Piel - Eiralabs

Transcription

Piel - Eiralabs

COLLAGENactive Antiox Belleza desde el interior.
Este complemento a base Péptidos de colágeno (Peptan®), Oxxynea®, PureWhey-C™ y
HyaMax® (sodio hialuronato) es el complemento alimenticio nutricosmético desarrollado
para sublimar la belleza del cutis desde el interior. Aquello que es bueno para nuestro interior
se refleja en nuestro aspecto exterior.
Más del 80% de las mujeres asocian el colágeno con el cuidado de la belleza y cosmética. El
colágeno es el componente estructural más importante de la piel constituyendo aproximadamente un 80 % de la masa seca de la piel. La epidermis de la piel es afectada tanto por la
estructura de la capa dermal así como por factores ambientales, incluidos el envejecimiento,
la radiación ultravioleta, hormonas y la nutrición.
Rev.: 05/12/2013
DESCRIPCIÓN PRODUCTO
Piel
COMPOSICIÓN
Peptan® es un colágeno hidrolizado
desarrollado y registrado por Rousselot. La
gama Peptan® está compuesta por
hidrolizados
de
colágeno
natural
procedente de 3 orígenes animales:
pescado, porcino y bovino.
El proceso está rigurosamente controlado
para obtener un preciso grado de hidrólisis,
conseguir el peso molecular óptimo y que
tenga
las
mejores
propiedades
organolépticas.
INDICACIONES
Peptan® es un producto recomendado para:
-
Mejorar el grado de hidratación de la
piel.
Aumentar la suavidad de la piel
reduciendo el número de surcos de
microrrelieve.
Evitar la formación de arrugas
profundas.
Mejorar la elasticidad de la piel.
Peptan® es ideal para:
• Alimentos funcionales
• Bebidas funcionales
• Suplementos alimenticios
• Barritas nutritivas
• Productos en polvo
• Cosméticos
Peptan® son péptidos de colágeno tipo I, el
mismo colágeno que se encuentra en los
huesos y la piel humanos. Peptan® es un
producto bioactivo natural de alta pureza
que contiene más del 97 % de proteína
(base seca), suministrado en una forma que
se puede utilizar y digerir fácilmente por el
cuerpo humano, como ya se ha demostrado
mediante análisis científicos.
Peptan® contiene 20 aminoácidos, incluidos
8 de los 9 aminoácidos esenciales. Se
caracteriza por el predominio de glicina,
prolina e hidroxiprolina, que representan
alrededor del 50% del contenido total de
aminoácidos. La concentración de glicina y
de prolina es de 10 a 20 veces superior a la
de otras proteínas. Esta composición
específica de aminoácidos proporciona a
Peptan® propiedades multifuncionales que
no se pueden encontrar en otras fuentes
proteicas. Una gran fuente de aminoácidos
típicos.
Peptan® tiene excelentes propiedades
organolépticas: es inodoro e insípido, por lo
Rev.: 05/12/2013
que no encubre el sabor ni el olor del
producto terminado, aun cuando se utilice
en elevadas concentraciones.
Peptan® presenta una elevada digestibilidad
como se ha demostrado clínicamente. Más
del 90 % de los hidrolizados son digeridos y
rápidamente absorbidos después de la
ingestión oral.
DESCRIPCIÓN
Actualmente, los péptidos de colágeno se
usan en varios campos, entre los que se
incluyen las comidas y bebidas funcionales
y los complementos dietéticos. Varios
estudios, entre ellos se incluyen ensayos in
vitro de Rousselot, han demostrado que los
péptidos de colágeno son altamente
asimilables.
Más del 90% de los péptidos hidrolizados
son digeridos y absorbidos rápidamente
tras su ingestión por vía oral. Para que sean
activos, los péptidos de colágeno deben
poseer una biodisponibilidad excelente.
Cuando los colágenos de tipo I son
digeridos por la colagenasa, los péptidos
resultantes son quimioatractivos para los
fibroblastos. Los péptidos de colágeno
pueden actuar como mensajeros y
desencadenar la síntesis y reorganización
de nuevas fibras de colágeno mediante la
estimulación de los fibroblastos.
SALUD PIEL
Los estudios clínicos realizados por
Rousselot muestran que Peptan® aumenta
la elasticidad de la piel, lo que podría
deberse a una mayor cohesión de las fibras
de colágeno.
Se ha demostrado que la ingesta oral de 5
a 10 gramos diarios de péptidos de
colágeno puede tener un efecto positivo
sobre los tejidos humanos que contienen
colágeno, como la piel.
Estos
resultados
se
correlacionan
perfectamente con los estudios clínicos de
Rousselot en los que se ha demostrado que
Peptan® mejora la hidratación y la suavidad
de la piel. Por lo tanto, es posible que
estimule el recambio de las células de la
epidermis, acelerando desplazamiento del
agua a través de la capa cutánea,
aumentando la capacidad de fijación del
agua de la porción más externa de la
epidermis y evitando la formación de
arrugas profundas mediante la estimulación
de la síntesis de colágeno.
Efecto del Peptan® sobre la elasticidad,
hidratación y tonicidad de la piel
En un estudio ex vivo utilizando explantes
obtenidos de una mujer caucásica de 49
años se añadieron al medio de cultivo
péptidos de colágeno Peptan®F. Los
explantes de piel se sumergieron en el
medio para simular la ingestión oral.
Únicamente la parte inferior de la dermis
estuvo en contacto con el medio.
Los resultados demostraron que al cabo de
9 días de cultivo, Peptan® F produjo un
aumento del grosor de la piel, así como de
la densidad de colágeno en la dermis
papilar, en lo que respecta a la morfología
general.
Peptan®F aumentó de un 5 a un 9% el
porcentaje de superficie ocupado por el
colágeno en la dermis papilar según la
dosis de Peptan®F utilizado en el cultivo. Un
aumento de un 5% de colágeno en la piel
corresponde a una actividad destacada
comparable a la de los mejores productos
cosméticos sometidos al mismo tipo de
prueba.
Rev.: 05/12/2013
Efecto antienvejecimiento de Peptan®
También se evaluó en contenido en ácidos
glucosaminoglicanos
(GAG)
en
la
epidermis. Dado que es el único ácido GAG
presente en la piel, el ácido hialurónico,
representa aproximadamente el 70% de los
GAG, se procedió a observar la variación
de este ácido mediante una técnica de
tinción que lo tiñe de azul en las células
cultivadas en el medio con Peptan®.
El incremento de la superficie ocupada por
el
azul
alciano
está
directamente
relacionado con la estimulación del ácido
hialurónico. Al cabo de 9 días de
tratamiento con Peptan®F se hizo patente el
incremento de los ácidos GAG, así como su
distribución regular en los espacios
intercelulares de la epidermis y muy
ligeramente en toda la dermis papilar.
Peptan®F produjo un incremento bastante
evidente de GAG ácidos en la epidermis,
que osciló entre un 625 a un 745%
dependiendo de la dosis de Peptan®F del
cultivo.
Las líneas finas, las arrugas y la pérdida de
elasticidad son síntomas que se asocian
con frecuencia al envejecimiento de la piel.
La exposición medioambiental acumulada y
la disminución natural de la renovación
celular contribuyen a la aparición de otros
signos asociados con menor frecuencia al
envejecimiento: piel ajada, áspera o seca.
Se ha puesto a prueba el efecto del
Peptan® sobre la hidratación de la piel y la
acción antienvejecimiento utilizando las
técnicas más avanzadas de investigación
en este campo.
En este sentido se han hecho diferentes
estudios con Peptan® en varios grupos de
mujeres con edades comprendidas entre
los 35 y los 59 años, a las que se les ha
administrado 10 g de Peptan® al día en una
sola toma o bien repartidos en dos tomas
de 5 g, y con periodos de tratamiento que
oscilaron entre las 8 y las 12 semanas.
Los resultados demostraron que Peptan®
aumentó de forma significativa en un 28%
la hidratación de la piel en el 91% de las
Rev.: 05/12/2013
voluntarias tratadas con Peptan® en
comparación con el grupo placebo al
término de las 8 semanas de tratamiento.
Respecto a la degeneración cutánea, es
decir la aparición de surcos y arrugas,
Peptan® también presentó un efecto
significativo.
El número de surcos de microrelieve
disminuyó un 26% después de 12 semanas
de tratamiento y el número de arrugas
profundas fue menor en el grupo Peptan®
que en el placebo en el que aumentó un
30%
Por último, en el mismo grupo de mujeres
que tomaron Peptan® durante 12 semanas
se observó un aumento de un 19% de la
elasticidad de la piel al término de dicho
periodo.
CONCLUSIONES
La hidratación de la piel, según refrieron las
voluntarias, aumentó significativamente en
el grupo tratado con Peptan®. El 68% de las
mujeres percibieron los efectos positivos del
Peptan® sobre la sequedad de la piel.
Rev.: 12/12/2013
DESCRIPCIÓN PRODUCTO
Salud articular
COMPOSICIÓN
Peptan® es un colágeno hidrolizado
desarrollado y registrado por Rousselot. La
gama Peptan® está compuesta por
hidrolizados
de
colágeno
natural
procedente de 3 orígenes animales:
pescado, porcino y bovino.
El proceso está rigurosamente controlado
para obtener un preciso grado de hidrólisis,
conseguir el peso molecular óptimo y que
tenga
las
mejores
propiedades
organolépticas.
INDICACIONES
Peptan® es un producto recomendado para:
- La reconstitución de la estructura de la
sustancia fundamental del tejido óseo.
- La prevención de la degeneración ósea y
articular: osteoporosis y artrosis.
- La mejoría de la movilidad articular:
artritis.
Peptan® es ideal para:
• Alimentos funcionales
• Bebidas funcionales
• Suplementos alimenticios
• Barritas nutritivas
• Productos en polvo
• Cosméticos
Peptan® son péptidos de colágeno tipo I, el
mismo colágeno que se encuentra en los
huesos y la piel humanos. Peptan® es un
producto bioactivo natural de alta pureza
que contiene más del 97 % de proteína
(base seca), suministrado en una forma que
se puede utilizar y digerir fácilmente por el
cuerpo humano, como ya se ha demostrado
mediante análisis científicos.
Peptan® contiene 20 aminoácidos, incluidos
8 de los 9 aminoácidos esenciales. Se
caracteriza por el predominio de glicina,
prolina e hidroxiprolina, que representan
alrededor del 50% del contenido total de
aminoácidos. La concentración de glicina y
de prolina es de 10 a 20 veces superior a la
de otras proteínas. Esta composición
específica de aminoácidos proporciona a
Peptan® propiedades multifuncionales que
no se pueden encontrar en otras fuentes
proteicas. Una gran fuente de aminoácidos
típicos.
Peptan® tiene excelentes propiedades
organolépticas: es inodoro e insípido, por lo
Rev.: 12/12/2013
que no encubre el sabor ni el olor del
producto terminado, aun cuando se utilice
en elevadas concentraciones.
Peptan® presenta una elevada digestibilidad
como se ha demostrado clínicamente. Más
del 90 % de los hidrolizados son digeridos y
rápidamente absorbidos después de la
ingestión oral.
DESCRIPCIÓN
Actualmente, los péptidos de colágeno se
usan en varios campos, entre los que se
incluyen las comidas y bebidas funcionales
y los complementos dietéticos. Varios
estudios, entre ellos se incluyen ensayos de
Rousselot, han demostrado que los
péptidos de colágeno son altamente
asimilables.
Más del 90% de los péptidos hidrolizados
son digeridos y absorbidos rápidamente
tras su ingestión por vía oral. Para que sean
activos, los péptidos de colágeno deben
poseer una biodisponibilidad excelente.
Cuando los colágenos de tipo I son
digeridos por la colagenasa, los péptidos
resultantes son quimioatractivos para los
fibroblastos. Los péptidos de colágeno
pueden actuar como mensajeros y
desencadenar la síntesis y reorganización
de nuevas fibras de colágeno mediante la
estimulación de los fibroblastos.
Durante la artritis esta regulación es
alterada por la expresión de moléculas proinflamatorias que proporcionan el estímulo
para la síntesis de enzimas que degradan la
matriz. Estas enzimas actúan sobre las
fibras de agrecan y colágeno, dando como
resultado la pérdida de cartílago y de
funcionalidad de la articulación.
