Patent Application: US-201715688917-A

Abstract:
a method for photoprotection of the retinal pigmentary epithelium in a mammal includes administering an effective amount of a composition containing an extract of seeds of bixa orellana . a process for preparing the extract and formulations of the composition are also provided .

Description:
one aspect of the invention therefore relates to a composition comprising an extract of urucum seeds for photoprotection cells of retinal pigment epithelium in a mammal . the urucum or achiote , or bixa orellana is a tree or shrub of tropical america . it produces red fruit filled with seeds thorns . in the context of the invention , the term “ seed extract of bixa orellana ” is an extract prepared from the outer coat of the seeds , that is to say , the waxy substance covering the seeds of bixa orellana . this waxy substance is known to be rich in bixin and carotenoids , as well as its use as a food coloring agent . it is known from wo 01 / 85183 a composition for the prevention and treatment of eye disorders , said composition may include an extract of bixa orellana as an inhibitor of aldose reductase . this extract comprises gallic acid and / or pyrogallol , thus it is probably a leaf extract of bixa orellana , as described by terashima et al . [ chem . pharm . bull . 39 ( 12 ), 3346 - 3347 ( 1991 )], which actually shows its activity as an inhibitor of aldose reductase . in addition , wo 01 / 85183 shows the inhibition of aldose reductase as part of a mechanism to protect against cataracts and diabetic retinopathy . wo 01 / 85183 does not therefore show the effectiveness of an extract from seeds of bixa orellana for photoprotection of rpe cells . according to one embodiment of the present invention , the composition of seeds of bixa orellana is for the treatment of macular degeneration related to age ( amd ) in the mammal . according to another embodiment of the invention , the composition is intended to treat stargardt &# 39 ; s disease and / or retinitis pigmentosa . stargardt &# 39 ; s disease or stargardt &# 39 ; s syndrome is a hereditary disease , involving a bilateral decrease in visual acuity due to atrophy of the macula . according to another embodiment of the invention , the composition is intended to prevent damage to the retina may be caused by exposure to blue radiation . blue rays by means of the radiation corresponding to the blue part of the spectrum of visible light , or wavelengths comprised between 435 and 490 nm . according to one embodiment of the invention , the composition further comprises a derivative of gallic acid and / or a compound of the anthocyanidin family . the gallic acid derivative can be ellagic acid , either pure or provided as a pomegranate extract . indeed , pomegranate contains ellagic acid in large quantities ( panichayupakarananta et al ., 2010 ). the compound of the anthocyanidin family may be cyanidin , either purie or provided in the form of an acai extract . this plant does indeed contain cyanidin glycosides . cyanidin may also be provided in the form of an extract of hibiscus . the composition of bixa orellana seeds can be used as a food , a dietary supplement or a medicament . dietary supplement means a product containing said compound or extract or enriched in said compound extracted intended to supplement the diet by providing nutrients beneficial to health as defined by the european directive 2002 / 46 / ec . for example , a food supplement can be a capsule or tablet to swallow or a powder or small ampulla to be mixed with food and providing beneficial effects on the retina . a drug means a product containing a precise dose of said compound or said extract as defined by european directive 65 / 65 / ce ie any substance or composition presented as having properties for treating or preventing the disease in human beings or animal . for example , the drug containing the compound at therapeutic doses may be administered orally in capsule or tablet form or injected intravitreally or administered by any other way to give beneficial effects on the retina . another aspect of the invention relates to a composition comprising a derivative of gallic acid and / or a compound of the anthocyanidin family , for photoprotection of the retinal pigmentary epithelium in the mammal . the gallic acid derivative is preferably ellagic acid , especially purified form or made of an extract of pomegranate . the compound of the anthocyanidin family is preferably cyanidin , either purie or provided in the form of an extract of acai or hibiscus . the applications of this alternative composition are the same as those of the previously mentioned composition comprising an extract of bixa orellana seeds . as well as the latter , the other compositions may be used as a food , a dietary supplement or a medicament . the invention will be better understood upon reading the following description and examining the accompanying figures . these are for information only and not limiting of the invention . fig1 illustrates the effect of an extract urucum of bixin and norbixin on the