 10 , -H, -OH or -CH 2 OR 10 ; and R 7 is R 10 , -OR 10 , -H,
- the invention provides an anthocyanin derivative as a malvidin comprising derivative, where the anthocyanin derivative comprises a compound according to formula (IIB), wherein: R 1 and R 3 are OCH 3 ; R 2 , R 8 and R 9 are -OH; R 4 is -OH; R 5 is -OH; and R 6 is -CH 2 OR 10 .
- formula (IIB) wherein: R 1 and R 3 are OCH 3 ; R 2 , R 8 and R 9 are -OH; R 4 is -OH; R 5 is -OH; and R 6 is -CH 2 OR 10 .
- the invention provides an anthocyanin derivative as a delphinidin comprising derivative, where the anthocyanin derivative comprises a compound according to formula (IIA), wherein: R 1 , R 2 , R 3 , R 8 and R 9 are -OH; R 4 is -H or -OH; R 5 is - H or -OH; R 6 is R 10 , -OR 10 , -H, -OH or -CH 2 OR 10 ; and R 7 is R 10 , -OR 10 , -H, -OH or - CH 2 OR 10 .
- formula (IIA) wherein: R 1 , R 2 , R 3 , R 8 and R 9 are -OH; R 4 is -H or -OH; R 5 is - H or -OH; R 6 is R 10 , -OR 10 , -H, -OH or -CH 2 OR 10 ; and R 7 is R 10 , -OR 10 , -H, -OH or
- the invention provides an anthocyanin derivative as a peonidin comprising derivative, where the anthocyanin derivative comprises a compound according to formula (IIA), wherein: R 1 is -OCH 3 ; R 2 , R 8 and R 9 are -OH; R 3 is -H; R 4 is -H or -OH; R 5 is -H or -OH; R 6 is R 10 , -OR 10 , -H, -OH or -CH 2 OR 10 ; and R 7 is R 10 , -OR 10 , -H, - OH or -CH 2 OR 10 .
- formula (IIA) wherein: R 1 is -OCH 3 ; R 2 , R 8 and R 9 are -OH; R 3 is -H; R 4 is -H or -OH; R 5 is -H or -OH; R 6 is R 10 , -OR 10 , -H, -OH or -CH 2 OR 10 ; and R 7 is
- the invention provides an anthocyanin derivative as a peonidin comprising derivative, where the anthocyanin derivative comprises a compound according to formula (IIB), wherein: R 1 is -OCH 3 ; R 2 , R 8 and R 9 are -OH; R 3 is -H; R 4 is - OH; R 5 is -OH; and R 6 is -CH 2 OR 10 .
- formula (IIB) wherein: R 1 is -OCH 3 ; R 2 , R 8 and R 9 are -OH; R 3 is -H; R 4 is - OH; R 5 is -OH; and R 6 is -CH 2 OR 10 .
- the invention provides an anthocyanin derivative as a cyanidin comprising derivative, where the anthocyanin derivative comprises a compound according to formula (IIA), wherein: R 1 , R 2 , R 8 and R 9 are -OH; R 3 is -H; R 4 is -H or -OH; R 5 is -H or -OH; R 6 is R 10 , -OR 10 , -H, -OH or -CH 2 OR 10 ; and R 7 is R 10 , -OR 10 , -H, -OH or - CH 2 OR 10 .
- formula (IIA) wherein: R 1 , R 2 , R 8 and R 9 are -OH; R 3 is -H; R 4 is -H or -OH; R 5 is -H or -OH; R 6 is R 10 , -OR 10 , -H, -OH or -CH 2 OR 10 ; and R 7 is R 10 , -OR 10 , -H
- the invention provides an anthocyanin derivative as a cyanidin comprising derivative, where the anthocyanin derivative comprises a compound according to formula (IIB), wherein: R 1 , R 2 , R 8 and R 9 are -OH; R 3 is -H; R 4 is -OH; R 5 is - OH; and R 6 is -CH 2 OR 10 .
- formula (IIB) wherein: R 1 , R 2 , R 8 and R 9 are -OH; R 3 is -H; R 4 is -OH; R 5 is - OH; and R 6 is -CH 2 OR 10 .
- the invention provides an anthocyanin derivative as a pelargonidin comprising derivative, where the anthocyanin derivative comprises a compound according to formula (IIA), wherein: R and R are -H; R , R and R are -OH; R is -H or - OH; R 5 is -H or -OH; R 6 is R 10 , -OR 10 , -H, -OH or -CH 2 OR 10 ; and R 7 is R 10 , -OR 10 , -H, -OH or -CH 2 OR 10 .
- formula (IIA) wherein: R and R are -H; R , R and R are -OH; R is -H or - OH; R 5 is -H or -OH; R 6 is R 10 , -OR 10 , -H, -OH or -CH 2 OR 10 ; and R 7 is R 10 , -OR 10 , -H, -OH or -CH 2 OR
- the invention provides an anthocyanin derivative as a pelargonidin comprising derivative, where the anthocyanin derivative comprises a compound according to formula (IIB), wherein: R 1 and R 3 are -H; R 2 , R 8 and R 9 are -OH; R 4 is -OH; R 5 is -OH; and R 6 is R 10 .
- formula (IIB) wherein: R 1 and R 3 are -H; R 2 , R 8 and R 9 are -OH; R 4 is -OH; R 5 is -OH; and R 6 is R 10 .
- the sugar component can comprise, for example, a glycoside derivative, including a ⁇ -D-glucose, a ⁇ - D-galactose, a ⁇ - D-arabinose, a ⁇ - a D-rhamnose or a ⁇ -D-xylose sugar derivative.
- R 11 , R , R , R 14 and R 15 are each independently -H, -OH or -O-(d-C 6 alkyl).
- the phenyl moiety of the group of formula (III) can have the following independent substitution patterns: (i) p-OH; (ii) m, p-di-OH; (iii) m,p-di-OCH 3 ; (iv) m-OCH 3 ; or (v) p-OH, wherein "p” represents a para substituent on the phenyl moiety of the group of formula (III) and "m” represents a meta substituent on the phenyl moiety of the group of formula (III).
- anthocyanin derivatives include:
- the present invention encompasses compositions, formulations and methods directed to Anthocyanin Derivatives. Any method, mixture or formulation directed to anthocyanins described herein is also meant to encompass Anthocyanin Derivatives.
- Anthocyanin Derivatives of Formulas (IIA) and (IIB) The Anthocyanin Derivatives of the present invention can be made using methodology known to those of ordinary skill in the art or by using the synthetic procedures outlined below in Schemes 1 and 2.
- Scheme 1 shows a method for making the Anthocyanin Derivatives of formulas (IIA) ) aanndd ((IIIIB) using an triazole ester coupling reaction to add group R 10 to the anthocyanin aglycone.
- Y is -CHR 5 - or-CHR 5 -CHR 6 -; and R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , R 9 and R 10 are as defined above for the compounds of formulas (IIA) and (IIB).
- An antioxidant compound having a carboxylic acid moiety (1) can be reacted with l,l'-carbonyldi(l, 2, 4-triazole) to provide a triazole substituted intermediate of formula 2.
- the intermediate of formula 2 can then be coupled with an anthocyanin aglycone of formula 3 in presence of l,8-diazabicyclo[5.4.0]undec-7-ene (DBU) to provide the corresponding anthocyanin derivative of formula (IIA) or (IIB).