Efecto del Peptan® sobre las células de
la articulación
Los resultados de los estudios de Rousselot
confirman el efecto positivo del Peptan®
sobre las células de la articulación
(condrocitos). Una dosis de 10 g de
péptidos de colágeno al día ha demostrado
un efecto positivo sobre la reducción del
dolor articular.
Se ha investigado el efecto del Peptan®
sobre los principales componentes de la
matriz extracelular del cartílago: agrecan y
colágeno de tipo II, y se ha evidenciado que
Peptan® aumenta significativamente la
expresión de marcadores específicos del
cartílago: agrecan (azul) y colágeno de tipo
II (verde) (Fig. 1).
SALUD ARTICULAR
El cartílago está constituido por un único
tipo celular, los condrocitos embebidos en
una matriz extracelular compuesta por dos
componentes principales: colágeno de tipo
II, que otorga resistencia a la tracción al
tejido y agrecan, que proporciona la
capacidad del cartílago para resistir a
fuerzas de compresión. Una síntesis y
renovación
orquestadas
aseguran
y
mantienen las características bioquímicas
del cartílago.
®
Fig.1 Efecto del Peptan sobre la expresión del
ARNm de agrecan y de colágeno de tipo II tras 8 días
de tratamiento (qPCR). *: significativo respecto al
control p < 0,05
Peptan® demuestra tener un efecto sobre
las células de las articulaciones similar al
del ibuprofeno. Estos datos confirman que
Rev.: 12/12/2013
Peptan® puede prevenir la degradación de
la matriz cartilaginosa aumentando la
producción de agrecan y colágeno de tipo
II, en línea con resultados de estudios
anteriores. Los datos también refuerzan la
hipótesis de que la presencia de péptidos
de colágeno hidrolizado puede ser
entendida por los condrocitos como una
señal de degradación del cartílago que, en
consecuencia, activan la síntesis de
agrecan y colágeno de tipo II como
respuesta.
Peptan® mejora el dolor articular
Mediante el uso de la escala de valoración
WOMAC, se demostró que Peptan®
produjo un descenso del dolor de la
articulación a los 3 y 6 meses, siendo dicho
resultado estadísticamente significativo al
término del estudio (Fig. 2).
Estos resultados muestran que Peptan®
puede ser usado para prevenir la
degradación del cartílago, previniendo así
las molestias y el dolor articular debido a
dicha degradación.
Efecto del Peptan® sobre la mejoría
funcional articular en mujeres con artritis
de rodilla
Los excelentes resultados de los estudios in
vitro a nivel celular promovieron a
Rousselot a corroborar dichos resultados a
nivel clínico.
Para ello, se llevó a cabo un estudio clínico
aleatorio a doble ciego comparado respecto
a placebo de 6 meses de duración, en el
que participaron 100 mujeres con edades
comprendidas entre los 40 y 70 años de
edad afectadas de gonartriris (artritis de
rodilla).
®
Fig.2 Efecto del Peptan sobre el dolor articular de la
rodilla en mujeres con gonartritis a los 3 y 6 meses de
tratamiento (p < 0,05 a los 6 meses)
Peptan® mejora la funcionalidad articular
La valoración del grado de funcionalidad y
movilidad de la rodilla (cojear, saltar,
caminar, subir) también demostró una
evolución positiva con el uso del Peptan®,
tanto a los 3 como 6 meses de tratamiento
(Fig. 3).
El objetivo fue evaluar la eficacia de un
tratamiento con 8 g de Peptan® o placebo
sobre el dolor y la funcionalidad de la
articulación de la rodilla a los 3 y 6 meses
de consumo del tratamiento.
Los resultados mostraron una mejoría
general de los dos parámetros evaluados
así como un altísimo grado de seguridad en
el consumo de Peptan®.
®
Fig.3 Efecto del Peptan sobre la funcionalidad de la
rodilla en mujeres con gonartritis a los 3 y 6 meses de
tratamiento (p < 0,05 a los 6 meses)
Rev.: 12/12/2013
SALUD OSEA
Así mismo se ha estudiado el beneficio del
consumo de Peptan® sobre la estructura
ósea y su efecto sobre las destrucción y
regeneración del hueso.
Efecto del Peptan® sobre la diferenciación de células en osteoblastos
En cultivos de osteoblastos y osteoclastos
se observa el aumento de los niveles de
fosfatasa alcalina, un marcador de
formación ósea, cuando Peptan® está
presente en el medio.
Dado que el número de células no aumenta
con Peptan® en comparación con el grupo
de control, esto significa que Peptan®
induce la diferenciación de células en
osteoblastos, en lugar de osteoclastos (Fig.
4).
Peptan® restaura la densidad mineral, el
tamaño y la solidez ósea
Para estudiar el efecto de Peptan® sobre la
estructura ósea, se usó el modelo animal de
ratones con histerectomía de ovario, ya que
causa una baja densidad de masa ósea:
osteopenia.
Los ratones se alimentaron con o sin
Peptan® durante 12 semanas y se
recogieron diversos datos. En los ratones
con histerectomía alimentados con Peptan®
las mediciones mostraron la restauración
del valor de densidad mineral ósea cerca
del nivel del grupo de control (Fig. 5).
Fig.4 Mediciones de la actividad de fosfatasa alcalina
®
(ALP) en cultivo de células óseas con Peptan B
®
(bovino), Peptan® P (porcino) y Peptan F (pescado)
durante 14 días, en comparación con BSA (proteínas
estándar).
Fig.5 Aumento de la Densidad Mineral Ósea (DMO)
de los ratones, en el grupo de control (Control), el
®
grupo ovariectomizado alimentado sin Peptan (Ovx),
y el grupo ovariectomizado alimentado con Peptan®
(Ovx + Peptan)
Además, cuando el cultivo se realiza en un
sistema que permite la medición de la
reabsorción ósea mediada por osteoclastos,
se observa que la presencia de Peptan®
reduce el área de reabsorción en
comparación con el control.
Además, se midió también el crecimiento de
la zona cortical del hueso del fémur y del
tamaño del hueso, encontrándose un
aumento significativo en los ratones con
histerectomía alimentados con Peptan®, en
comparación con el control (Fig. 6a).
Rev.: 12/12/2013
Los péptidos de Peptan® presentan una
elevada digestibilidad y biodisponibilidad, lo
que garantiza su acción sobre las zonas del
organismo en las que llevan a cabo su
función.
Peptan® ha demostrado que actúa sobre los
procesos articulares, reduciendo el dolor y
evitando la degeneración del cartílago
articular.
Debido a esta restauración, la carga
máxima de rotura de los huesos (fuerza
requerida para producir una rotura) fue
significativamente mayor en los ratones con
histerectomía de ovario alimentados con
Peptan® (Fig. 6b).
Así mismo, Peptan® posee una acción
específica sobre los dos procesos
implicados en el mantenimiento de la
estructura ósea. Por un lado Peptan® inhibe
la acción de los osteclastos y por tanto
reduce la degradación de masa ósea, al
mismo
tiempo
que
estimula
la
diferenciación de las células en osteoblastos contribuyendo a la reconstrucción del
hueso. Esta doble acción se manifiesta en
el aumento de la densidad mineral ósea y el
tamaño y solidez de los huesos.
Gracias a estas propiedades sobre la salud
articular y ósea, Peptan® es un gran aliado
para aquellos grupos de personas más
susceptibles de padecer inflamación y/o
degeneración articular y ósea, como son las
personas
ancianas,
mujeres
menopáusicas y los deportistas.
DOSIS RECOMENDADA
Se recomiendan de 8 a 10 g Peptan® al día.
Fig.6 Medición del área cortical (a) y la carga máxima
de rotura (b), en el grupo de control (Control), el
®
grupo ovariectomizado alimentado sin Peptan (Ovx),
®
y el grupo ovariectomizado alimentado con Peptan
(Ovx + Peptan).
CONCLUSIONES
Peptan® es un colágeno de elevada calidad
con muy buenas características gracias a
sus
propiedades
organolépticas
y
funcionales sobre el organismo.
El colágeno hidrolizado Peptan® se
considera un ingrediente alimentario
seguro.
Rev.: 05/12/2013
Las voluntarias valoraron una mejora de la
pérdida de hidratación que percibían tras la
limpieza y la aplicación de maquillaje. Aquí
se confirmó el efecto positivo de la
administración por vía oral del Peptan® en
la rutina diaria del cuidado de la piel.
Así mismo, Peptan® fue percibido por las
voluntarias como más eficaz que el placebo
en cuanto a la elasticidad, la tonicidad y el
brillo de la piel.
Por tanto Peptan®
clínicamente que:
-
ha
demostrado
Mejora el grado de hidratación de la
piel.
Aumenta la suavidad de la piel
reduciendo el número de surcos de
microrrelieve.
Evita la formación de arrugas profundas.
Mejora la elasticidad de la piel
La tolerancia del producto fue evaluada
mediante el examen clínico y el cuestionario
que realizaron las voluntarias. No se
comunicó ninguna reacción adversa en el
grupo Peptan®.
Todos los resultados llevan a la misma
conclusión: Peptan® mejora significativamente las sequedad de la piel y los
síntomas asociados.
Estos estudios demuestran que Peptan®
administrado diariamente hasta 12 semanas, produce un efecto positivo en la
estructura de la piel, haciendo que las
células sinteticen el colágeno y el ácido
hialurónico, componentes clave de la piel
relacionados con el tono, la elasticidad y la
hidratación de la piel.
DOSIS RECOMENDADA
Gracias a estos resultados y a su situación
legal, Peptan® constituye un ingrediente
muy
potente
para
el
mercado
nutricosmético.
Se recomiendan 10 g Peptan® al día
repartidos en varias tomas.
El colágeno hidrolizado Peptan® se
considera un ingrediente alimentario
seguro.
Mediterranean
Polyphenol Concentrate
SCIENCE PACK
Executive summary
White paper
Published efficacy study
Technical data sheets
page 2
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page 22
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The health benefits of the Mediterranean diet
The evidence for a protective effect of fruit & vegetables on CVDs has been
shown in several meta-analyses and this protective effect has been
strengthened by recent results from a large-scale study using data from the
European Prospective Investigation into Cancer and nutrition (EPIC) cohort
launched for 15 years between 1992 and 1999 [1]. Authors found that
consumption of fruit & vegetables was inversely associated with all-cause
mortality with a hazard ratio of 0.90 and a confidence interval of 0.86 to 0.94
for the highest quartile of consumption. The PREDIMED survey launched in
2003 confirmed health benefits of the Mediterranean diet in a long-term
nutritional intervention study as a primary prevention of CVDs and on all other
causes of mortality and incidence of heart failure, diabetes, cancer,
dementia & other neurodegenerative diseases [2]. In particular, this study
demonstrated a remarkable decrease of abdominal circumference, as well
as an increase in high density lipoprotein (HDL-cholesterol), a decrease in
triglycerides, a lowering of blood pressure and a decrease in blood glucose
concentration. This was mainly due to the plentiful use of fruits, olive oil, bread,
pasta, vegetables, herbs, garlic, red onions, and other foods of vegetable
origin, all fruit & vegetables rich in phenolic compounds.
The ‘5-a-day’ model
Although traditional recipes vary within the 19 countries bordering the
Mediterranean Sea, a common dietary pattern is characteristic from that
area. The typical Mediterranean diet is high in fruit, vegetables, cereals,
beans, nuts & seeds and includes olive oil as an important monounsaturated
fat source.
4
The discovery of the health benefits of the Mediterranean Diet is attributed to
the American scientist Ancel Keys who pointed out the correlation between
cardiovascular diseases (CVDs) and diet for the first time in 1975 [3]. Starting
from Keys’s studies, many other researchers have analyzed the association
between dietary habits and non-communicable chronic diseases (NCDs) [4].
It is now possible to say that there is a consensus around the beneficial
qualities of the Mediterranean way of eating, which in 1990 led the World
Health Organization (WHO) to issue recommendations for a minimum daily
intake of 400 g of fruit and vegetables based on evidence that higher
consumption was protective against CVDs and various NCDs [5]. This was the
beginning
of
the
‘5-a-day’ fruit
&
vegetables
campaign
which
recommended varying numbers of 75-80 g servings of fruit & vegetables.
