Chemistry">
Carotenoides e Compostos Fenólicos de Spondias Do Nordeste Brasileiro - Composição e Bioacessibilidade
Carotenoides e Compostos Fenólicos de Spondias Do Nordeste Brasileiro - Composição e Bioacessibilidade
Carotenoides e Compostos Fenólicos de Spondias Do Nordeste Brasileiro - Composição e Bioacessibilidade
CAMPINAS
2018
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CAMPINAS
2018
3
4
Comissão Examinadora:
________________________________________________________________
Profª Drª Adriana Zerlotti Mercadante (Orientadora)
Faculdade de Engenharia de Alimentos – UNICAMP
________________________________________________________________
Profª Drª Leila Queiroz Zepka (Membro Titular)
Universidade Federal de Santa Maria – UFSM
________________________________________________________________
Profª Drª Lilian Regina Barros Mariutti (Membro Titular)
Faculdade de Engenharia de Alimentos – UNICAMP
________________________________________________________________
Prof. Drª Renata Aparecida Soriano Sancho (Membro Titular)
Pesquisadora
________________________________________________________________
Profª Drª Solange Guidolin Canniatti Brazaca (Membro Titular)
Departamento de Agroindústria, Alimentos e Nutrição – ESALQ, USP
AGRADECIMENTO
À minha esposa, Patricia de kássia Coutinho Pereira Costa, que com amor entendeu
e participou de forma intensa na concretização desse plano de Deus em nossas
vidas. Te Amo!
À minha orientadora, Profª Drª Adriana Zerlotti Mercadante, pela amizade,
conhecimentos, profissionalismo e suporte cedidos para que esse trabalho fosse
uma realidade. Peço a Deus que continue a te abençoar com força e vigor para que
a Ciência em nosso País continue a crescer.
À Unicamp, em particular, ao corpo docente e funcionários da Faculdade de
Engenharia de Alimentos pela contribuição indispensável. Aos colegas e
funcionários do Laboratório de Química de Alimentos, pelo agradável convívio diário
e pela troca de experiências. Levarei para sempre a recordação.
À Fundação de Amparo à Pesquisa do Estado de São Paulo, que de forma inderita
tem uma participação em todo o trabalho aqui realizado.
À banca examinadora pela disponibilidade, atenção e sugestões.
Às pessoas mais importantes da minha vida, minha família. Meus pais, Antônio Viera
da Costa e Linadaura Alves da Costa, a meus irmãos, Gilson Alves da Costa,
Juliana Alves da Costa e Édipo de Brito Alves, e sobrinhos, Julia Vitória Alves
Dantas e Bruno Alves Souza, e a minha segunda família, Jose Pereira, Terezinha
Martins e Diego Coutinho, e aos meus Tios, a todos esses quero agradecer pela
torcida e vibração nas conquistas e pela constante presença nas dificuldades. Pelo
amor sem medida, dedicação, confiança, conselhos, orações e muitas vezes até
renúncias. Muito Obrigado! Amo Vocês!
Aos amigos que fizeram parte da minha caminhada até aqui. Obrigada por estarem
sempre presentes e compartilhando comigo todos os momentos. Conexão CV novos
e antigos, Mafia, Amigos da Mari, Dr. Gilsandro Professor, DGTA, Bonde do Gil,
Chacara Barão, GB BC I, Amo vocês!
Agradeço a Deus, acima de tudo, pela vida, força, coragem, oportinidade e pelas
pessoas especiais que colocou em meu caminho.
7
RESUMO
ABSTRACT
The Brazilian Northeast is a region that presents favorable conditions for the
development of some species of flora that are considered exotic, among which we
can highlight the fruits of the genus Spondias. The fruits of the genus Spondias
present high potential not only for in natura consumption but also for the use in the
processing of pulps, juices, jellies, ice-creams and sweets. From the phytochemical
point of view, the species of the genus Spondias stand out because they are rich in
bioactive compounds; however, a large number of bioactive substances in these
species are still unknown. Thus, this work aims to separate, identify and quantify
carotenoids, carotenoid esters and phenolic compounds using HPLC-DAD-MS MS in
cajá (Spondias mombin L.), umbu (Spondias tuberosa), umbu-cajá (Spondias spp.)
and seriguela (Spondias purpurea), as well as to evaluate the in vitro bioaccessibility
of carotenoids and their esters in beverages prepared with cajá pulp. In general, the
evaluated Spondias species presented the same chromatographic profile of
carotenoids and carotenoid esters, except for seriguela. The total carotenoids
contents were: 192.39 ± 8.20 (μg.g-1dw) in cajá, 74.58 ± 1.73 (μg.g-1dw) in
seriguela, 28.07 ± 2.04 (μg. g-1dw) in umbu and 125.76 ± 4.21 (μg.g-1dw) in umbu-
cajá. Considering all fruits of the genus Spondias, the major carotenoids were (all-E)-
lutein, (all-E)-β-cryptoxanthin, (all-E)-β-carotene, (all-E)-β-cryptoxanthin laurate and
(all-E)-β-cryptoxanthin myristate. The phenolic compound rutin was the major one in
Spondias mombin, Spondias tuberosa and Spondias spp., and galoyl-HHDP-
hexoside 1 was the major in Spondias purpurea. The quantification of the total
phenolic compounds was carried out by HPLC-DAD through analytical curves of
gallic acid, rutin and quercetin, with 405.51 ± 10.26 μg.g-1 dw, 202.64 ± 3.79 μg.g-1
dw, 179.77 ± 6.65 μg.g-1 dw and 179.55 ± 0.57 μg.g-1 dw in cajá, seriguela, umbu
and umbu-cajá, respectively. The increased bioaccessibility of carotenoids in cajá
beverage accompanied the increase in the amount of lipids in the milk added to the
beverage; the addition of sugar increased the bioaccessibility in a similar extent for all
carotenoids in the order water> skimmed milk> whole milk.