- DBU l,8-diazabicyclo[5.4.0]undec-7-ene
- Scheme 2 shows a method for making the Anthocyanin Derivatives of formulas (IIA) and (IIB) using an olefin metathesis reaction to add group R 10 to the anthocyanin aglycone.
- anthocyanin derivatives of formula (IIA) or (IIB) A hydroxy group of an anthocyanin aglycone of formula 3 can be derivatized using acryloyl chloride to provide an anthocyanin acrylate intermediate of formula 4.
- an antioxidant compound having a hydroxy moiety (5) can be reacted with allyl bromide to provide an allylic ether intermediate of formula 6.
- the intermediate of formula 4 can then be coupled with an intermediate of formula 6 via an olefin metathesis reaction using a ruthenium catalyst of formula 7 to provide the co ⁇ esponding anthocyanin derivative of formula (IIA) or (IIB).
- RPE retinal pigment epithelial
- lipofuscin is not a distinct chemical compound, but a generic name given to an autofluorescent intracellular material that is distributed widely amongst the post-mitotic cells of different organs of the body. With advancing age, and with various eye diseases and disorders, there is a marked increase in the lipofuscin granule content in RPE cells.
- RPE cell lipofuscin contains lipids, proteins and a heterogeneous mixture of different fluorophores.
- A2E The major fluorophore in RPE cell lipofuscin is A2E, and the chemical name for A2E is 2- ⁇ 2-[2,6- Dimethyl-8-(2,6,6-trimethyl-cyclohex-l-enyl)-octa-l,3,5,7-tetraenyl]-4-[4-methyl-6-(2,6,6- trimethyl-cyclohex-l-enyl)-hexa-l,3,5-trienyl]-pyridin-l-yl ⁇ -ethanol.
- A2E is a pyridinium bisretinoid compound that is generated as a byproduct of the visual cycle (see Figure 4).
- the cells involved in visual transduction are the rod and cone photoreceptor cells. Rods and cones are specialized for responding to dim light and color vision respectively.
- the outer segments of the photoreceptor cells are replete with light-capturing proteins consisting of rod or cone opsin bound through a Schiff base linkage to the chromophore 1 l-cts-retinal. It is the isomerization of the retinal chromophore form the ll-cis to the ⁇ dl-trans form by a photon of light that initiates phototransduction.
- Photoreceptor cells in turn are dependent on cooperative interactions with the underlying retinal pigment epithelium.
- the RPE cells contain the machinery to regenerate 11-czs-retinal from all-tra s-retinal, the latter being released from opsin after photoisomerization.
- RPE cells also phagocytose and degrade outer segment membrane that is discarded daily by the photoreceptor cells to balance the continual production of new disks at the base of the photoreceptor segments.
- ATR all-tr ⁇ /zs-retinal
- the visual cycle enzymes i.e., all-tr ⁇ r ⁇ -retinol dehydrogenase, isomerase and l l-cts-retinol dehydrogenase.
- A2E is generated from two molecules of ATR and ethanolamine, where ATR is released from photoactivated rhodopsin and ethanolamine being the head group of phosphatidylethanolamine (PE), an abundant membrane phospholipid.
- PE phosphatidylethanolamine
- A2E-induced cellular damage also encompass lipofuscin induced damage and other lipofuscin-component induced damage.
- A2E Ophthalmol. Vis. Sci, (2002) 43: 1222-1227.
- the photoexcixation of A2E leads to the generation of singlet oxygen and the addition of the latter to carbon-carbon double bonds along the retinoid-derived side-arms of A2E such that epoxides are formed.
- A2E is converted to a mixture of compounds (A2E-epoxides) bearing epoxides of varying numbers.
- These highly reactive epoxides have been shown mediate cell damage (Spa ⁇ ow, J.R. et al, J. Biol. Chem., (2003) 278: 18207-18213).
- the photochemical events provoked by the i ⁇ adiation of A2E in RPE cells initiates cell death (Spa ⁇ ow et al, (2000)) by way of a pathway that involves the participation of cysteine-dependent proteases (caspases) to cleave cellular substrates and that is modulated by the mitochondrial protein bcl-2 (Spa ⁇ ow et al, (2002)).
- caspases cysteine-dependent proteases
- the present invention provides methods of administering anthocyanins and/or Anthocyanin Derivatives to subjects to treat eye diseases and disorders where lipofuscin or lipfuscin component related damage (i.e., A2E-related damage) may be involved.
- A2E-related damage includes iso-A2E related damage (see, Figure 5).
- Eye diseases and disorders where lipofuscin or A2E-related damage may be involved include, but are not limited to, age-related macular degeneration (AMD), juvenile macular degeneration, retinitis pigmentosa, choroidal neovascular membrane (CNVM), macular hole, pattern dystrophy, cataracts or glaucoma.
- AMD age-related macular degeneration
- CNVM choroidal neovascular membrane
- Types of juvenile macular degeneration that are contemplated for treatment include, but are not limited to, cone-rod dystrophy, comeal dystrophy, Fuch's dystrophy, Sorsby's macular dystrophy, Best disease, Stargadt's disease, and juvenile retinoschisis.
- Types of pattern dystrophy that are contemplated for treatment include, but are not limited to, adult-onset foveomacular vitelliform dystrophy, butterfly-shaped pigment dystrophy, reticular dystrophy of the retinal pigment epithelium, multifocal pattern dystrophy simulating fundus flavimaculatus and fundus pulverulentus.
- One method of treatment relates to conferring resistance to blue-light damage to retinal pigment epithelial (RPE) cells in a subject comprising administering to the subject an amount of one or more anthocyanins and/or Anthocyanin Derivatives effective to quench singlet oxygen in the RPE cells, thereby increasing resistance to blue-light damage in the RPE cells.
- Another method is directed to reducing RPE cell death in a subject comprising administering to the subject an amount of one or more anthocyanins and/or Anthocyanin Derivatives effective to quench singlet oxygen in the RPE cells, thereby reducing RPE cell death in the subject.
- Yet another method is directed to reducing or preventing lipofuscin or A2E accumulation in a RPE cell in a subject comprising administering to the subject an amount of one or more anthocyanins and/or Anthocyanin Derivatives effective to prevent or reduce lipofuscin or A2E levels in the RPE cell. Additionally a method is provided for a stabilizing an RPE cell membrane which comprises contacting the RPE cell with an amount of one or more anthocyanins and/or Anthocyanin Derivatives.
- the present invention also contemplates administering anthocyanins anthocyanins and/or Anthocyanin Derivatives using the above-mentioned methods in combination with one or more of the following entities: isotretinoin (13-c ⁇ -retinoic acid), dehydrogenases (including all-tr ⁇ «s-retinol- dehydrogenase), a agents that target dehydrogenases (i.e., agents that repress activity of 11- czs-retinol dehydrogenase (see, US Patent Application Publication No.
- the present invention also provides a method for treating or preventing an eye disease in a subject comprising contacting a cell in vitro with one or more anthocyanins and/or Anthocyanin Derivatives; and implanting the cell into an eye of the subject, thereafter treating or preventing an eye disease in the subject.
- the cell can be a retinal pigment epithelial cell obtained from donors, from the subject itself or from cell lines.
- the cell can be a stem cell or a retinal progenitor cell, such that after implantation, the cell can help to regenerate damaged tissue, such as damaged retinal pigment epithelium.
- the cell Prior to implantation, the cell can be treated in vitro to accumulate anthocyanins and/or Anthocyanin Derivatives and to be transfected with a gene, such as an isomerase gene, a dehydrogenase gene, an abcr gene, or any combination thereof.