The role of polyphenols in antioxidant homeostasis
The importance of antioxidants in human health has become increasingly
clear and some epidemiological studies showed the potential benefits of
dietary antioxidants. Although health authorities have not yet recognized the
importance of total antioxidant capacity in the bloodstream, combined
results from multiple studies around the world demonstrate that an antioxidant
homeostasis exists. These studies also point out that this equilibrium is deeply
linked to health and that this is balanced with both endogenous antioxidant
defenses (SOD, GPX, Catalase…) and exogenous bioactive antioxidants
delivered from various foods (polyphenols, carotenoids, minerals…).
Fruit & vegetables are the largest contributors (beverages not included) to
total dietary antioxidant capacity (TDAC), which is considered a dietary
quality indicator [6]. Several bioactive components of the Mediterranean diet
may explain the benefits of fruit & vegetables such as olive oil, red wine
polyphenols (antioxidants), or whole-grain phenolic acids, flavonoids, tannins,
5
lignans, and other active compounds. Although the antioxidant capacity of
food is derived from the accumulative and synergistic antioxidant power of
vitamins,
polyphenols,
carotenoids
and
other
minor
constituents
[7],
polyphenols are the main antioxidants present in fruit & vegetables (> 90% of
TDAC) [8]. To elucidate the significance of polyphenols in human health, it is
essential to know the amount of polyphenols consumed in the diet and their
bioavailability. Polyphenols constitute a very heterogeneous group of
compounds, with over 5,000 different molecules that have different properties
and levels of bioavailability [9]. Polyphenols are divided into five main groups
according to their structure profile: phenolic acids, flavonoids, stilbenes,
lignans and others (such as secoiridoids) [10-11]. This diversity should be
considered when studying the health effects of these compounds and
hampers the estimation of their content in foods.
Proprietary composition of Oxxynea®
OXXYNEA® is a Mediterranean diet-based innovative ingredient which when
taken daily fulfills the 5-a-day recommendation in terms of antioxidant
potency. Oxxynea® is developed from a selection of 22 natural extracts and
concentrates from fruit & vegetables*, providing an amount of total
polyphenols higher than 75 % with an ORAC 5.0™ value (Brunswick
Laboratories, Inc – MA USA) higher than 45,000 µmol trolox equivalent
(TEQ)/day and bringing a representative fingerprint of bioactive polyphenols
(Figure 1) acting synergistically to offer the best health benefits from the
traditional Mediterranean diet.
11 fruit: red & white grape, bilberry, grapefruit, papaya, pineapple,
strawberry, apple, apricot, cherry, orange, blackcurrant.
11 vegetables: green tea, carrot, tomato, broccoli, green cabbage,
onion, garlic, wheat germ, asparagus, olive, cucumber.
6
Figure 1: The Oxxynea® HPLC fingerprint at 280 nm
An analysis of total polyphenols following the Folin Ciocalteu methodology
[13] showed that a daily intake of Oxxynea® delivers the same quantity of
antioxidants as the consumption of 5 typical servings of fruit & vegetables
selected among the most consumed in France (5 F&V) [12] (Figure 2).
However, the synergistic formulation of 22 natural extracts and concentrates
of fruits & vegetables* demonstrates better antioxidant benefits from Oxxynea
than from 5 F&V (Figures 4 and 5)
Measured by Folin-Ciocalteu (mg Gallic Acid Equivalent)
Figure 2: Total polyphenol amount in both Oxxynea® and 5 F&V
7
Mechanism of action of Oxxynea®
High levels of reactive oxygen species (ROS) have been associated with the
initiation and progression of atherosclerosis, the underlying cause of CVDs.
Well-established CVDs risk factors such as smoking and aging are associated
with increased ROS production, as are obesity, hypertension, diabetes, and
excessive alcohol consumption [14]. The core action of all polyphenols is their
capacity to eliminate ROS with their aptitude to directly and indirectly
cleanse oxidant species such as superoxide anion (O2·-), hydrogen peroxide
(H2O2), hydroxyl radical (OH·) and various peroxyradical (ROO), and
subsequently to prevent organs & tissues from oxidative damages (Figure 3).
In the organism, polyphenol benefits from Oxxynea® might be able to boost
scavenging antioxidant defenses as demonstrated by their particularly high
ORAC 5.0™ value, and to balance endogenous oxidative stress as
demonstrated by the KRL ex vivo assay of blood protection against induced
oxidation(Kirial International, France) [15]. Besides, Oxxynea® is able to
indirectly provide additional antioxidant benefits as demonstrated on
atherogenic Golden Syrian Hamsters [16] with an action on both the
expression and the activity of the pro-oxidant endogenous enzyme, the
NADPH oxidase.
Figure 3: Antioxidant mechanism of action of Oxxynea®
8
Ex vivo and in vitro antioxidant benefits
KRL ex vivo assay
During the submission of a blood sample to a radical aggression in a
controlled and standardized assay, all biological enzymatic systems and
bioactive compounds mobilize to protect the integrity of the cells from
lysis. Measuring the decrease in absorbance is used to monitor the progressive
destruction of the cells. The resistance of the blood to radical attack is
expressed by the time required for lysis of 50% of blood cells (hemolysis half
time). In the KRL assay, Oxxynea® protects blood cells at 214% of the level of
antioxidant protection obtained with 5 F&V (Figure 4).
Measured by KRL Testing (mg Gallic Acid Equivalent)
Figure 4: Oxxynea® and 5 F&V blood cell protection against oxidative stress
ORAC 5.0™ in vitro assay
ORAC 5.0™ consists of five types of assays that evaluate the antioxidant
capacity against five primary different ROS occurring in human metabolism.
The assay is based on the capacity of a sample to protect a probe from
damages by those ROS. The degree of probe preservation indicates the
antioxidant capacity based on the equivalence of a water soluble form of
vitamin E, the Trolox.
9
ORAC 5.0™ includes the following:
ORAC against Peroxyl Radical both hydrophilic and lipophilic medium
HORAC: ORAC against Hydroxyl Radical
NORAC: ORAC against Peroxynitrite
SORAC: ORAC against Superoxide Anion
SOAC: ORAC against Singlet Oxygen
In the ORAC 5.0™ assay, Oxxynea® demonstrates an antioxidant protection
equivalent to 182% of the protection obtained with 5 F&V (Figure 5).
Measured by ORAC 5.0™ assay (µmol Trolox Equivalent)
Figure 5: Oxxynea® and 5 F&V ORAC 5.0™ antioxidant capacity
Preclinical investigation: Oxxynea® benefits on atherogenic-fed Hamsters [16]
The regular consumption of Oxxynea (equivalent daily dose of 800 mg for
Humans) during 12 weeks by atherogenic-fed hamsters demonstrates a
significant decrease -45.5% (p < 0.05) of ROS production in the heart as
assessed with O2·- release from cardiac tissue (Figure 6). O2·- has been widely
described to participate to the first steps of the initiation of atherosclerosis in
Humans [17].
Figure 6: Cardiac superoxide anion production is reduced with Oxxynea®
10
This beneficial result is supported by an increase in total plasma antioxidant
capacity (TPAC) in animals taking Oxxynea®: +10% (p < 0.05) compared to
placebo, which demonstrates the bioavailability (absorption + efficacy) of
polyphenols from the product.
Figure 7: TPAC increase demonstrates polyphenols bioavailability from
Oxxynea®
The regular consumption of polyphenols from Oxxynea® during 12 weeks is
able to significantly limit the atherosclerotic process as demonstrated with the
highly significant reduction -77% (p < 0.05) of average fatty streak formation in
aortic arch of atherogenic-fed hamsters (Figure 8) which corresponds to the
first step of the atheroma development in Humans.
Figure 8: Polyphenols from Oxxynea® deeply limit fatty streak formation in
aortic arch
11
The Mediterranean diet health benefits from Oxxynea®
Polyphenols from Oxxynea® have been demonstrated to play a key role in
scavenging and reducing various ROS as demonstrated in vivo, in vitro and ex
vivo, to such an extent that despite a similar level of polyphenols as in 5
typical servings of fruit & vegetables, Oxxynea® brings higher antioxidant
protection thanks to its synergistic formulation from 22 natural extracts and
concentrates of fruit & vegetables*.
In preclinical application the antioxidant benefits of Oxxynea® validate the
capacity of polyphenols acting in cooperation to mitigate the early
atherosclerotic process. Because modern lifestyles and eating habits
increasingly tend to aggravate health condition, the resulting risen stress is
liable to weaken antioxidant defenses, leading at turn to an increased risk of
suffering from unbalanced ROS levels and free radicals-mediated long term
NCDs such as diabetes, cancers and CVDs. It is now well accepted that the
cornerstone of the benefits of a regular consumption of fruit and vegetables
within the Mediterranean diet is the wide and complementary source of
antioxidant activities from polyphenols which main action is to scavenge ROS
and other free radicals. Oxxynea® has been specifically designed to fulfill this
function.
12
Literature cited:
[1] Leenders M, et al. 2013. Fruit and Vegetable Consumption and Mortality
European Prospective Investigation into Cancer and Nutrition. American Journal of
Epidemiology. Online publication
[2] Estruch R, et al. 2013. Primary Prevention of Cardiovascular Disease with a
Mediterranean Diet. The New England Journal of Medicine. 368(14): 1279-90
[3] Keys A, Keys M. 1975. How to Eat Well and Stay Well the Mediterranean Way.
Doubleday Eds. 488 p
[4] World Health Organization. 2013. Health topics on No Communicable Diseases
[5] World Health Organization. 2004. Report of a joint FAO/WHO workshop on fruit &
vegetables for health
[6] Saura-Calixto F, Goñi I. 2009. Definition of the Mediterranean diet based on
bioactive compounds. Critical Reviews in Food Science and Nutrition. 49: 145–52
[7] Liu D, Colina-Ibarra J, Kakuda A, Hue S J. 2008. The scavenging capacity and
synergistic effects of lycopene, vitamin E, vitamin C and β-carotene mixtures on the
DPPH free radical. LWT Food Science and Technology. 41:1344–49
[8] Saura-Calixto F, Pérez-Jiménez J, Goñi I. Dietary fiber and associated antioxidants
in fruit and vegetables. De la Rosa L A, Álvarez-Parilla E, González-Aguilar G A (Eds.),
2010. Fruit and vegetable phytochemicals, Wiley-Blackwell, Iowa. pp. 223–34
[9] Neveu V, et al. 2009. Phenol-Explorer: an online comprehensive database on
polyphenol contents in foods. The Journal of Biological Databases and Curation. ID
Bap024
[10] Manach C, Scalbert A, Morand C, Remesy C, Jimenez L. 2004. Polyphenols: food
sources and bioavailability. American Journal of Clinical Nutrition. 79(5): 727–47
[11] Perez-Jimenez J, Neveu V, Vos F, Scalbert A. 2010. Identification of the 100
richest dietary sources of polyphenols: an application of the Phenol-Explorer
database. European Journal of Clinical Nutrition. 64 (S3): S112–20.
[12]
Source:
Kantar
Worldpanel
market
data.
Available
at:
www.lesfruitsetlegumesfrais.com/filiere-et-metiers/les-chiffres-cles/les-produits-lesplus-consommes
[13] Georgé S, Brat P, Alter P, Amiot MJ. 2005. Rapid determination of polyphenols
and vitamin C in plant-derived products. Journal of Agricultural Food and Chemistry.
53(5): 1370-3
13
[14] Pitale S, Sahasrabuddhe A. 2011. Fetal origin of vascular aging. Indian Journal of
Endocrinology and Metabolism. 15(8): 289-97
[15] Caspar-Bauguil S, et al. 2009. Evaluation of whole antioxidant defenses of human
mononuclear cells by a new in vitro biological test: lack of correlation between
erythrocyte and mononuclear cell resistance to oxidative stress. Clinical Biochemistry.