protection of the epr tested for phototoxicity . fig2 illustrates the effect of chlorogenic acid , rutin and of ellagic acid on the protection of the epr tested for phototoxicity . fig3 illustrates the effect of cyanidin , cyanidin - 3 - glucoside or delphinidin - 3 - sambubioside and 20 - hydroxyecdysone on the protection of the epr tested for phototoxicity . extract a is made by stirring the seeds urucum in absolute ethanol ( 3 l per kg of seeds ) for 16 hours . the agitation in alcohol has the effect of detaching the waxy film on the surface lying seeds . a suspension is obtained . it is sieved to remove urucum seeds . this suspension is then reduced to ⅛th then decanted . a heavy suspension is formed . the lipid - rich supernatant is eliminated . maltodextrin is added to the solid deposit and the mixture is spray dried . the extract contains 16 % by weight of bixin . in the following examples , the concentration of the extract is expressed in bixin equivalents . extract a is also rich in carotenoids . it also contains other terpenic compounds such as geranylgeraniol and tocotrienols ( 90 % δ and 10 % β ) and several flavonoids . the extract has the following features for 100 g ( table 1 ): according to one embodiment of the invention , the extract a can be subjected to saponification , so as to turn all or part of bixin to norbixin . the inventors have tested 15 natural substances and extract a on a rpe cellular model of phototoxicity described below ( table 2 ). to test the photoprotective effect of the test substances , the inventors used a cellular model of induced phototoxicity by treatment with a2e followed by illumination with blue radiation . this model was produced from primary cultures of adult porcine rpe cells . cell survival was determined by the ratio between the number of living cells and the total number of cells ( alive + dead , respectively quantified using specific staining ). image acquisition was performed using a fluorescence microscope controlled by metamorph software and quantifications were made by processing images acquired by a program dedicated quantification . the experiments were performed in 96 - well microplates in quadruplicate and the experiment was repeated at least four times . cells were treated for 48 hours with these compounds , the last 24 hours in the presence of a2e before induction of phototoxicity . three concentrations ( 0 . 1 , 1 and 10 μm ) were tested for each compound . some compounds were further tested at 20 μm to achieve a range of concentrations . the results , presented as averages and standard deviations are expressed as percentage of survival compared to the control without a2e . the experiments did not show a protective effect of 15 compounds or extract a at concentrations of 0 . 1 and 1 μm ( data not shown for lutein , zeaxanthin , resveratrol , crocetin , naringenin and orientin , and results shown for urucum extract a , cyanidin and ellagic acid in fig1 - 3 ). in a series of tests ( n = 5 ), extract a allowed a significant protection , with cell survival for 20 μm of the order of 93 % of the control without a2e , to be compared with that of control + a2e , which is only 45 % ( fig1 ). among the 15 compounds tested at 10 μm , two provide a cellular protection against phototoxicity . notably , 10 μm cyanidin provides a protection in the order of 87 % survival compared to the control without a2e . at 20 μm , the same compound provides almost complete protection ( fig2 ). ellagic acid also provides protection in the order of 68 % compared to the control without a2e . at 20 μm , this compound provides protection similar to that measured at 10 μm , but with a better reproducibility ( fig3 ). photoprotective effects expected for lutein , zeaxanthin and resveratrol (“ positive controls ”) were not observed with these experimental conditions . bellmann c , sahel j - 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637 . finnemann s c , leung l w , rodriguez - boulan e . 2002 . the lipofuscin component a2e selectively inhibits phagolysosomal degradation of photoreceptor phospholipid by the retinal pigment epithelium . proc . nat . acad . sci . usa 99 : 3842 - 2847 . kim s r , nakanishi k , itagaki y , sparrow j r . 2006 . photooxidation of a2 - pe , a photoreceptor outer segment fluorophore , and protection by lutein and zeaxanthin . exp . eye res . 82 , 828 - 839 . lecerf j - m . 2009 . μnutriments : l &# 39 ; exemple de la dégénérescence maculaire liée à l &# 39 ; âge ( dmla ). médecine maladies métaboliques 3 ( 5 ), 496 - 501 . lecerf j - m , desmettre t . 2010 . nutrition et dégénérescence maculaire liée à l &# 39 ; âge . j . fr . ophtalmol . 33 , 749 - 757 . panichayupakarananta p , issuriya a , sirikatitham a , wang w . 2010 . antioxidant assay - guided purification and lc determination of ellagic acid in pomegranate peel . j . chrom . sci . 48 , 456 - 459 . souied e , le tien v , coscas g , soubrane g . 2007 . vers la prévention de la dégénérescence maculaire liée à l &# 39 ; âge . j . fr . ophtalmol . 30 ( 5 ), 449 - 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