- the present invention provides both in vitro and in vivo methods to screen or test anthocyanins anthocyanins and/or Anthocyanin Derivatives in relation to the various types of lipofuscin or A2E-mediated damage.
- the present invention provides methods for and a cell culture model to identify whether or not a particular anthocyanin and/or an AD, mixture, dose amount, or formulation can protect against A2E-mediated cell damage.
- Figure 6 shows the basic methodology of the cell culture testing/screening method.
- a human RPE cell line, ARPE-19 is grown in cell culture.
- ARPE-19 cells do not have endogenous A2E, ARPE-19 cells that are not incubated with A2E provides a negative control for testing the effects of anthocyanins and/or Anthocyanin Derivatives on preventing or reducing A2E mediated damage.
- Figures 7 and 8 show that ARPE-19 cells accumulate A2E in a manner that is consistent with how RPE cells accumulate A2E in vivo.
- Figures 7 and 8 show that A2E accumulates in the lysosomal compartment of ARPE-19 cells.
- the cell culture model system can test the therapeutic effects of anthocyanins and/or Anthocyanin Derivatives with other cell types, including primary cells that are isolated from animal donors.
- the cell culture model system can be modified such that anthocyanin testing is conducted on cells obtained from donors that are afflicted with an eye disease or disorder such that these cells do not have to be incubated with lipofuscin, A2E or some other lipofuscin component.
- the cell types can include, but are not limited to, primary RPE cells, RPE progenitor cells, stem cells, ARPE-19 cells and any RPE cell line.
- the present invention provides a method to test whether anthocyanins and/or Anthocyanin Derivatives (for the purposes of the present invention, testing anthocyanins and/or Anthocyanin Derivatives refers to testing individual anthocyanins and/or Anthocyanin Derivatives, mixtures, doses or formulations) can protect against the deleterious effects that lipofuscin, A2E or other lipofuscin components have on membrane integrity.
- cultured cells such as ARPE-19 cells, are incubated with A2E (see, Figure 9).
- A2E When A2E accumulates in cells, it perturbs membrane integrity as indicated by the leakage of the cytoplasmic enzyme lactate dehydrogenase (LDH) out of the cells.
- LDH lactate dehydrogenase
- This leakage can be detected by a variety of methods known in the art, such as by testing samples of the cell culture media for the presence of LDH by absorbance ( Figure 9B), ELISA or Western or by testing for ability of dyes, which are otherwise not permeable, to enter the cell and label the nucleus. As shown in Figure 9B, incubation with increasing amounts of A2E results in increasing amounts of LDH leakage.
- a method for testing whether anthocyanins can protect against A2E-mediated cell membrane damage comprises: incubating cells with or without anthocyanins and/or Anthocyanin Derivatives, incubating cells with A2E, and assaying for leakage of LDH.
- Particular anthocyanins and/or Anthocyanin Derivatives, mixtures, doses or formulations that decrease the leakage of LDH indicates a potential therapeutic composition.
- the present invention provides a method to test whether anthocyanins and/or Anthocyanin Derivatives can protect against A2E mediated blue light-induced damage.
- anthocyanins and/or Anthocyanin Derivatives can protect against A2E mediated blue light-induced damage.
- blue light-exposed A2E-laden RPE cells exhibit a propensity for apoptosis (Spa ⁇ ow et al, J. Biol. Chem. (2003) 278(20): 18207-18213; Spa ⁇ ow et al, Invest. Ophth. & Vis.
- the present invention provides methods to test whether anthocyanins and/or Anthocyanin Derivatives can reduce A2E-mediated blue-light damage, as indicated by a reduction of A2E epoxidation.
- A2E Upon blue-light i ⁇ adiation of cells containing A2E, A2E self-generates singlet oxygen with the singlet oxygen in turn reacting with A2E to generate epoxides at carbon-carbon double bounds, such that the reduction of A2E levels upon blue-light i ⁇ adiation indicates A2E- epoxidation and subsequent apoptosis.
- FIG 15 and Example 2 cells incubated with various anthocyanins reduce the loss of A2E by blue-light induced A2E- epoxidation.
- Figure 16 and Example 3 present an alternative method to test anthocyanins and/or Anthocyanin Derivatives protection, as A2E-epoxidation is catalyzed by endoperoxide.
- the results in Figure 16 are similar, as the anthocyanins repress the formation of A2E-epoxides.
- Anthocyanin repression of A2E-epoxidation is shown to be linked to preventing blue-light induced A2E-mediated apoptosis ( Figure 17 and Example 4).
- cultured cells such as ARPE-19 cells
- A2E incubated with A2E and with or without anthocyanins and/or Anthocyanin Derivatives (different types, mixtures, doses, formulations).
- the incubated cells are then exposed to blue-light (i.e., around 430 nm) or endoperoxide and are assayed for A2E levels and/or for the percentage of cells that are viable or non-viable.
- Anthocyanins and/or Anthocyanin Derivatives that reduce A2E-epoxidation or the percent of non-viable cells indicate that they are therapeutic candidates. Further, doses or formulations of anthocyanins and/or Anthocyanin Derivatives that reduce A2E-epoxidation or the percent of non-viable cells indicate potential therapeutically effective amounts or formulations.
- the present invention provides a method to test whether anthocyanins and/or Anthocyanin Derivatives can protect against A2E cellular accumulation.
- This method comprises the incubation of cells with A2E and with or without anthocyanins and/or Anthocyanin Derivatives, followed by assaying the cells for their intracellular levels of A2E.
- del-gal and cya-gal anthocyanins reduce the accumulation of A2E (and iso-A2E) in cells.
- the above cell culture models can be used as assay methods in conjunction with animal models to test whether anthocyanins and/or Anthocyanin Derivatives can have a therapeutic effect against A2E-related damage in vivo.
- abcX the knock-out mouse, abcX " is deficient in both copies of the abcr gene and manifests symptoms of Stargadt's disease and other lipofuscin-accumulation related diseases/disorders: (i) slow photoreceptor degeneration, (ii) delayed recovery of rod sensitivity following light exposure, (iii) elevated all-tr ⁇ ws-retinal and reduced all-trfms-retinol in photoreceptor outer-segments following a photobleach assay, (iv) constitutively elevated phosphatidylethanolamine (PE) in outer- segments, and (v) accumulation of lipofuscin (and A2E) in RPE cells elevated levels of Schiff base conjugate between all-trans-retinal and phosphat
- the present invention provides a method to test whether anthocyanins and/or Anthocyanin Derivatives (including compounds, mixtures, doses and formulations) can protect against lipofuscin related damage in vivo.
- This method comprises administering anthocyanins and/or Anthocyanin Derivatives to a first abcr gene deficient animal; administering a negative control to a second abcr gene deficient animal; recovering retinal pigment epithelial cells (or other eye cells) from the first and second abcr gene deficient animals; and comparing the health of the recovered retinal pigment epithelial cells from the first and second abcr gene deficient animals, whereby a therapeutically effective composition of anthocyanins and/or Anthocyanin Derivatives is determined.
- Comparing the health of the cells recovered from abcr gene deficient animals comprises the various cell culture methods discussed supra and additional methods.
- the methods to compare the health of the cells include, but are not limited to, comparing intracellular A2E levels, comparing intracellular lipofuscin levels, comparing A2E subcellular localization, comparing percent of cell viability within the recovered populations, comparing sensitivity to blue-light mediated apoptosis, comparing sensitivity to A2E- epoxidation, and comparing LDH leakage.