42(6): 510-4
[16] Sutra T, Décordé K, Riss J, Dallas C, Cristol J P, Rouanet J M. 2007. A commercial
extract of fruits and vegetables, Oxxynea®, acts as a powerful antiatherosclerotic
supplement in an animal model by reducing cholesterolemia, oxidative stress, and
NADPH oxidase expression. Journal of Agricultural and Food Chemistry. 55(10): 425863
[17] Cathcart M K. 2004. Regulation of superoxide anion production by NADPH
oxidase in monocytes/macrophages: contributions to atherosclerosis. Atherosclerosis,
Thrombosis, and Vascular Biology. 24(1): 23-8
14
Oxxynea®
Published Efficacy Study
15
4258
J. Agric. Food Chem. 2007, 55, 4258−4263
A Commercial Extract of Fruits and Vegetables, Oxxynea, Acts
as a Powerful Antiatherosclerotic Supplement in an Animal
Model by Reducing Cholesterolemia, Oxidative Stress, and
NADPH Oxidase Expression
THIBAULT SUTRA,†,‡ KELLY DEÄ CORDEÄ ,† JEÄ ROME RISS,† CONSTANT DALLAS,§
JEAN-PAUL CRISTOL,‡ AND JEAN-MAX ROUANET*,†
EA 3762 Nutrition & Aliments, Place E. Bataillon, Université Montpellier 2, 34095 Montpellier,
France, EA 2993 Nutrition Humaine & Athérogénèse, Université Montpellier 1, CHU La Peyronie,
34925 Montpellier, France, and Nutraceutic Business Consulting (NB Consulting),
24 rue René Caillie, 34500 Béziers, France
The effects of fruit and vegetable extract (Oxxynea) on plasma cholesterol, early atherosclerosis,
cardiac production of superoxide anion, and NAD(P)H oxidase expression were studied in an animal
model of atherosclerosis. Thirty six hamsters were divided into two groups of 18 and fed an atherogenic
diet for 12 weeks. They received by gavage either water or Oxxynea in water at a human dose
equivalent of 10 fruits and vegetables per day. Oxxynea lowered plasma cholesterol and non-HDL
cholesterol, but not HDL-cholesterol, and increased plasma antioxidant capacity. It also strongly
reduced the area of aortic fatty streak deposition by 77%, cardiac production of superoxide anion by
45%, and p22phox subunit of NAD(P)H oxidase expression by 59%. These findings support the view
that chronic consumption of antioxidants supplied by fruits and vegetables has potential beneficial
effects with respect to the development of atherosclerosis. The underlying mechanism is related mainly
to inhibiting pro-oxidant factors and improving the serum lipid profile.
KEYWORDS: Atherosclerosis; hamsters; fruits and vegetables; antioxidant compounds; NADPH oxidase
INTRODUCTION
Mortality from cardiovascular disease is the leading cause
of death in the industrialized world. Diet is believed to play a
major role in the development of this disease, and much research
is being focused on identifying ways to prevent it through
changes in dietary habits. Oxidation of low-density lipoproteins
(LDL) is traditionally accepted as initiating processes leading
to the development of atherosclerosis. The earliest events in
the development of the pathology are endothelial dysfunction
and oxidative stress in the vascular cell wall, activation of
inflammatory cells, and migration of vascular smooth muscle
cells to the intima with the modification of the extracellular
matrix, leading to the artery remodeling (1). Development of
atherosclerosis is thought to be closely dependent upon increased
oxidative stress, that is, an imbalance between reactive oxygen
species (ROS) generation (chiefly superoxide anions, hydrogen
peroxide, hydroxyl radicals) and natural cell antioxidant capacity
in favor of the former (2). ROS can regulate many signaling
pathways, such as infiltration of monocytes in intima and
vascular smooth muscle cell proliferation. The cause of oxidative
* Author to whom correspondence should be addressed [telephone/fax
33 (0)4 67 14 35 21; e-mail [email protected]].
† Université Montpellier 2.
‡ Université Montpellier 1.
§ NB Consulting.
stress observed in atherosclerosis awaits clarification. Recent
findings have suggested that the major source of ROS in the
vascular wall, and also in vascular smooth muscle cells, is the
NAD(P)H oxidase system. This is a membrane-associated
enzyme, composed of five subunits, catalyzing the one-electron
reduction of oxygen, using NADH or NADPH as the electron
donor. NAD(P)H oxidase generates significant amounts of
superoxide radicals, and an association between enzymatic
activity and clinical risk factors in atherosclerosis has been
shown (3). Moreover, expression of membrane subunits of
NAD(P)H oxidase, such as p22phox, Nox 1, and Nox 4, is
modulated in atherosclerotic arteries (4) and in vascular injury
(5) by various cytokines like interferon (IFN)-γ and transforming
growth factor (TGF)-β1. Azumi et al. (6) found that the severity
of atherosclerotic lesion correlated with p22phox overexpression
in coronary arteries. Excessive generation of superoxide anion
by phagocyte NADPH oxidase is responsible for LDL oxidation,
which is the key factor in the initation and progression of
atherosclerosis (1, 7). The contribution of NADPH oxidase to
the pathogenesis of atherosclerosis overshoots LDL oxidation
process. NAD(P)H oxidase induces the expression of adhesion
molecules in endothelial cells for recruitment of monocytederived macrophages (8), leading to an amplification system
(9) and vascular smooth muscle cells proliferation (10).
10.1021/jf070029n CCC: $37.00 © 2007 American Chemical Society
Published on Web 04/20/2007
Oxxynea as a Powerful Antiatherosclerotic Supplement
The importance of antioxidants in human health has become
increasingly clear, and some epidemiological studies showed
the potential health benefits of dietary antioxidants (11). Fruits
and vegetables consumption is inversely related to cancer and
coronary heart disease mortality, and this appears not to be due
exclusively to high levels of vitamins and fibers (12, 13). Several
studies have shown that flavonoids also contribute to the overall
antioxidant capacity of fruits and vegetables and also to the
beneficial effects, a view supported by recent research demonstrating that dietary flavonoids protect against vascular diseases
and reduce the risk of myocardial infaction (13). There is
growing interest in flavonoids and phenolic compounds because
they are potent antioxidants and inhibit low-density lipoprotein
(LDL) oxidation in vitro (14), properties that are associated with
their ability to scavenge free radicals and chelate metals. An
increased consumption of phenolics has been correlated with a
reduced risk of cardiovascular diseases and certain types of
cancers (15, 16). Moreover, polyphenols have been shown to
directly interact with NAD(P)H oxidase, inhibiting most of the
ROS production in the vessel wall (17). Cumulatively or
synergistically, these dietary antioxidants provide bioactive
mechanisms to reduce oxidative stress.
With the exception of recent research by Adams et al. (18)
with a transgenic mice model, few studies have investigated
the effect of plant material on atherosclerosis and oxidative stress
in rodents (19, 20); moreover, these studies were only focused
on the effect of vegetables. Golden Syrian hamsters fed a fatrich diet develop dyslipidemia and atherosclerotic plaques,
similar in many respects to human atheroma (21-23). Hamsters
were selected for this study because of their responsiveness to
plasma cholesterol lowering and anti-atherogenic interventions
(24). Moreover, hamster has a plasma lipoprotein distribution
similar to that of humans and LDL as the major plasma
cholesterol carrier. To induce an oxidative stress, their high
cholesterol and high fat diet was rendered deficient in vitamin
C and E and in selenium. This study was designed to trigger
the arterial wall response to such a stress (fatty streak formation
and aortic atherosclerosis emergence) and then to look at the
modulation of this effect by a commercial fruit and vegetable
extract, Oxxynea. In addition, for the first time, modulation of
oxidative stress parameters including cardiac production of
superoxide anions and NAD(P)H oxidase expression was
measured in this model.
MATERIALS AND METHODS
Fruits and Vegetable Extract. According to the manufacturer (NB
Consulting, Béziers, France), the powdered Oxxynea extract was
obtained from 22 varieties of antioxidant-rich fruits and vegetables
including apple, asparagus, bilberry, apricot, black currant, broccoli,
carrot, cherry, cucumber, garlic, grapefruit, green cabbage, olive, onion,
orange, papaya, pineapple, red and white grapes, strawberry, tea, tomato,
and wheat germ. Oxxynea contains high level of catechins, that is, sum
of procyanidin dimers B1, B2, B3, and B4 (1.14 g/100 g) and
monomeric catechins (catechin, 0.55 g/100 g; epicatechin, 3.08 g/100
g; epichatechin-3-O-gallate, 4.10 g/100 g; epigallocatechin, 4.17 g/100
g; epigallocatechin-3-O-gallate, 21.33 g/100 g). Other phenolic compounds such as gallic acid and anthocyanins were detected in lower
amounts (0.15 and 0.6 g/100 g, respectively). The extract also contained
low levels of lycopene (28 mg/100 g) and vitamin C (4.92 mg/100 g).
Oxygen Radical Absorbance Capacity (ORAC) Value. The
ORAC-fluorescein assay was based on the method of Ou et al. (25)
that was subsequently modified by Davalos et al. (26). Briefly, the
reaction was performed in 75 mM phosphate buffer (pH 7.4), and the
final assay mixture (200 µL) contained fluorescein (120 µL, 70 nM
final concentration) as oxidizable substrate, 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH, 60 µL, 12 mM final concentration) as
J. Agric. Food Chem., Vol. 55, No. 10, 2007
4259
oxygen radical generator, and antioxidant (20 µL, either trolox [1-8
µM, final concentration] or Oxxynea [at different concentrations]). The
reaction was performed at 37 °C, and fluorescence was recorded every
minute for 80 min. ORAC values was expressed as trolox equivalents
by using the standard curve calculated for each experiment. The final
ORAC value for Oxxynea was 6100 µmol of trolox equiv/g.
Animals. Male golden Syrian hamsters (Janvier, Le Genest-St-Isle,
France) weighing 85-95 g were randomly divided into groups with
approximately equal mean group body weights. The animals were
housed in plastic cages in a temperature-controlled room (23 ( 1 °C)
subjected to a 12 h light:dark cycle (lights on at 0700 h) with free
access to both food and water. Hamsters were handled according to
the guidelines of the Committee on Animal Care at the University of
Montpellier and NIH guidelines (27).
Diets and Feeding Procedures. Two experiments were carried out
concomitantly. Experiment 1 was used to determine at what point
hamsters on the atherogenic diet exhibited oxidative stress and
hypercholesterolemia. In such a way, four groups of six hamsters each
received either a standard or an atherogenic diet for 15, 30, 45, and 84
days. The standard diet consisted of 200 g/kg casein and 3 g/kg
L-methionine, 447 g/kg corn starch, 175 g/kg sucrose, 50 g/kg cellulose,
80 g/kg vegetable oil (corn oil/sunflower oil, 1/1), mineral mix (35
g/kg), and vitamin mix (10 mg/kg). The atherogenic diet has been
previously described (22) and consisted of 200 g/kg casein and 3 g/kg
L-methionine, 393 g/kg corn starch, 154 g/kg sucrose, 50 g/kg cellulose,
150 g/kg lard, 5 g/kg cholesterol, mineral mix (35 g/kg), and vitamin
mix (10 mg/kg). Vitamin and mineral mixes were formulated according
to AIN-93 guidelines (28) and supplied by Scientific Animal Food &
Engineering (SAFE, Augy, France). The atherogenic diet did not contain
selenium, vitamin C, and vitamin E.
In experiment 2, two groups of 18 hamsters were fed the atherogenic
diet for 84 days. The hamsters of each group were fed daily by gavage
either tap water (group 1; control) or a solution of Oxxynea in water
(group 2; experimental). The volume of the solutions force-fed was
adjusted daily to the weight of the hamsters and was established by
extrapolating 500 mL/d water drinking for a 70 kg human. This
represents a volume of 7.14 mL/(kg of body weight‚d). Based upon
the ORAC value of fruits and vegetables starting material and issuing
Oxxynea, and according to a recommended consumption of 10 servings
of fruits and vegetables/d for a human, that is, ∼800 g/d, hamsters
from the experimental group received 21.4 mg of Oxxynea/(kg body
weight‚d) dissolved in water.
Analytical Procedures. At the end of each experimental period,
hamsters were deprived of food for 18 h and were anesthetized with
an IP injection of pentobarbital (60 mg/mL at a dosage of 60 mg/kg
body weight). In experiment 1, only plasma cholesterol, cardiac
superoxide anion production, and NADPH oxidase expression were
measured as described below. In experiment 2, blood was drawn by
cardiac puncture with heparin moistened syringes, and plasma was
prepared by centrifugation at 2000g for 10 min at 4 °C, then stored at
-80 °C prior to analysis. Plasma concentrations of total cholesterol
(TC) and HDL cholesterol (HDL-C) were measured by an enzymatic
technique (Konelab, Thermo Electron Corp., Vantaa, Finland). Plasma
nonHDL-C was calculated from the difference between TC and HDLC.
The antioxidant capacity of plasma was measured as trolox
equivalent, that is, a quantitative value for general antioxidant levels
in biological samples (29, 30), which was assayed in plasma with a
quantitative colorimetric technique according to the kit supplier’s
instructions (Kit NX2332; Randox, Mauguio, France). The assay is
based on the incubation of a peroxidase and H2O2 with 2,2′-azino-di(3-ethylbenzthiazoline sulfonate) (ABTS) to produce the radical cation
ABTS°+. This has a relatively stable blue-green color, which is
measured at 600 nm. Antioxidants (albumin, uric acid, ascorbic acid,
R-tocopherol, glutathione, beta-carotene, etc.) in the sample suppressed
ABTS°+ color production to a degree proportional to their concentration.
Aortic Tissue Processing. Following blood collection and liver
removal, the intact aorta from 12 hamsters was first perfused with
phosphate buffered saline containing 1 mmol/L CaCl2 and 15 mmol/L
glucose for 5 min, then with 0.1 mmol/L sodium cacodylate buffer pH
7.4 containing 2.5 mmol/L CaCl2, 2.5% paraformaldehyde, and 1.5%
4260
glutaraldehyde for the fixation of the vasculature. The aorta was
carefully dissected between sigmoid valves and 3-4 cm after the aortic
arch and thoroughly cleaned of loose adventitial tissue; the aortic arch
was cut free, open longitudinally along the outside of the arch, pin
corked, immersed in fresh fixative solution, and stored at 4 °C until
staining. The aortic arches were then first rinsed for 48 h in 0.1 mol/L
sodium cacodylate buffer pH 7.4 containing 30 mmol/L CaCl2 and 250
mmol/L sucrose. The arches were then rinsed in distilled water, stained
for 40 s in Harris hematoxylin, rinsed in distilled water, and then quickly
in 70% isopropyl alcohol; finally, they were stained in Oil red O for
30 min according to Nunnari et al. (31), rinsed in 70% isopropyl alcohol,
and back to distilled water. Each aortic arch was then directly displayed
on a glass slide, endothelium side up, covered with Aquamount
mounting medium and cover slips, and observed en face by light
microscopy. All segments were photographed using a video digitizer.
A computerized image analysis system (ImageJ, Scion Corp., Frederick,
MD) attached to a compound light microscope was used to measure
the total Oil Red O stained area of each aortic arch. The area covered
by foam cells (aortic fatty streak lesion area or AFSA) was expressed
as a percentage of the total area surveyed.
Determination of Superoxide Anion Production. Superoxide anion
production was evaluated in hamsters that were not used for AFSA
measurement (n ) 6 per group). Briefly, the left ventricle (150 mg)
(41) was placed in Krebs buffer containing 250 µM of lucigenin, and
the intensity of luminescence was recorded on a luminometer (PerkinElmer Wallac, Victor, Turku, Finland). Results were expressed as count/
mg of protein.
Immunoblotting. Proteins were extracted as previously described
(32) from the frozen left ventricles of six hamsters previously used for
measurement of superoxide anion production. Samples were homogenized using an ultra turrax T25 basic (Irka-Werke) in an ice cold
extraction buffer containing 120 mM NaCl, 25 mM KCl, 2 mM CaCl2,
15 mM Tris-Cl pH 7.5, 0.5% Triton X-100, 1 mM PMSF, 0.1 mM
DTT, 10 M leupeptin, and 1 M pepstatin. Protein concentrations in
sample were determined by Bio-Rad Dc protein assay using BSA as a
standard. Proteins (50 µg) were separated with 12% SDS-PAGE and
then transferred to a nitrocellulose membrane (45 min, 100 V).
Membranes were incubated for 2 h with primary antibody against
p22phox (1/200, Santa Cruz Biotechnology, Santa Cruz, CA) in blocking
buffer. After six washes (6 °C, 5 min) in TBS/Tween under gentle
agitation, blots were incubated for 45 min with horseradish peroxidaselabeled antibody (1/5000). After further washes, blots were treated with
enhanced chemiluminescence detection reagents (ECL, Amersham), and
areas (mm2) were measured using the BIO-Profil 1D software (Fisher
Bioblock).
Statistical Analyses. Data are shown as the means ( SEM. Data
were subjected to logarithmic transformation where necessary to achieve
homogeneity of variances. Statistical analysis of the data was carried
out using the Stat View IV software (Abacus Concepts, Berkeley, CA)
by one-way ANOVA followed by Fisher’s protected least significant
difference test. Differences were considered significant at P < 0.05.
RESULTS
Evolution of Cholesterol Concentration, Superoxide Production, and NADPH Oxidase Expression during Early
Development of Atherosclerosis (Experiment 1). Plasma
cholesterol significantly increased in the hamsters fed the
atherogenic diet just from the first 15 days as compared to the
controls animals. No alteration in cholesterol was observed in
the control hamsters fed the standard diet, whereas in hamsters
fed the atherogenic diet, plasma cholesterol level significantly
increased from day 30 (7.76 ( 0.46 mmol/L) to day 45 (10.18
( 1.47 mmol/L) and leveled to 84 days (Figure 1a).
The time course of cardiac superoxide production was also
established. Whereas the cardiac superoxide level was constant
during 84 days in the control hamsters, we noted an increase
of superoxide production in atherogenic hamsters compared at
15, 30, 45, and 84 days by 18.5% (not significant, NS), 21.5%
(NS), 21.8% (p ) 0.0548), and 94.1% (p < 0.0001), respec-
Sutra et al.
Figure 1. Time course experiment of plasma cholesterol concentration
(A) and superoxide anion production (B) in hamsters fed a standard diet
(white bars) and in hamsters fed an atherogenic diet (black bars) during
experiment 1. Values are expressed as mean ± SEM of triplicate wells
(n ) 6). For each dietary treatment, bars with different index letters differ
(P < 0.05).
tively, such differences being only significant at 84 days (Figure
1b). In addition, cardiac superoxide levels increased by 20.5%
(NS) from day 15 to day 45 and were highest at 84 days (Figure
1b) in hamsters fed atherogenic diet. In agreement with the
cardiac superoxide production at 84 days of atherogenic diet,
the measure of cardiac NADPH oxidase expression by western
blot showed that cholesterol diet triggered a significant expression of p22phox (Figure 3) by 146% (p ) 0.001).
Oxxynea Improves Blood Lipid Profile. Nutritional parameters are shown in Table 1. No significant difference appeared
in food intake and final body weight between the two groups.
Plasma lipids are summarized in Table 2. Oxxynea significantly
reduced plasma total cholesterol by 11.7% (p < 0.0001) and
non-HDL cholesterol by 14% (p ) 0.0066), but not HDLcholesterol, as compared to the control group. Consequently,
the atherogenic index calculated as total cholesterol/HDLcholesterol was lowered by 8.3% (p ) 0.0139) in hamsters
receiving Oxxynea.
Oxxynea Improves Antioxidant Status and Decreased
O2°- by Preventing NADPH Oxidase Expression. In experiment 2, Oxxynea significantly increased by 10% the plasma
antioxidant capacity induced by the atherogenic diet (p )
0.0244) (Table 2). As shown in Figures 2 and 3, superoxide
4261
Table 2. Effects of Ingestion of a Fruit and Vegetable Extract
(Oxxynea) on Plasma Lipid Concentrations and on Plasma Antioxidant
Capacity (PAC) in Hamsters Fed an Atherogenic Dieta (Experiment 2)
group
atherogenic diet
atherogenic diet
+ Oxxynea
TCb (mmol/L)
HDLCc (mmol/L)
non-HDLC (mmol/L)
atherogenic indexd
PAC (mmol/L)
9.54 ± 0.20a
6.01 ± 0.27a
3.12 ± 0.15a
1.56 ± 0.04a
1.29 ± 0.06a
8.42 ± 0.16b
5.90 ± 0.16a
2.68 ± 0.17b
1.43 ± 0.03b
1.42 ± 0.10b
a Values are means ± SEM, n ) 18. Data were analyzed by one-way ANOVA
followed by the least significant difference test. For each dietary treatment, means
in a column with different letters differ, P < 0.05. b TC : total cholesterol. c HDLC
: high-density lipoprotein cholesterol. d Total cholesterol/HDL-cholesterol.
Figure 2. Cardiac superoxide anion production in hamsters fed a standard
or an atherogenic diet with (OXX) or without (ATH) Oxxynea. Values are
expressed as mean ± SEM of triplicate wells (n ) 6). For each dietary
treatment, bars with different index letters differ (P < 0.05).
Figure 4. Effects of ingestion of water (ATH) or a fruit and vegetable
extract Oxxynea (OXX), on aortic fatty streak area in hamsters fed an
atherogenic diet for 84 days (experiment 2). Values are expressed as
mean ± SEM (n ) 12). Bars with different index letters differ (P < 0.05).
Figure 3. Expression of the cardiac p22phox subunit of NAD(P)H oxidase
in hamsters fed a standard diet (STD) or an atherogenic diet with (OXX)
or without (ATH) Oxxynea during 84 days. The densitometric measurement
shows arbitrary area units. Values are expressed as mean ± SEM (n )
6). For each dietary treatment, bars with different index letters differ (P <
0.05).
Table 1. Effects of Ingestion of a Fruit and Vegetable Extract
(Oxxynea) on Body Weight and Food Intake of Hamsters Fed an
Atherogenic Dieta (Experiment 2)
group
atherogenic diet
atherogenic diet
+ Oxxynea
initial body weight, g
final body weight, g
food intake, g/d
91.3 ± 2.1a
130.9 ± 9.7a
3.47 ± 0.90a
86.7 ± 5.8a
129.6 ± 1.8a
3.49 ± 0.60a
a Values are means ± SEM, n ) 18. Data were analyzed by one-way ANOVA
followed by the least significant difference test. For each dietary treatment, means
in a column with different letters differ, P < 0.05.
anion production (Figure 2) and expression of p22phox (Figure
3) decreased by 45.5% (p < 0.0001) and 59.1% (p ) 0.001),
respectively, in hamsters receiving Oxxynea.
Oxxynea Powerfully Inhibits Lipid Deposition in Aortic
Arch. Average aortic fatty streak accumulation (AFSA),
measured as the percentage of Oil Red O staining relative to
the total area surveyed (Figure 4), was not detected in hamsters
fed the standard diet (experiment 1). In addition, AFSA was
significantly reduced by 77% (p ) 0.001) in the hamsters
receiving Oxxynea as compared to control animals on the
atherogenic diet (experiment 2).
DISCUSSION
This study reported the protective effect of fruit and vegetable
antioxidants supplementation against diet-induced oxidative
stress and atherosclerosis in hypercholesterolemic golden Syrian
hamsters.
The golden Syrian hamster is a good nutritional rodent model
of atherosclerosis, which could mimic the early stages of human
atherosclerosis, that is, fatty streak (33). As previously reported,
a high fat diet led to an early increase in total and non-HDL
cholesterol after 15 days of diet leading to lipid deposition on
aortic arch at 84 days. Interestingly, the atherogenic diet-induced
hypercholesterolemia is in parallel accompanied by a tendency
of superoxide anion overproduction, which reaches the significance at 45 days. In agreement with oxidative hypothesis of
atherosclerosis, it could be postulated that NADPH oxidase
expression and activity conspire with high non-HDL cholesterol
level to induce foam cells and fatty streak.
As reported in other rodent models of atherosclerosis, such
as insulin resistance (32) or hypertension (34), oxidative stress
could be a key event in diet-induced atherosclerosis and cardiac
remodelling. Hypercholesterolemia has been previously involved
in enhanced ROS production by NADPH oxidase activity (35)
in a model of cholesterol-fed mice. It has been further suggested
that ROS overproduction could be linked to an induction of
NADPH oxidase subunit in particular gp91phox in neutrophils
from hyperlipidemic guinea pig (36). Our observation of a
hypercholesterolemic diet-induced NADPH oxidase expression
(+146%) in cardiac tissue extends these observations. Superoxide anion and further ROS generation by monocyte-derived
macrophages could oxidize LDL, being in turn responsible for
4262
amplification loops by stimulation of phagocyte NADPH
oxidase. Beyond generation of foam cells and cholesterol
deposition (37), cholesterol-induced ROS generation could
participate in left ventricle remodelling as suggested by the
enhanced expression of p22phox in the infarcted sites.