- Intracellular A2E levels can be quantitatively determined by HPLC analysis, and qualitatively determined by microscopy, including fluorescent confocal microscopy and electron microscopy.
- Histological methods can also be utilized to determine the relative levels of cell viability in the retinal pigment epithelium. Additionally, the visual function of abcr gene deficient mice treated and not treated with anthocyanin can be tested by ERG analysis (Radu, R. et al, Proc. Natl. Acad. Sci. USA, (2003) 100(8): 4742-4747).
- anthocyanins and/or Anthocyanin Derivatives relates to at least: the prevention or reduction of lipofuscin accumulation, iso-A2E accumulation, A2E accumulation, A2E-epoxidation, cell membrane integrity, cell viability, or visual function.
- the methods can further comprise testing the health of cells treated or untreated with anthocyanins and/or Anthocyanin Derivatives in combination with isotretinoin (13-cis- retinoic acid), dehydrogenases (including all-tra/zs-retinol-dehydrogenase, l l-cts-retinol dehydrogenase), isomerase, RmP protein, an anti-phosphatidylethanolamine moiety, vitamin E, zinc, an anti-vascular endothelial growth factor moiety, or any combination thereof.
- isotretinoin 13-cis- retinoic acid
- dehydrogenases including all-tra/zs-retinol-dehydrogenase, l l-cts-retinol dehydrogenase
- isomerase isomerase
- RmP protein an anti-phosphatidylethanolamine moiety
- vitamin E zinc
- anthocyanins and Anthocyanin Derivatives contemplate essentially any method of delivery, including topical ophthalmic delivery, an oral delivery, a nasal delivery, an implant delivery, or an injection delivery.
- Topical ophthalmic delivery can include, for example, delivery of anthocyanins and/or derivatives by eye drops or a cream/ointment.
- the injection can be into the vitreous or subretinal compartment of an eye.
- the purpose of injection into the vitreous or subretinal compartment is to maximize or enhance the amount of anthocyanins and Anthocyanin Derivatives that come into contact with posterior areas of the eye, such as the retinal pigment epithelium.
- posterior areas of the eye such as the retinal pigment epithelium.
- repetitive insertion into the eye can cause complications such as retinal detachment, posterior dislocation, endophthalmitis, vitreous hemo ⁇ hage and cataract formation.
- a prolonged or controlled delivery can be accomplished by administering intravitreous polymer implants or microspheres containing anthocyanins and/or Anthocyanin Derivatives.
- anthocyanins and/or derivatives in the intravitreous polymer implants is made according to size. For example, as the internal limiting membrane of the retina prevents diffusion of substances larger than about 40,000 Daltons to about 70,000 Daltons from diffusing into the retina from the vitreous, anthocyanins and derivatives smaller than at least about 70,000 Daltons are selected for intravitreous polymer implant delivery and for intravitreous injection delivery. Alternatively, anthocyanins and derivatives smaller than about 40,000 Daltons are selected for intravitreous polymer implant and injection delivery.
- Injectable or implantable formulations can comprise biodegradable polymer microspheres that are attached to anthocyanins and Anthocyanin Derivatives and provide a controlled or prolonged/sustained release of the anthocyanins and Anthocyanin Derivatives.
- the biodegradable polymer microspheres can comprise poly(L-lactic acid) and poly(glycolic acid), wherein the poly(glycolic acid) can comprise poly(D,L-lactide-co-glycolide)(PLGA).
- the polymer microspheres can be injected or implanted into the vitreous (Moritera, T. et al, Invest. Ophthalmol. Vis.
- subretinal injection a device, such as a glass micropipette, is inserted transvitreally through a sclerotomy and stereotactically manipulated through the subretinal space.
- the subretinal delivery allows direct delivery of the microspheres to the retinal pigment epithelial because the RPE cells will phagocytose the microspheres and its attached anthocyanin cargo.
- Such a delivery may be favorable in certain circumstances, because anthocyanins will be delivered directly to lipofuscin, and hence, A2E.
- the delivery brings anthocyanins and A2E into direct contact because membrane vesicles carrying phagocytosed substances merge and localize to the lysosomal compartment, where lipofuscin accumulates.
- Biodegradable polymers can also be implanted into the sclera (Kunou, N. et al, J. Biomed. Mater. Res., (2000), 51: 635-641).
- Another formulation of the present invention comprises liposomes that encapsulate anthocyanins and/or Anthocyanin Derivatives.
- a benefit of a liposome formulation is that it allows for non-ocular injection. Liposomes can be injected to the arm of a subject, for example, whereby the liposomes travel throughout the bloodstream. Targeted delivery of the anthocyanins by the liposomes can be accomplished by light-targeted delivery (LTD). With LTD, anthocyanins and/or Anthocyanin Derivatives are encapsulated in heat- sensitive liposomes. The liposomes do not disassemble or degrade under normal physiological temperatures.
- a light beam i ⁇ adiates the choroidal neovascularization (CNV) and su ⁇ ounding tissues at a wavelength strongly absorbed by blood.
- CNV choroidal neovascularization
- the RPE, choriocapillaris and CNV are the first to reach the temperature necessary to melt the circulating liposomes (about 41 °C) (Zeimer, R. and Goldberg, M.F., Adv. Drug Deliv. Reviews, (2001), 52: 49-61).
- the formulation can comprise a saline solution.
- a saline solution can be an eye drop formulation, where an anthocyanin or an anthocyanin derivative compound can be esterified to a isopropyl-ester prodrug such that it can then be hydrolyzed in the cornea by esterases to a free hydroxyl form.
- the formulation can comprise a carbomer or a polymer such that the anthocyanin and/or derivatives are tableted, or are stabilized in a solution, syrup, ointment or cream.
- Bilberry extract was from Nature's Resource Premium Herb
- Bilberry extract (10 g) was dissolved in 250 ml of deionized water, and fractionated between the organic and aqueous phases by extracting an aqueous solution of bilberry with 250 ml of ethyl acetate three times.
- the fractions were assayed for their ability to inhibit A2E-epoxidation by exposing preparations of A2E (20 ⁇ M in DPBS) with and without each fraction (250 ⁇ g/ml), to 430nm light for 10 min (0.36 mW/mm2).
- Anthocyanins were isolated by preparative HPLC of the aqueous layer of bilberry extract. Each peak was collected for the mass spectroscopy.
- HPLC A HP 1100 Series HPLC equipped with photodiode a ⁇ ay detector was used with a reverse phase C18 column (150 mm x 4.6 mm, Cosmosil 5C18, Nacalai Tesque, Japan) for quantitation studies of anthocyanins and a Vydac C18 (250 mm x 22 mm, 10 ⁇ m, Grace Vydac, CA) for anthocyanin separation. Detection was at 520 nm.
- A2E was quantitated with the following gradient of acetonitrile in water (containing 0.1 % TFA): 85- 95 % (10 min), 95-100 (5 min), 100 % (5 min); flow rate 0.8 ml/min with monitoring at 430 nm.
- A2E was extracted from RPE cells with CHC13 after homogenizing with glass tissue homogenizer.
- A2E was used as external standard for quantitation and A2E levels were normalized to cellular protein concentration after protein assay by BioRad Bradford method.
- Anthocyanins were quantitated with the following gradient of 5 % formic acid in acetonitrile: 95-90 % (5 min), 90-85 % (10 min); flow rate 0.8 ml/min at 520 nm.