Oxxynea, a fruit and vegetable antioxidant extract, prevented
the progression of early atherosclerosis in aortic arch of
cholesterol-fed hamsters (<10% foam cell coverage of aorta).
In agreement with the recent report by Adams et al. (18) that a
diet rich in green and yellow vegetables inhibits atherosclerosis
in transgenic mice, we have shown that Oxxynea extract
prevents fatty streak formation in aortic arch of cholesterol fed
hamster. This effect could be in part due to a slight, but
significant, decrease in total and non-HDL cholesterol, without
affecting HDL cholesterol. The resulting improvement of
atherogenic index obtained with fruit and vegetable extract
extends the previous observation on the beneficial effects in
lipid parameters obtained with grape polyphenols (23). On the
other hand, nutritional antioxidants supplied from Oxxynea
could act throughout the improvement of antioxidant defenses
as demonstrated by significant increase in plasma antioxidant
capacity. This free radical scavenging capacity evidenced in
plasma is in agreement with the ORAC value observed in vitro
and could account in part for protection against LDL oxidation
reported for numerous polyphenols such as catechin, epicatechin,
epicatechin-3-O-gallate, epigallocatechin, and epigallocatechin3-O-gallate (38).
Furthermore, our findings suggest for the first time that the
fruit and vegetable antioxidant extract could prevent both NAD(P)H oxidase expression and O2°- overproduction in the heart
from hypercholesterolemic hamster. Here again, NAD(P)H
oxidase inhibition could be involved in prevention of LDL
oxidation and further atherosclerosis steps. Beyond the vicious
circles linked to LDL oxidation, the inhibition of ROS production by NAD(P)H oxidase system could also prevent other early
events in cardiovascular diseases such as endothelial dysfunction
or arterial remodelling. A recent study has shown that endothelium-dependent vasorelaxation is impaired in the high lipidfed golden syrian hamster (39). Our current results showing that
the fruit and vegetable extract inhibits the overproduction of
O2°- by NAD(P)H system strongly suggest that Oxxynea may
prevent the endothelial dysfunction. Indeed, an overproduction
of superoxide anion that could react with NO° to produce
peroxinitrite has been involved in the hypercholesterolemiainduced impairment of vasorelaxant system (40). On the other
hand, we have shown that an overproduction of ROS is strongly
associated with cardiac remodelling, suggesting a pathogenic
role of oxidative stress in its constitution (41). Pharmacological
or nutritional intervention could prevent both NAD(PH) oxidase
expression and activity and cardiac hypertrophy (41). Our results
showing that hypercholesterolemic diet activates and that
vegetable and fruit extracts inhibit NAD(P)H expression and
activity in the heart reinforce the hypothesis of a nutritional
modulation of ROS enzymatic producing systems.
Finally, improvement of plasma lipid profile, increase in PAC,
and decrease in superoxide anion production and reduction of
NAD(P)H oxidase expression (p22phox subunit) by Oxxynea
were associated with a total prevention of aortic fatty streak
lesion area. The relative contribution of each parameter such
as lipid profile, plasma antioxidant defenses, and overproduction
of ROS is difficult to establish. However, it is tempting to
speculate on a specific role of tissular oxidative stress. Indeed,
in a previous paper, it has been shown that the wine polyphenols-induced aortic fatty streak lesion area prevention was
Sutra et al.
associated with lipid and plasma antioxidant capacity improvement without any effect on plasma oxidative stress markers such
as MDA, AOPP, and AGEs (21). Taken together, these results
suggest a specific role of polyphenol in vascular tissue mediated
by NAD(P)H oxidase.
All of these results suggest that this extract acted by
mechanisms operating both inside and outside a hypolipemic
effect, especially an antioxidant effect. Although the constituent(s) responsible for these effetcs remain(s) unclear, candidates
such as vitamin C, vitamin E, carotenoids, selenium, and
polyphenols could act synergistically or additively to prevent
atherosclerosis in the hamster model. These promising results
obtained in a diet-induced atherosclerosis animal model give
rise to further studies in clinical fields.
LITERATURE CITED
(1) Ross, R. Atherosclerosis - an inflammatory disease. N. Engl.
J. Med. 1999, 340, 115-26.
(2) Frei, B. Reactive oxygen species and antioxidant vitamins: mechanisms of action. Am. J. Med. 1994, 97, 5S-13S, 22S-8S.
(3) Guzik, T. J.; West, N. E.; Black, E.; McDonald, D.; Ratnatunga,
C.; Pillai, R.; Channon, K. M. Vascular superoxide production
by NAD(P)H oxidase: association with endothelial dysfunction
and clinical risk factors. Circ. Res. 2000, 86, E85-90.
(4) Lassegue, B.; Clempus, R. E. Vascular NAD(P)H oxidases :
specific features, expression, and regulation. Am. J. Physiol.:
Regul., Integr. Comp. Physiol. 2003, 285, R277-97.
(5) Szöcs, K.; Lassegue, B.; Sorescu, D.; Hilenki, L. L.; Valppu,
L.; Couse, T. L.; Wilcox, J. N.; Quinn, M. T.; Lambeth, J. D.;
Griendling, K. K. Upregulation of Nox-based NAD(P)H oxidases
in restenosis after carotid injury. Arterioscler., Thromb., Vasc.
Biol. 2002, 22, 21-7.
(6) Azumi, H.; Inoue, N.; Takeshita, S.; Rikitake, Y.; Kawashima,
S.; Hayashi, Y.; Itoh, H.; Yokoyama, M. Expression of NADH/
NADPH oxidase p22phox in human coronary arteries. Circulation 1999, 100, 1494-8.
(7) Aviram, M. Modified forms of LDL and atherosclerosis.
Atherosclerosis 1993, 98, 1-9.
(8) Aviram, M.; Rosenblat, M.; Etzioni, A.; Levy, R. Activation of
NADPH oxidase required for macrophage-mediated oxidation
of low-density lipoprotein. Metabolism 1996, 45, 1069-79.
(9) Heinloth, A.; Heermeyer, K.; Raff, U.; Wanner, C.; Galle, J.
Stimulation of NADPH oxidase by oxidized low-density lipoprotein induces proliferation of human vascular endothelial cells.
J. Am. Soc. Nephrol. 2000, 11, 1819-25.
(10) Zhao, G. F.; Sen, J. J.; Zhang, H.; She, M. P. Effects of oxidized
low density lipoprotein on the growth of human artery smooth
muscle cells. Chin. Med. J. (Beijing, Engl. Ed.) 2005, 118, 19738.
(11) Block, G. The data support a role for antioxidants in reducing
cancer risk. Nutr. ReV. 1992, 50, 207-13.
(12) Parodi, P. W. The French paradox unmasked: the role of folate.
Med. Hypotheses 1997, 49, 313-8.
(13) Hertog, M. G.; Kromhout, D.; Aravanis, C.; Blackburn, H.;
Buzina, R.; Fidanza, F.; Giampaoli, S.; Jansen, A.; Menotti, A.;
Nedeljkovic, S. Flavonoid intake and long-term risk of coronary
heart disease and cancer in the seven countries study. Arch.
Intern. Med. 1995, 155, 381-6.
(14) Frankel, E. N.; Kanner, J.; German, J. B.; Parks, E.; Kinsella, J.
E. Inhibition of oxidation of human low-density lipoprotein by
phenolic substances in red wine. Lancet 1993, 341, 454-7.
(15) Bravo, L. Polyphenols: chemistry, dietary sources, metabolism,
and nutritional significance. Nutr. ReV. 1998, 56, 317-33.
(16) Duthie, G.; Crozier, A. Plant-derived phenolic antioxidants. Curr.
Opin. Lipid 2000, 11, 43-7.
(17) Orallo, F.; Alvarez, E.; Camina, M.; Leiro, J. M.; Gomez, E.;
Fernandez, P. The possible implication of trans-Resveratrol in
the cardioprotective effects of long-term moderate wine consumption. Mol. Pharmacol. 2002, 61, 294-302.
(18) Adams, M. R.; Golden, D. L.; Chen, H.; Register, T. C.; Gugger,
E. T. A diet rich in green and yellow vegetables ihibits
atherosclerosis in mice. J. Nutr. 2006, 136, 1886-89.
(19) Nicolle, C.; Cardinaut, M.; Aprikian, O.; Busserolle, J.; Grolier,
P.; Rock, E.; Demigné, C.; Mazur, A.; Sclabert, A.; Amouroux,
P.; Rémézy, C. Effect of a carrot intake on cholesterol metabolism and on anti-oxidant status in cholesterol-fed rat. Eur. J. Nutr.
2003, 42, 254-61.
(20) Nicolle, C.; Cardinaut, M.; Gueux, E.; Jaffrelo, L.; Rock, E.;
Mazur, A.; Amouroux, P.; Rémézy, C. Health effect of vegetablebased diet : lettuce consumption improves cholesterol metabolism and antioxidant status in the rat. Clin. Nutr. 2004, 23, 60514.
(21) Auger, C.; Teissèdre, P. L.; Gérain, P.; Lequeux, N.; Bornet,
A.; Serisier, S.; Besançon, P.; Caporiccio, B.; Cristol, P.;
Rouanet, J. M. Dietary wine phenolics catechin, quercetin and
resveratrol efficiently protect hypercholesterolemic hamsters
against aortic fatty streak accumulation. J. Agric. Food Chem.
2005, 53, 2015-21.
(22) Auger, C.; Caporiccio, B.; Landrault, N.; Teissèdre, P. L.;
Laurent, C.; Cros, G.; Besançon, P.; Rouanet, J. M. Red wine
phenolic compounds reduce plasma lipids and apolipoprotein B,
and prevent early aortic atherosclerosis in hypercholesterolemic
Golden Syrian hamsters (Mesocricetus auratus). J. Nutr. 2002,
132, 1207-13.
(23) Vinson, J. A.; Teufel, K.; Wu, N. Red wine, dealcoholized red
wine, and espacially grape juice, inhibit atherosclerosis in a
hamster model. Atherosclerosis 2001, 156, 67-72.
(24) Kowala, M. C.; Nunnari, J. J.; Durham, S. K.; Nicolosi, R. J.
Doxazosin and cholestyramine similarly decrease fatty streak
formation in the aortic arch of hyperlipidemic hamsters. Atherosclerosis 1991, 91, 35-49.
(25) Ou, B.; Hampsch-Woodill, M.; Prior, R. L. Development and
validation of an improved oxygen radical absorbance capacity
assay using fluorescein as the fluorescent probe. J. Agric. Food
Chem. 2001, 49, 4619-26.
(26) Davalos, A.; Gomez-Cordoves, C.; Bartolomé, B. Extending
applicability of oxygen radical absorbance capacity (ORACFluorescein) assay. J. Agric. Food Chem. 2004, 52, 48-54.
(27) National Research Council. Guide for the Care and the Use of
Laboratory Animals. Publication no. 85-23(reV.); National
Institutes of Health: Bethesda, MD, 1985.
(28) Reeves, P. G.; Nielsen, F. H.; Fahey, G. C., Jr. AIN-93 purified
diets for laboratory rodents: final report of the American Institute
of Nutrition ad hoc writing committee on the reformulation of
the AIN-76 rodent diet. J. Nutr. 1993, 123, 1939-51.
(29) Rice-Evans, C.; Miller, N. J. Total antioxidant status in plasma
and body fluids. Methods Enzymol. 1994, 234, 279-93.
(30) Koechlin, C.; Couillard, A.; Cristol, J. P.; Chanez, P.; Hayot,
M.; Le Gallais, D.; Prefaut, C. Does systemic inflammation
trigger local exercise-induced oxidative stress in COPD? Eur.
Respir. J. 2004, 23, 538-44.
4263
(31) Nunnari, J. J.; Zand, T.; Joris, I.; Majno, G. Quantification of
Oil red O staining of the aorta in hypercholesterolemic rats. Exp.
Mol. Pathol. 1989, 51, 1-8.
(32) Delbosc, S.; Paizanis, E.; Magous, R.; Araiz, C.; Dimo, T.;
Cristol, J. P.; Cros, G.; Azay, J. Involvement of oxidative stress
and NADPH oxidase activation in the development of cardiovascular complications in a model of insulin resistance, the
fructose-fed rat. Atherosclerosis 2005, 179, 43-9.