- a solvent system of 5 % formic acid in acetonitrile was used as the mobile phase with the following gradient: 93 % (v/v, 30 min), 93-90 % (10 min), 90-87 % (30 min), 87-77 % (20 min), 77-75 % (10 min), 75-30 % (20 min); flow rate 4.0 ml/min at 520 nm; injection volume 100 ⁇ l (40mg/ml).
- EXAMPLE 2 Anthocyanins Protect Against A2E-Epoxidation in a Cell-Free System
- anthocyanins are purported to have antioxidant properties, anthocyanins were tested as to whether they could inhibit light-induced epoxidation of A2E.
- A2E in aqueous media was exposed to 430 nm illumination in the presence and absence of the anthocyanins del-gal, del-glc, cya-gal and cya-glc and the mixtures were subsequently analyzed by quantifying the consumption of A2E as A2E- epoxidation occu ⁇ ed. Blue light i ⁇ adiation resulted in an HPLC profile that revealed a decrease in the absorbance of the A2E peak as compared with the control non-illuminated sample (Figure 15).
- singlet oxygen mediates the epoxidation of A2E, it is advantageous to determine whether the anthocyanins inhibit A2E-epoxidation by quenching singlet oxygen.
- singlet oxygen was generated from the endoperoxide of 1,4- dimethylnaphthalene; the latter decomposes to release singlet oxygen. It has been previously demonstrated that for the generation of A2E-epoxides, the endoperoxide can substitute for blue light i ⁇ adiation. Accordingly, mixtures of A2E, 1,4-dimethyl naphthalene endoperoxide and del-gal or cya-gal in methanol were incubated for 14-15 hours at room temperature. As shown in Fig.
- ARPE-19 cells were incubated with del-gal and cya-gal ( 500 ⁇ M) for 2-5 days. Incorporation of anthocyanins into the cells was confirmed by light microscopy (Fig 17A). Separation of membrane, mitochondrial and cytosolic fractions, showed that anthocyanins were not only incorporated into the plasma membrane ( ⁇ 90%), but also penetrated into the cell cytosol ( ⁇ 10%). When incubated with anthocyanins for up to 5 days, the ARPE-19 cells showed no evidence of cell toxicity (data not shown).
- ARPE-19 cells that had accumulated A2E were incubated with 100 ⁇ M of the following anthocyanins: del-gal, del-glc, del-ara, cya-gal, cya-glc, cya-ara, pet-gal, pet- glc, pet-ara, mal-gal, mal-glc, mal-ara, and the aglycones themselves without sugar substituents (i.e., delphinidin, cyaniding, petunidin, and malvidin). The cells were then exposed to blue-light, and cell viability was assessed.
- anthocyanins del-gal, del-glc, del-ara, cya-gal, cya-glc, cya-ara, pet-gal, pet- glc, pet-ara, and the aglycones themselves without sugar substituents (i.e., delphinidin, cyaniding, petuni
- the % protection refers to the percentage of cells that survived the blue-light induced A2E epoxidation mediated apoptosis. As can be observed, the percent protection of anthocyanins can vary according to which aglyclone (and which sugar is attached to the aglyclone (or no sugar)) is incubated with the cells.
- delphinidin species may possess the most potent antioxidant activity due to the fact that is has three OH groups at the 3', 4' and 5' positions (as opposed to the other aglycones having one or two OH groups at these positions - see brief description for Figure 20 and Figure 20); in this example, the malvidin species were most effective in protecting the cells against A2E-epoxidation mediated death, potentially due a greater stability of malvidin as compared to the other aglyclone species.
- EXAMPLE 5 Anthocyanins Inhibit Lipid Peroxidation [00145] Anthocyanins reduced cellular incorporation of A2E.
- EXAMPLE 6 Anthocyanins Reduce Cellular Incorporation of A2E
- cellular localization of incorporated anthocyanins was measured by HPLC quantitation of anthocyanin in different cellular compartment. Most of incorporated anthocyanins were located at cellular membrane fraction (Fig. 6), as is supposed by previous reports about the cellular localization of anthocyanin and its affinity on membrane bound carrier protein (ref).
- anthocyanins were pretreated to RPE cells before A2E (20 ⁇ M) treatment, and then quantitated intracellular A2E by HPLC. Cellular incorporation of A2E was significantly blocked by pretreatment of anthocyanin (Fig. 18).
- EXAMPLE 7 Anthocyanins Reduce A2E-Mediated RPE Cell Death [00148] A2E has detergent-like properties that can critically damage cell membranes.
- EXAMPLE 9 Anthocyanin Derivative with Improved Antioxidant Properties (see Figure 24) [00151] As shown in Example 4, anthocyanins have antioxidant properties as they could inhibit light-induced epoxidation of A2E. Thus, an anthocyanin derivative (formula (V)) was tested to measure its antioxidant properties. For these experiments, A2E in aqueous media was exposed to 430 nm illumination in the presence and absence of anthocyanins (see Figure 24, "Cy") and the anthocyanin derivative ("AcylCy"). The mixtares were subsequently analyzed by quantifying the consumption of A2E as A2E-epoxidation occu ⁇ ed.
- reaction solution is concentrated and purified with reverse phase HPLC (H 2 O and CH 3 CN with TFA as solvent), such that an anthocyanin derivative (delphinidin-3-O-(6-O-caffeoyl-glucoside)) according to formula (VT) below is isolated.
- reaction solution is concentrated and purified with reverse phase HPLC (H 2 O and CH 3 CN with TFA as solvent), such that an anthocyanin derivative (delphinidin-3-O-(6-O-caffeoyl-galactoside)) according to formula (VII) below is isol.
- HPLC HPLC
- an anthocyanin derivative delphinidin-3-O-(6-O-caffeoyl-galactoside)
- reaction solution is concentrated and purified with reverse phase HPLC (H 2 O and CH 3 CN with TFA as solvent), such that an anthocyanin derivative (delphinidin-3-O-(5-O-caffeoyl-arabinoside)) according to formula (VIII) below is isolated.
- reverse phase HPLC H 2 O and CH 3 CN with TFA as solvent
- reaction solution is concentrated and purified with reverse phase HPLC (H 2 O and CH 3 CN with TFA as solvent), such that an anthocyanin derivative (cyanidin-3-O-(6-O-caffeoyl-galactoside)) according to formula (IX) below is isolated.
- reaction solution is concentrated and purified with reverse phase HPLC (H 2 O and CH 3 CN with TFA as solvent), such that an anthocyanin derivative (cyanidin-3-O-(5-O-caffeoyl-arabinoside)) according to formula (X) below is isolated.
- reverse phase HPLC H 2 O and CH 3 CN with TFA as solvent
- reaction solution is concentrated and purified with reverse phase HPLC (H 2 O and CH 3 CN with TFA as solvent), such that an anthocyanin derivative (petunidin-3-O-(6-O-caffeoyl-glucoside)) according to formula (XI) below is isolated.
- reaction solution is concentrated and purified with reverse phase HPLC (H 2 O and CH 3 CN with TFA as solvent), such that an anthocyanin derivative (petanidin-3-O-(6-O-caffeoyl-galactoside)) according to formula (XII) below is isolated.
- reaction solution is concentrated and purified with reverse phase HPLC (H 2 O and CH 3 CN with TFA as solvent), such that an anthocyanin derivative (petanidin-3-O-(5-O-caffeoyl-arabinoside)) according to formula (XIII) below is isolated.
- reaction solution is concentrated and purified with reverse phase HPLC (H 2 O and CH 3 CN with TFA as solvent), such that an anthocyanin derivative (malvidin-3-O-(6-O-caffeoyl-glucoside)) according to formula (XIV) below is isolated.
- an anthocyanin derivative malvidin-3-O-(6-O-caffeoyl-glucoside)
- reaction solution is concentrated and purified with reverse phase HPLC (H 2 O and CH 3 CN with TFA as solvent), such that an anthocyanin derivative (malvidin-3-O-(6-O-caffeoyl-galactoside)) according to formula (XV) below is isolated. (XV).
- reaction solution is concentrated and purified with reverse phase HPLC (H 2 O and CH 3 CN with TFA as solvent), such that an anthocyanin derivative (malvidin-3-O-(5-O-caffeoyl-arabinoside)) according to formula (XVI) below is isolated.
- reaction solution is concentrated and purified with reverse phase HPLC (H 2 O and CH 3 CN with TFA as solvent), such that an anthocyanin derivative (peonidin-3-O-(6-O-caffeoyl-glucoside)) according to formula (XVTI) below is isolated.
- reverse phase HPLC H 2 O and CH 3 CN with TFA as solvent
- reaction solution is concentrated and purified with reverse phase HPLC (H 2 O and CH 3 CN with TFA as solvent), such that an anthocyanin derivative (peonidin-3-O-(6-O-caffeoyl-galactoside)) according to formula (XVIII) below is isolated.
- reverse phase HPLC H 2 O and CH 3 CN with TFA as solvent
- reaction solution is concentrated and purified with reverse phase HPLC (H 2 O and CH CN with TFA as solvent), such that an anthocyanin derivative (peonidin-3-O-(5-O-caffeoyl-arabinoside)) according to formula (XIX) below is isolated.
- reverse phase HPLC H 2 O and CH CN with TFA as solvent
- reaction solution is concentrated and purified with reverse phase HPLC (H 2 O and CH 3 CN with TFA as solvent), such that an anthocyanin derivative (pelargonidin-3-O-(6-O-caffeoyl-glucoside)) according to formula (XX) below is isolated.
- XX anthocyanin derivative
- reaction solution is concentrated and purified with reverse phase HPLC (H 2 O and CH 3 CN with TFA as solvent), such that an anthocyanin derivative (pelargonidin-3-O-(6-O-caffeoyl-galactoside)) according to formula (XXI) below is isolated.
- XXI anthocyanin derivative
- reaction solution is concentrated and purified with reverse phase HPLC (H 2 O and CH 3 CN with TFA as solvent), such that an anthocyanin derivative (pelargonidin-3-O-(5-O-caffeoyl-arabinoside)) according to formula (XXII) below is isolated.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Molecular Biology (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US54371604P | 2004-02-11 | 2004-02-11 | |
US60/543,716 | 2004-02-11 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2005077176A1 true WO2005077176A1 (fr) | 2005-08-25 |
Family
ID=34860452
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2005/004553 WO2005077176A1 (fr) | 2004-02-11 | 2005-02-11 | Composes anthocyaniniques et procedes d'utilisation correspondants |
Country Status (1)
Country | Link |
---|---|
WO (1) | WO2005077176A1 (fr) |
Cited By (31)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2007023774A1 (fr) * | 2005-08-22 | 2007-03-01 | Hirosaki University | Agent d’amélioration de la diminution du débit sanguin artériel ophthalmique |
WO2007103960A1 (fr) * | 2006-03-08 | 2007-09-13 | Allergan, Inc. | Traitement oculaire a base d'agents activateurs de la sirtuine |
JP2007302577A (ja) * | 2006-05-09 | 2007-11-22 | National Agriculture & Food Research Organization | 茶葉抽出組成物 |
WO2008054639A2 (fr) * | 2006-10-30 | 2008-05-08 | Insite Vision Incorporated | Réactifs et procédés de traitement de maladies et d'infections oculaires |
US7432307B2 (en) | 2004-11-04 | 2008-10-07 | Sirion Therapeutics, Inc. | Modulators of retinol-retinol binding protein (RBP)-transthyretin (TTR) complex formation |
JP2009126810A (ja) * | 2007-11-22 | 2009-06-11 | Kagoshima Univ | ツバキ由来のアントシアニン色素、その製造方法及び用途、並びにツバキの品種識別方法 |
WO2009108577A3 (fr) * | 2008-02-25 | 2009-11-19 | Liu, Wan-Tzu Liao | Utilisation de soja noir pour traiter des maladies ophtalmiques |
WO2012013792A1 (fr) | 2010-07-29 | 2012-02-02 | Visiotact Pharma | Composition transmucosale contenant des anthocyanines pour l'atténuation d'une gêne visuelle |
JP2012062258A (ja) * | 2010-09-14 | 2012-03-29 | Oriza Yuka Kk | 血管新生抑制剤及びそれを用いた眼疾患予防・治療剤 |
US8314152B2 (en) | 2004-06-23 | 2012-11-20 | Acucela, Inc. | Methods and compositions for treating ophthalmic conditions with retinyl derivatives |
AU2011239238B2 (en) * | 2006-03-08 | 2013-10-10 | Allergan, Inc. | Ocular therapy using sirtuin-activating agents |
WO2014027865A1 (fr) * | 2012-08-17 | 2014-02-20 | 주식회사 파마킹 | Composition contenant comme principe actif de la s-allyl-l-cystéine et destinée à prévenir ou à traiter des maladies oculaires, et formulation pharmaceutique contenant cette composition |
FR2996773A1 (fr) * | 2011-05-13 | 2014-04-18 | Inst Biophytis Sas | Utilisation de composes et composition pour le traitement de la degenerescence maculaire liee a l'age (dmla) |
CN105124759A (zh) * | 2015-06-17 | 2015-12-09 | 湖北中烟工业有限责任公司 | 含黄酮类化合物香精的烟用嘴棒及其制备方法 |
WO2016189260A1 (fr) | 2015-05-27 | 2016-12-01 | Biophytis | Utilisation de 3-désoxyanthocyanidines pour le traitement de maladies oculaires |
US20170150883A1 (en) * | 2015-11-26 | 2017-06-01 | Hon Hai Precision Industry Co., Ltd. | Lens for indicating intraocular pressure of eye of user |
CN108094408A (zh) * | 2018-01-06 | 2018-06-01 | 济宁市第二人民医院 | 一种病理组织的保存固定液及其制备方法 |
US10016388B2 (en) * | 2016-08-26 | 2018-07-10 | Hon Hai Precision Industry Co., Ltd. | Ophthalmic lens, intraocular lens, and ophthalmic lens package having ophthalmic lens |