(33) Nikkari, S. T.; Salokivi, T.; Jaakkola, O. The hyperlipidemic
hamster as an atherosclerosis model. Artery 1991, 18, 285-90.
(34) Delbosc, S.; Cristol, J. P.; Descomps, B.; Nimran, A.; Jover, B.
Simvastatin prevents angiotensin II-induced cardiac alteration
and oxidative stress. Hypertension 2002, 40, 142-7.
(35) Matsumoto, T.; Miyamori, K.; Kobayashi, T.; Kamata, K.
Apocynin normalizes hyperreactivity to phenylephrine in mesenteric arteries from cholesterol-fed mice by improving endothelium-derived hyperpolarizing factor response. Free Radical
Biol. Med. 2006, 41, 1289-303.
(36) Maeda, K.; Yasunari, K.; Sato, E. F.; Inoue, M. Enhanced
oxidative stress in neutrophils from hyperlipidemic guinea pig.
Atherosclerosis 2005, 181, 87-99.
(37) Witzum, J. L.; Steinberg, D. Role of oxidized lipoprotein in
atherogenesis. J. Clin. InVest. 1991, 88, 1785-92.
(38) Shafiee, M.; Carbonneau, M. A.; Urban, N.; Descomps, B.;
Leger, C. L. Grape and grape seed extract capacities at protecting
LDL against oxidation generated by Cu2+, AAPH or SIN-1 and
at decreasing superoxide THP-1 cell production. A comparison
to other extracts or compounds. Free Radical Res. 2003, 37,
573-84.
(39) Georgescu, A.; Alexandru, M.; Constantinescu, E.; Popo, B.
Effect of gap junction uncoupler heptanol on resistance arteries
reactivity in experimental model of diabetes, hyperlipemia and
hyperlipemia-diabetes. Vasc. Pharmacol. 2006, 44, 513-8.
(40) Wagner, A. H.; Kohler, T.; Ruckschloss, U.; Just, I.; Hecker,
M. Improvement of nitric oxide-dependent vasodilatation by
HMGCoA reductase inhibitor through attenuation of endothelial
superoxide anion formation. Arterioscler., Thromb., Vasc. Biol.
2000, 20, 61-9.
(41) Al-Awwadi, N. A.; Araiz, C.; Bornet, A.; Delbosc, S.; Cristol,
J. P.; Linck, N.; Azay, J.; Teissedre, P. L.; Cros, G. Extracts
enriched in different polyphenolic families normalize increased
cardiac NADPH oxidase expression while having differential
effects on insulin resistance, hypertension, and cardiac hypertrophy in high-fructose-fed rats. J. Agric. Food Chem. 2005, 53,
151-7.
Received for review January 4, 2007. Revised manuscript received
March 6, 2007. Accepted March 12, 2007.
JF070029N
Oxxynea®
Technical data sheets
Oxxynea FP – for tablets and capsules
Oxxynea WS – for instant powder and beverages
www.fytexia.com
Oxxynea FP
SPECIFICATIONS
Product description
Fruit & vegetable extracts & concentrates standardized in ORAC value
Properties
Specification
Frequency Methods
Color
Aspect
Taste
Odor
Brown
Fine powder
Characteristic
Typical
Each batch
Each batch
Each batch
Each batch
Internal method
Internal method
Internal method
Internal method
Total Polyphenols (%, Catechin eq. UV)
ORAC Value (µmol TE/g)
≥ 75%
≥ 5000
Each batch
Each batch
Internal method
Dr CAO method
Loss on drying (%)
Ash (%)
Solubility (1% W/V)
pH (10% W/V)
Bulk density
Tapped density
Mesh size
≤ 10%
≤ 10%
Moderate in water
3.5-5.5
≥ 0.40
≥ 0.55
95% through 40mesh
Each batch
Each batch
Each batch
Each batch
Each batch
Each batch
Each batch
EC 71/393
DE 08/09/1977
Internal method
Internal method
Internal method
Internal method
Internal method
≤ 2ppm
≤ 1ppm
≤ 0.1ppm
≤ 3ppm
Comply with Eur. Ph.
& Reg. (EC) 396/2005
1/year*
1/year*
1/year*
1/year*
1/year*
1/year*
1/year*
ISO11885 / ISO17294-2
ISO11885 / ISO17294-2
DIN EN 13806 / ASU L 00.00-19/4
ISO11885 / ISO17294-2
Internal method
Internal method
Internal method
< 10 000 CFU/g**
< 100 CFU/g**
< 10 CFU/g
Absence/1 g
Absence/25g
Absence/1g
Each batch
Each batch
Each batch
Each batch
Each batch
Each batch
NF V08-051 / V08-100
NF V08-059
3M 01/02-09/89
NF ISO 7251
BKR 23/07-10/11
NF EN ISO 6888-3
Physico-chemical values
Contaminants
Arsenic (As, ppm)
Cadmium (Cd, ppm)
Mercury (Hg, ppm)
Lead (Pb, ppm)
Methanol (ppm, GC-FID)
Ethanol (ppm, GC-FID)
Pesticides (ppm, GC-MS / LC-MS-MS)
Microbiological analysis
Total plate count
Yeasts & moulds
Coliforms
E.Coli
Salmonella
Staphylococcus aureus
* Control plan defined annually.
** Acceptable maximal count: 5 times the acceptance criterion according to Eur. Ph. VII° Ed 5.1.8 Category B.
Packaging: 25kg in a double polyethylene bag & box.
Storage: 24 months in the original unopened bag under cool (15-25°C) and dry conditions away from light.
Handling: handle powder in a room with good ventilation.
23
Oxxynea WS
SPECIFICATIONS
Product description
Blend of fruit & vegetable extracts & concentrates standardized in ORAC value
Properties
Specification
Frequency Methods
Color
Aspect
Taste
Odor
Red purple brown
Fine powder
Characteristic
Typical
Each batch
Each batch
Each batch
Each batch
Internal method
Internal method
Internal method
Internal method
Total Polyphenols (%, Catechin eq. UV)
ORAC Value (µmol TE/g)
≥ 40%
≥ 3500
Each batch
Each batch
Internal method
Dr CAO method
Loss on drying (%)
Ash (%)
Solubility (1% W/V)
pH (10% W/V)
Bulk density
Tapped density
Mesh size
≤ 10%
≤ 10%
Moderate in water
3.5-5.5
≥ 0.30
≥ 0.50
95% through 70mesh
Each batch
Each batch
Each batch
Each batch
Each batch
Each batch
Each batch
EC 71/393
DE 08/09/1977
Internal method
Internal method
Internal method
Internal method
Internal method
≤ 2ppm
≤ 1ppm
≤ 0.1ppm
≤ 3ppm
& Reg. (EC) 396/2005
1/year*
1/year*
1/year*
1/year*
1/year*
1/year*
1/year*
ISO11885 / ISO17294-2
ISO11885 / ISO17294-2
DIN EN 13806 / ASU L 00.00-19/4
ISO11885 / ISO17294-2
Internal method
Internal method
Internal method
< 10 000 CFU/g**
< 100 CFU/g**
< 10 CFU/g
Absence/1 g
Absence/25g
Absence/1g
Each batch
Each batch
Each batch
Each batch
Each batch
Each batch
NF V08-051 / V08-100
NF V08-059
3M 01/02-09/89
NF ISO 7251
BKR 23/07-10/11
NF EN ISO 6888-3
Physico-chemical values
Contaminants
Arsenic (As, ppm)
Cadmium (Cd, ppm)
Mercury (Hg, ppm)
Lead (Pb, ppm)
Methanol (ppm, GC-FID)
Ethanol (ppm, GC-FID)
Pesticides (ppm, GC-MS / LC-MS-MS)
Microbiological analysis
Total plate count
Yeasts & moulds
Coliforms
E.Coli
Salmonella
Staphylococcus aureus
* Control plan defined annually.
** Acceptable maximal count : 5 times the acceptance criterion according to Eur. Ph. VII° Ed 5.1.8 Category B.
Packaging: 25kg in a double polyethylene bag & box.
Storage: 24 months in the original unopened bag under cool (15-25°C) and dry conditions away from light.
Handling: handle powder in a room with good ventilation.
24
For further information, please contact:
Fytexia
Fytexia Corp.
ZAE Via Europa – 3, rue d’Athènes
1251 Avenue of the Americas
34350 VENDRES – France
NEW YORK, NY 10020 - USA
Tel: +33 (0)4 67 21 90 98
Tel: +1 (973) 204 - 0460
[email protected]
[email protected]
25
Take a Healthy F&V
Mediterranean concentrate
11 Superfruits &
11 Vegetables
extract
Take the essence of
Mediterranean diet
in your daily routine
Fytexia developed Oxxynea® as a next-generation concentrate with all
of the benefits of the Mediterranean Diet and Lifestyle extracted from
an exclusive combination of 11 superfruits & 11 vegetables.
11 superfruits : Grape (White&Red), Orange, Grapefruit, Billberry,
Papaya, Pineapple, Strawberry, Apple, Apricot, Cherry, Blackcurrant
11 vegetables : Tomato, Carrot, Green Tea, Olive, Broccoli, Green Cabbage,
Onion, Garlic, Wheat Germ, Cucumber, Asparagus
Oxxynea® is the ultimate ‘full spectrum’ polyphenolic concentrate (flavanols,
flavonoids, catechins,naringin), lycopene, carotenoids, vitamins. With an Orac
Value of 5000 in a single gram (powder) and polyphenol levels of >75%.
The ultimate “Safety Net”
There is no doubt on fruits and vegetables benefits on health, nowadays
people take care of their health. So why don’t men and women consume
the amount of fruits and vegetables recommended by health professionals (5
by day)? It’s a matter of time and convenience. The current way of life do not
let time to shop fresh fruits and vegetables, to cook healthy meals every day.
Oxxynea® is the solution.
- High ORAC value (equivalent of 5 portions of F&V (400g))
- Easy to use
- No added calories (2.8kcal-3.8kcal/daily dose)
- Proven bioavailability
Oxxynea® is the “safety net” we all need to help protect,
maintain and improve our health and vitality.
Example of Oxxynea HPLC profile
(wavelength: 280nm)
2 capsules
of 400 mg
the equivalent of 5 servings
of fruits daily.
It’s well known in scientific world that Mediterranean diet has been associated with a reduction in
coronary heart disease1,2,3. In 2003, the WHO concluded that there was convincing evidence that
fruits and vegetables lower the risk of coronary heart disease and recommended an intake of 400500 g /day - the equivalent to five or six portions of about 80 grams each4.
Health benefits of
Mediteranean Lifestyle & diet
Scientific evidence
5
Potent and effective, Oxxynea® is born from an uncompromising dedication to scientific methods and innovative product development.
A placebo controlled study using 36 animals was conducted and after 12 weeks of treatment more parameters were measured:
- Bioavailability5 : Plasmatic antioxidant capacity (+10%)
- Anti-aging study5 : Plasmatic Free Radical Scavenger (-45.5%)
- Cardiovascular study5 : Total cholesterol levels (-11.7%) and aortic fatty streak accumulation (-77%)
range of natural & scientifically
5 - T. SUTRA et al. J. Agric. Food Chem. 2007, 55, 4258-4263
proven ingredients
Endless Possibilities for your Next Formulation
There are polyphenols, then there’s Oxxynea® : developed to amplify the number of health benefits
that formulators can attach to their products. And more functionality means higher visibility, more
marketing opportunities and greater consumer demand.
Simply put - Oxxynea® is the single most important component in your new formulation. With
Oxxynea® as the foundation, the possibilities are almost endless.
Available as a dry powder extract for inclusion in tablets and capsules (Oxxynea-FP), and in a
water soluble form - Oxxynea-ws for “supercharging” beverages and dairy products, Oxxynea
is already a key constituent in some of the world’s most popular consumer health and vitality
products.