US10328097B2 (en) * | 2015-04-07 | 2019-06-25 | Hyalblue S.R.L. | Glycosaminoglycan esters, processes for their preparation and their use in formulations for ophthalmic use |
CN110143986A (zh) * | 2019-06-05 | 2019-08-20 | 吉林农业大学 | 一种稳态化锦葵3-o-葡萄糖苷衍生物及其制备方法与应用 |
CN110713506A (zh) * | 2019-11-22 | 2020-01-21 | 长春中医药大学 | 一种稳态化花翠素-3-o-葡萄糖苷衍生物及制备方法 |
CN111562340A (zh) * | 2020-06-29 | 2020-08-21 | 中国农业科学院蔬菜花卉研究所 | 一种快速对番茄果实中花青素进行种类解析和含量测定的方法 |
CN112526045A (zh) * | 2019-09-17 | 2021-03-19 | 云南中医药大学 | 一种同时检测或鉴别舒心降脂片中有效成分的方法 |
CN113391003A (zh) * | 2021-07-14 | 2021-09-14 | 云南中医药大学 | 一种同时检测生脉饮中有效成分含量的方法 |
CN113716996A (zh) * | 2021-08-17 | 2021-11-30 | 河南省林业科学研究院 | 一种红叶杨品种树干施肥增效的方法 |
CN113876706A (zh) * | 2021-09-27 | 2022-01-04 | 李青 | 一种花青素混悬滴眼液及其制备方法 |
CN115581702A (zh) * | 2022-12-12 | 2023-01-10 | 汤臣倍健股份有限公司 | 芍药素-3-o-阿拉伯糖苷在制备药物或保健食品的应用 |
CN115598255A (zh) * | 2021-10-12 | 2023-01-13 | 广州白云山陈李济药厂有限公司(Cn) | 陈皮的检测分级方法 |
CN115590874A (zh) * | 2022-12-12 | 2023-01-13 | 汤臣倍健股份有限公司(Cn) | 锦葵素-3-o-葡萄糖苷在制备药物或保健食品的应用 |
CN115645430A (zh) * | 2022-12-12 | 2023-01-31 | 汤臣倍健股份有限公司 | 飞燕草素-3-o-半乳糖苷在制备药物或保健食品的应用 |
CN115645429A (zh) * | 2022-12-12 | 2023-01-31 | 汤臣倍健股份有限公司 | 一种花色苷组合物及其应用 |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5925620A (en) * | 1990-08-20 | 1999-07-20 | Ohlenschlaeger; Gerhard | Therapeutically active mixture of glutathione and anthocyanin compounds |
US5955102A (en) * | 1998-09-04 | 1999-09-21 | Amway Corporation | Softgel capsule containing DHA and antioxidants |
-
2005
- 2005-02-11 WO PCT/US2005/004553 patent/WO2005077176A1/fr active Application Filing
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5925620A (en) * | 1990-08-20 | 1999-07-20 | Ohlenschlaeger; Gerhard | Therapeutically active mixture of glutathione and anthocyanin compounds |
US5955102A (en) * | 1998-09-04 | 1999-09-21 | Amway Corporation | Softgel capsule containing DHA and antioxidants |
Cited By (46)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US8314152B2 (en) | 2004-06-23 | 2012-11-20 | Acucela, Inc. | Methods and compositions for treating ophthalmic conditions with retinyl derivatives |
US8410168B2 (en) | 2004-06-23 | 2013-04-02 | Acucela, Inc. | Methods and compositions for treating ophthalmic conditions with retinyl derivatives |
US7432307B2 (en) | 2004-11-04 | 2008-10-07 | Sirion Therapeutics, Inc. | Modulators of retinol-retinol binding protein (RBP)-transthyretin (TTR) complex formation |
WO2007023774A1 (fr) * | 2005-08-22 | 2007-03-01 | Hirosaki University | Agent d’amélioration de la diminution du débit sanguin artériel ophthalmique |
AU2007223057B2 (en) * | 2006-03-08 | 2011-07-21 | Allergan, Inc. | Ocular therapy using sirtuin-activating agents |
WO2007103960A1 (fr) * | 2006-03-08 | 2007-09-13 | Allergan, Inc. | Traitement oculaire a base d'agents activateurs de la sirtuine |
JP2014028822A (ja) * | 2006-03-08 | 2014-02-13 | Allergan Inc | サーチュイン活性化剤を用いる眼処置 |
JP2009529538A (ja) * | 2006-03-08 | 2009-08-20 | アラーガン、インコーポレイテッド | サーチュイン活性化剤を用いる眼処置 |
AU2011239238B2 (en) * | 2006-03-08 | 2013-10-10 | Allergan, Inc. | Ocular therapy using sirtuin-activating agents |
JP2007302577A (ja) * | 2006-05-09 | 2007-11-22 | National Agriculture & Food Research Organization | 茶葉抽出組成物 |
WO2008054639A2 (fr) * | 2006-10-30 | 2008-05-08 | Insite Vision Incorporated | Réactifs et procédés de traitement de maladies et d'infections oculaires |
WO2008054639A3 (fr) * | 2006-10-30 | 2008-08-07 | Insite Vision Inc | Réactifs et procédés de traitement de maladies et d'infections oculaires |
JP2009126810A (ja) * | 2007-11-22 | 2009-06-11 | Kagoshima Univ | ツバキ由来のアントシアニン色素、その製造方法及び用途、並びにツバキの品種識別方法 |
WO2009108577A3 (fr) * | 2008-02-25 | 2009-11-19 | Liu, Wan-Tzu Liao | Utilisation de soja noir pour traiter des maladies ophtalmiques |
US8883230B2 (en) | 2008-02-25 | 2014-11-11 | National Yang-Ming University | Use of black soybean for treating ophthalmic diseases |
JP2011513316A (ja) * | 2008-02-25 | 2011-04-28 | ワン−ツ・リアオ・リュー | 眼疾患の治療のための黒大豆の使用 |
EP2417967A1 (fr) | 2010-07-29 | 2012-02-15 | Visiotact Pharma | Composition transmucosale contenant des anthocyanines pour soulager une gêne visuelle |
WO2012013792A1 (fr) | 2010-07-29 | 2012-02-02 | Visiotact Pharma | Composition transmucosale contenant des anthocyanines pour l'atténuation d'une gêne visuelle |
JP2012062258A (ja) * | 2010-09-14 | 2012-03-29 | Oriza Yuka Kk | 血管新生抑制剤及びそれを用いた眼疾患予防・治療剤 |
FR2996773A1 (fr) * | 2011-05-13 | 2014-04-18 | Inst Biophytis Sas | Utilisation de composes et composition pour le traitement de la degenerescence maculaire liee a l'age (dmla) |
WO2014027865A1 (fr) * | 2012-08-17 | 2014-02-20 | 주식회사 파마킹 | Composition contenant comme principe actif de la s-allyl-l-cystéine et destinée à prévenir ou à traiter des maladies oculaires, et formulation pharmaceutique contenant cette composition |
KR101423631B1 (ko) | 2012-08-17 | 2014-07-25 | 주식회사파마킹 | 에스-알릴엘-시스테인을 유효성분으로 포함하는 안질환 예방 또는 치료용 조성물 및 이를 포함하는 의약제제 |
US9326960B2 (en) | 2012-08-17 | 2016-05-03 | Pharmaking Co., Ltd. | Composition for preventing or treating eye diseases, containing S-allyl-L-cysteine as active ingredient, and pharmaceutical formulation containing same |
US10328097B2 (en) * | 2015-04-07 | 2019-06-25 | Hyalblue S.R.L. | Glycosaminoglycan esters, processes for their preparation and their use in formulations for ophthalmic use |
WO2016189260A1 (fr) | 2015-05-27 | 2016-12-01 | Biophytis | Utilisation de 3-désoxyanthocyanidines pour le traitement de maladies oculaires |
US10513503B2 (en) | 2015-05-27 | 2019-12-24 | Biophytis | Use of 3-deoxyanthocyanidins for treating occular diseases |
CN105124759A (zh) * | 2015-06-17 | 2015-12-09 | 湖北中烟工业有限责任公司 | 含黄酮类化合物香精的烟用嘴棒及其制备方法 |