Commercial name
Oxxynea-FP
Oxxynea-WS
Active compounds
ORAC Value ≥ 5000
Total Polyphenols ≥ 75%
ORAC Value ≥ 3500
Total Polyphenols ≥ 40%
Recommended dose*
0.8g/day
In one or several doses
1g/day
In one or several doses
Solubility (1%)
Moderately
100% water soluble
Applications
Food supplements
(capsules/tablets)
Functional foods
(beverages/dairy products/bakery)
Bibliography
1 - Trichopoulou et al. N Engl J Med. 2003 ;348 :2599-2608
2 - Knoops KTB et al. JAMA. 2004 ; 292 :1433-1439
3 - De Logeril M et al. Circulation. 1999 ;99 :779-785
4 - F.L. Crowe et al. European Heart Journal published online
www.fytexia.com - www.oxxynea.com
ZAE Via Europa - 34350 VENDRES - FRANCE - Tel. 33 (0)4 67 21 90 98 - [email protected]
*A l l thes e i nf orm ati on’s
has not b e ev al uated
b y Europ ean or FD A
legislation. It remains to
the industrial customer
to
comply
with
all
applicable
laws
and
regulations.
Move
your body!
Mangosteen extract
Joint Flexibility
Less Pain…
More Flexibility,
No side effect!
Millions of men and women worldwide suffer from serious joint and
connective tissue pain. Prescription medications, such as well known
NSAID (Non Steroidal Anti Inflammatory Drug) so called Nimesulide,
can lead to serious side effects.
…Less Pain, …More flexibility, …less stomach ache.
What has been missing in the market place is a safe, natural, and effective
ingredient, for reducing pain and inflammation in tendons, ligaments and
joints.
Fytexia®’s R&D filled the gap and created MangoSelect®, a
branded vegetable ingredient that provides quick and proven
results after only five days (at 600mg/day) .
MangoSelect® is a dry extract of the tropical fruit mangosteen (Garcinia
mangostana L.) standardized in polyphenols (> 45%) and and - mangosteen
(xanthones family)
Example of
Mangoselect® HPLC profile
(wavelength: 254nm)
A natural way to fight Joint Pain
MangoSelect® is caracterized by a unique composition of
polyphenols and xanthones ( - and -mangosteen), well known
for its antioxidant1 and anti-inflammatory2,3 properties.
MangoSelect® reduces joint pain in two ways:
* as a COX-2 inhibitor 4,5
* by decreasing the release of inflammatory cytokines
such as TNF , IL-6 and IL-10.
As a COX-2 inhibitor, MangoSelect® works to calm inflammatory
pathways… by blocking the enzyme that is partially responsible
for inflammation (and ultimately, pain).
MangoSelect® really acts through the same mechanism
as Common NSAIDS (ibuprofen, Nimesulide) or powerful
corticosteroids like Dexamethasone, but does not generate any
risks nor side effects.
g
2 capsules
of 300 mg
= -37% joint
pain reduction
100% Natural
& Vegetable
2.5
5.0
7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 40.0 42.5 45.0
Human study6
Scientific evidence
A clinical study has been carried out on 24 subjects (9 elderly
individuals and 15 athletes) who had joint and connective tissue
injury. They were randomly split into two groups of 12 to receive
either the active extract (600 mg/d) or a positive control (100 mg
/d Nimesulide) for 5 days. The use of Visual Analog Scales (VAS) in
assessment of pain has been well validated. The scale ranges from
0 (no pain) to 10 (intense pain). Pain has been measured both at
baseline and after 5 days of supplementation.
Results6
After 5 days of treatment, a significant overall pain reduction for the 24 subjects (both groups) was noted.
MangoSelect® helped reducing pain in joint and connective tissues by -37,4% (p<0.05) whereas Nimesulide by -54% (p<0.05).
For the elderly subjects, the pain relief was as good with MangoSelect (50% reduction) as compared to Nimesulide (52.5%).
For the 15 athletes, MangoSelect® helped reducing pain by -31% (p<0,05) whereas Nimesulide by -55%.
range of natural & scientifically
proven ingredients
Animal study6
TNF blood levels (ng/ml)
as a function of treatment
-69%
LPS
alone
LPS
+Nime
Systemic inflammation was initiated in 25 mice with the injection of bacterial
lipopolysaccharide (LPS). Various treatments were then given to alleviate the
inflammation : MangoSelect®, Nimesulide and Dexamethasone. TNF which is
the main inflammatory mediator was measured after 90 minutes. TNF not only
directly affects tissues where it is released, it also turns on COX-2 enzyme that
induces the production of further inflammatory mediators.
-83%
Results
Treatment with MangoSelect® significanty decreases by 69%
blood TNF-alpha level (p<0.05). As expected the glucocorticoid
(Dexamethasone) significantly decreases by 83% the level of blood
TNFalpha as compared to the control animals (only LPS) (p<0.005)
LPS
LPS
+Mangos +Dexamet
Commercial name
MangoSelect®
Description
Natural Mangostana fruit dry extract
Active compounds
Total Polyphenols
≥ 45%
- mangosteen
≥10%
- mangosteen
≥ 1%
Recommended dosage
600mg/day in one or more doses
Application
MangoSelect® can be used in any food supplements
Bibliography
1- Kondo et al .; J Agric. Food Chem. 2009, 57:8788-8792
2- Tewtrakul et al .; J of Ethnopharmacology. 2009, 121: 379-382
3- Chen et al .; Food Chemical Toxicology. 2007, 10: 1016
4- Nakatani et al .; Biochemical Pharmacology. 2002, 63: 73-79
5- Nakatani et al .; Mol Pharmacology. 2004, 66: 667-674
6- Fytexia- Unpublished internal data 2008
www.fytexia.com - www.mangoselect.com
ZAE Via Europa - 34350 VENDRES - FRANCE - Tel. 33 (0)4 67 21 90 98 - [email protected]
*All these information’s
h a s n ot b e e v a lu a t e d
b y E u rop e a n or FD A
legislation. It remains
to
the
industrial
customer to comply with
all applicable laws and
regulations.
Pharmacokinetic study of HyaMax
Absorption, distribution, and excretion
examinations of 99mTechnetium labelled
hyaluronic acid (HyaMax™) after single dose
per oral administration
FENCHEM BIOTEK LTD
Distributed by PHARMAFOODS
JULY 2013
Background
z
Hyaluronic acid (HA) is being listed as an ingredient in
an ever-increasing number of dietary supplements
targeted to joint health and skin health
z
No published reports on uptake of HA after oral
administration were found in the scientific literature
z
The aim of the study:
To examine the absorption, excretion and distribution of
HA (HyaMax™) after a single oral administration
Design of study
z
Study group:
99mTc
z
Control group:
99mTc
•
•
HyaMax
HyaMax™ sodium hyaluronate
MW: 1.0-1.5 MDa
99mTc: gamma emitting isotope 99mtechnetium (Tc)
Methods
z
z
z
z
z
z
Tissue biodistribution
Urinary/faeces excretion examinations
Blood & urinary clearance
Scintigraphic examinations
Nano SPECT/CT scans
Autoradiography
Results
z
HyaMax™ was labeled with 99mTechnetium at 85%
efficiency and was stable for at least 48 hours
z
After a single oral application, a proportion (< 5%) of
99mTc-HyaMax™ has been absorbed from the
gastrointestinal tract
z
The absorbed part of 99mTc-HyaMax™ was distributed
to various organs, especially joints and skins. All tissues
examined showed incorporation of radioactivity starting
at 15 minutes and persisting for 48 hours.
z
The main part of applied 99mTc-HyaMax™ is
excreted via the faeces (around 90 % between 0-72
hs) and a small proportion is excreted via the urine
(< 5% between 0-72 hs)
z
The control material behave differently – a faster
excretion has been seen by the urinary tract from the
body
z
Scintigraphic imaging, nano SPECT/CT scans,
autoradiography have illustrate the presence of
labeled HyaMax™ in joints, connective tissue and
skin samples.
Radioactivity distribution
99mTc
HyaMax
Stomach
Small intestine
Large intestine
86.42% excreted by faeces
99mTc
Kidney
Stomach
Urinary bladder
93.2% excreted by urines
z
The control(99mTc) is different from the test
study(99mTc-HyaMax™) in the radioactivity
distribution
-The difference is due to the Sodium hyaluronate
-The metabolism of 99mTc-HyaMax™ has little connection
with free 99mTc
z
Approximately 5% of the orally ingested dose of 99mTcHyaMax™ had potential for systemic distribution.
z
Measurable amounts of radioactivity reached skins and
joints after oral administration of 99mTc-HyaMax™
z
There are difference of radioactivity distribution between
labeled HyaMax™ and label itself in the joints and skins.
The label(99mTc) is still
attached to HyaMax™ in the
body after oral consumption
The radioactivity reached
joints and skins is generated
by 99mTc-HyaMax™ instead
of free 99mTc
HyaMax™ gets into joints and skins after oral consumption
0,6
0,30
0,5
0,25
0,4
0,20
I.D.% / organ
I.D.% / organ
Pharmacokinetic and internal dosimetry of 99mTc-HyaMax™
0,3
0,15
0,10
tbio = 0,12 hs
0,2
tbio = 0,10 hs
0,05
0,1
0,00
0
10
20
30
40
50
60
Time (hours)
Pharmacokinetic properties of
99mTc-HA in knee after oral
application
70
80
0
10
20
30
40
50
60
70
Time (hours)
Pharmacokinetic properties of
99mTc-HA in skin after oral
application
80
Conclusion
z
HyaMax™ is presented to be the first commercial
HA with clinical evidence for uptake and distribution
to connective tissues and skins after orally
consumption
z
The findings support a rationale for biological
actions seen in animal and human studies
z
The findings support the application of HA in dietary
supplements or functional foods designed for joint
and skin health.
Acknowledgement
z
Schiff® Nutrition International, Salt Lake City, Utah, USA
z
Research Institute for Radiobiology and Radiohygiene, “Fodor
József” National Center of Health, Budapest, Hungary
z
Life Sciences Division, American Institute for Biosocial and
Medical Research, Inc
Fenchem Biotek Ltd.
Data Sheet
Tel: 0086-25-84574708
Fax: 0086-25-84574987
E-mail:[email protected]
www.fenchem.com
PRODUCT NAME
HyaMax® sodium hyaluronate LMW
DESCRIPTION
Hyamax® Sodium Hyaluronate（1.0-1.5 Million Da）is the standardized natural Hyaluronic
acid in sodium salt. It is a stable form of hyaluronic acid composed by hydrophilic
molecules of glucoronic acid and N-acetyl glycosamine. Hyaluronic Acid is present in
every tissue of the body. It is most concentrated in the synovial fluid which bathes the
joints, in the vitreous fluid in the eye, and in the skin.
PRODUCT IDENTIFICATION
Chemical name: Hyaluronic Acid, Sodium Salt
CAS number: 9067-32-7
EINECS: 232-678-0
Formula: （C14H20NO11Na）n
Structure:
The hyaluronic acids (HA) are a class of macromolecular proteoglycans characterized by
a highly polymerized chain of glucuronic acid and N-acetylglucosamine units bonded to
protein.
SPECIFICATION
ITEMS
SPECIFICATION
Appearance
White Powder
Transparency
Penetrate Light Rate 99.0%Min
Assay (by UV)
90.0% Min
Glucuronic Acid
44.0% Min
Fenchem Biotek Ltd.
Data Sheet
Tel: 0086-25-84574708
Fax: 0086-25-84574987
E-mail:[email protected]
www.fenchem.com
Molecular Weight
LMW: 1.0-1.5 MDa
pH Value
6.0 - 7.5
Loss On Drying
5.0% Max
Protein (Dry Wt. Basis)
0.1% Max
Heavy Metals (As Pb)
20ppm Max
As
2ppm Max
Total Plate Count
1000 cfu/g Max
Mold & Yeast
100 cfu/g Max
E.Coli
Negative
Salmonella
Negative
CERTIFICATION
Kosher certified, Halal certified
STORAGE
The product may be stored for 24 months from the date of manufacture in the unopened
original container. Keep container tightly closed under cool(2℃-10℃) and light proof
conditions. Once opened, use contents quickly.
APPLICATION
Hyaluronic acid is widely used in health food, dietary supplement and cosmetic industries.
In health food fields, hyaluronic acid has been used for skin moisturizing senility treatment,
and improving vigor. It is responsible for lubricating the cartilage in the joint areas. With
hyaluronic acid, joints have the mobility to bend with ease. Hyaluronic acid also aids in
lubrication of the eyes. In cosmetic field, hyaluronic acid has perfect molding moisture
action and is used in many products, including cosmetics, shampoo, bath liquid etc.

Piel - Eiralabs

Transcription

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