TWI630437B (zh) * | 2015-11-26 | 2018-07-21 | 鴻海精密工業股份有限公司 | 眼用鏡片材料及眼用鏡片 |
US20170150883A1 (en) * | 2015-11-26 | 2017-06-01 | Hon Hai Precision Industry Co., Ltd. | Lens for indicating intraocular pressure of eye of user |
US10016388B2 (en) * | 2016-08-26 | 2018-07-10 | Hon Hai Precision Industry Co., Ltd. | Ophthalmic lens, intraocular lens, and ophthalmic lens package having ophthalmic lens |
CN108094408A (zh) * | 2018-01-06 | 2018-06-01 | 济宁市第二人民医院 | 一种病理组织的保存固定液及其制备方法 |
CN110143986B (zh) * | 2019-06-05 | 2022-02-18 | 吉林农业大学 | 一种稳态化锦葵3-o-葡萄糖苷衍生物及其制备方法与应用 |
CN110143986A (zh) * | 2019-06-05 | 2019-08-20 | 吉林农业大学 | 一种稳态化锦葵3-o-葡萄糖苷衍生物及其制备方法与应用 |
CN112526045A (zh) * | 2019-09-17 | 2021-03-19 | 云南中医药大学 | 一种同时检测或鉴别舒心降脂片中有效成分的方法 |
CN112526045B (zh) * | 2019-09-17 | 2023-03-31 | 云南中医药大学 | 一种同时检测或鉴别舒心降脂片中有效成分的方法 |
CN110713506A (zh) * | 2019-11-22 | 2020-01-21 | 长春中医药大学 | 一种稳态化花翠素-3-o-葡萄糖苷衍生物及制备方法 |
CN111562340A (zh) * | 2020-06-29 | 2020-08-21 | 中国农业科学院蔬菜花卉研究所 | 一种快速对番茄果实中花青素进行种类解析和含量测定的方法 |
CN113391003A (zh) * | 2021-07-14 | 2021-09-14 | 云南中医药大学 | 一种同时检测生脉饮中有效成分含量的方法 |
CN113716996A (zh) * | 2021-08-17 | 2021-11-30 | 河南省林业科学研究院 | 一种红叶杨品种树干施肥增效的方法 |
CN113876706A (zh) * | 2021-09-27 | 2022-01-04 | 李青 | 一种花青素混悬滴眼液及其制备方法 |
CN115598255A (zh) * | 2021-10-12 | 2023-01-13 | 广州白云山陈李济药厂有限公司(Cn) | 陈皮的检测分级方法 |
CN115598255B (zh) * | 2021-10-12 | 2023-12-12 | 广州白云山陈李济药厂有限公司 | 陈皮的检测分级方法 |
CN115581702A (zh) * | 2022-12-12 | 2023-01-10 | 汤臣倍健股份有限公司 | 芍药素-3-o-阿拉伯糖苷在制备药物或保健食品的应用 |
CN115590874A (zh) * | 2022-12-12 | 2023-01-13 | 汤臣倍健股份有限公司(Cn) | 锦葵素-3-o-葡萄糖苷在制备药物或保健食品的应用 |
CN115645430A (zh) * | 2022-12-12 | 2023-01-31 | 汤臣倍健股份有限公司 | 飞燕草素-3-o-半乳糖苷在制备药物或保健食品的应用 |
CN115645429A (zh) * | 2022-12-12 | 2023-01-31 | 汤臣倍健股份有限公司 | 一种花色苷组合物及其应用 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
WO2005077176A1 (fr) | Composes anthocyaniniques et procedes d'utilisation correspondants | |
Jang et al. | Anthocyanins Protect Against A2E Photooxidation and Membrane Permeabilization in Retinal Pigment Epithelial Cells¶ | |
Del Rio et al. | Berry flavonoids and phenolics: bioavailability and evidence of protective effects | |
Hribar et al. | The metabolism of anthocyanins | |
US5521215A (en) | NMDA-blocking pharmaceuticals | |
Wenzel et al. | Metabolism and bioavailability of trans‐resveratrol | |
Szumny et al. | Application of Cornelian Cherry Iridoid‐Polyphenolic Fraction and Loganic Acid to Reduce Intraocular Pressure | |
Miyazawa et al. | Direct intestinal absorption of red fruit anthocyanins, cyanidin-3-glucoside and cyanidin-3, 5-diglucoside, into rats and humans | |
Vanzo et al. | Uptake of grape anthocyanins into the rat kidney and the involvement of bilitranslocase | |
Ichiyanagi et al. | Absorption and metabolism of delphinidin 3-O-β-d-glucopyranoside in rats | |
US20090156668A1 (en) | Ginkgolide Compounds, Compositions, And Extracts, And Uses Thereof | |
Ferreira et al. | Flavonoid compounds as reversal agents of the P-glycoprotein-mediated multidrug resistance: biology, chemistry and pharmacology | |
PL213644B1 (pl) | Zastosowanie glikozydów mono- i diacyloglicerolu do wytwarzania leku do leczenia, lagodzenia lub profilaktyki stanów zapalnych | |
Alaimo et al. | Toxic effects of A2E in human ARPE-19 cells were prevented by resveratrol: a potential nutritional bioactive for age-related macular degeneration treatment | |
CA3232521A1 (fr) | Composes efficaces pour traiter l'hepatotoxicite et des steatoses hepatiques, et utilisations de ceux-ci | |
Čvorović et al. | Bioavailability of flavonoids: the role of cell membrane transporters | |
US20100286255A1 (en) | Method for extracting secoisolariciresinol and dihydroquercetin from wood | |
Cheng et al. | Light-induced generation and toxicity of docosahexaenoate-derived oxidation products in retinal pigmented epithelial cells | |
Wang et al. | Protective effect of total flavones from Hippophae rhamnoides L. against visible light-induced retinal degeneration in pigmented rabbits | |
WO2006076387A2 (fr) | Cyanidine-3-glucoside utilise en tant qu'agent neoplasique | |
Khamchun et al. | Dual modulatory effects of diosmin on calcium oxalate kidney stone formation processes: Crystallization, growth, aggregation, crystal-cell adhesion, internalization into renal tubular cells, and invasion through extracellular matrix | |
Wagner | Phenolic compounds in plants of pharmaceutical interest | |
US10251842B1 (en) | Nanocapsule containing a bioactive compound, and a method of reducing toxicity resulting from cancer therapy | |
Mahdinloo et al. | Synthesis and preparation of vitamin A coupled butein-loaded solid lipid nanoparticles for liver fibrosis therapy in rats | |
Penissi et al. | Novel anti-ulcer α, β-unsaturated lactones inhibit compound 48/80-induced mast cell degranulation |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A1 Designated state(s): AE AG AL AM AT AU AZ BA BB BG BR BW BY BZ CA CH CN CO CR CU CZ DE DK DM DZ EC EE EG ES FI GB GD GE GH GM HR HU ID IL IN IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MA MD MG MK MN MW MX MZ NA NI NO NZ OM PG PH PL PT RO RU SC SD SE SG SK SL SY TJ TM TN TR TT TZ UA UG US UZ VC VN YU ZA ZM ZW |
|
AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): BW GH GM KE LS MW MZ NA SD SL SZ TZ UG ZM ZW AM AZ BY KG KZ MD RU TJ TM AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IS IT LT LU MC NL PL PT RO SE SI SK TR BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
NENP | Non-entry into the national phase |
Ref country code: DE |
|
WWW | Wipo information: withdrawn in national office |
Country of ref document: DE |
|
122 | Ep: pct application non-entry in european phase |