9
SUMÁRIO
CAPITULO I
INTRODUÇÃO GERAL E OBJETIVOS
CAPITULO II
REVISÃO BIBLIOGRÁFICA
CAPITULO III
Identification and Quantification of Carotenoid Esters in Fruits of the Genus
Spondias
ABSTRACT .......................................................................................................... 60
1. Introduction..................................................................................................... 61
2. Materials and methods................................................................................... 62
2.1. Materials ........................................................................................................ 62
2.2. Samples......................................................................................................... 62
2.3. Carotenoid extraction .................................................................................... 63
2.4. HPLC-DAD-MS/MS analysis ......................................................................... 63
3. Results and discussion ................................................................................. 64
3.1. Identification of free carotenoids and carotenoid esters in Spondias ............. 64
3.2. Identification of chlorophyll derivatives .......................................................... 72
3.3. Quantitative composition of carotenoids ........................................................ 72
4. Literature cited................................................................................................ 76
CAPITULO IV
IDENTIFICAÇÃO E QUANTIFICAÇÃO POR HPLC-DAD-MS/MS DE COMPOSTOS
FENÓLICOS EM ESPÉCIES DE SPONDIAS
RESUMO ............................................................................................................. 82
INTRODUÇÃO ..................................................................................................... 83
MATERIAL E MÉTODOS .................................................................................... 85
10
CAPITULO V
In vitro bioaccessibility of free and esterified carotenoids in cajá frozen pulp-
based beverages
CAPÍTULO I
INTRODUÇÃO GERAL E OBJETIVOS
12
1. INTRODUÇÃO GERAL
até o uso farmacológico e medicinal das frutas deste gênero (Gouvêa et al.,
2017; Muñoz-López et al., 2017; Neiens et al., 2017; Mohiuddin et al., 2016;
Giuffrida et al., 2015; Gomes et al., 2013; Silva et al., 2005; Ceva-Antunes et
al., 2003).
Os compostos bioativos dos frutos do gênero Spondias foram
estudados, porém a maioria dos estudos apresentam a quantidade total de
carotenoides ou a quantidade total de compostos fenólicos. Poucos trabalhos
descrevem a identificação desses compostos, a qual foi realizada com base em
comparações com os poucos padrões disponíveis comercialmente e espectros
UV-vis (Giuffrida et al., 2015; Moreira et al., 2012; Almeida et al., 2011; Melo &
Andrade, 2010; Gonçalves et al., 2010; Genovese et al., 2008; Hamano &
Mercadante, 2001).
Compostos bioativos provenientes da dieta humana contribuem para a
diminuição dos riscos no desenvolvimento de inúmeras doenças, como as
crônicas degenerativas, o câncer, as cardiovasculares, a catarata, a
degeneração macular e inflamações como a aterosclerose, entre outras. Uma
das hipóteses para tais ações benéficas se deve ao fato de que essas
substâncias bioativas funcionam como antioxidantes que desativam os radicais
livres, e dessa forma retardam o envelhecimento celular (Stanner et al., 2004;
Braca et al., 2002; Lampe, 1999; Serdula et al., 1996; Maxwell, 1995).
Os carotenoides são pigmentos responsáveis pela cor em frutas e
vegetais, que varia do amarelo ao laranja (Buena et al., 2014; Britton, 1995).
Os carotenoides mono ou di-hidroxilados (xantofilas) estão esterificados com
ácidos graxos na maioria das frutas (Breithaupt & Bamedi, 2001).
Os compostos fenólicos são metabólitos secundários produzidos pelas
plantas como resposta a situações críticas e de estresse. Os compostos
fenólicos podem ser encontrados em todas as partes das plantas como caules,
folhas e frutos. No entanto, as maiores concentrações podem estar disponíveis
em partes específicas da planta, e no caso dos frutos, a casca pode apresentar
maior teor de compostos fenólicos em relação à polpa (Shahidi & Naczk, 2011;
Andrés-Lacueva et al., 2010).
A bioacessibilidade de carotenoides é um termo usado para se referir à
fração de carotenoides que é liberada do alimento ingerido na dieta, tornando-
se acessível para ser absorvido no organismo (Fernández-García et al., 2012).
14
2. OBJETIVOS
3. REFERÊNCIAS BIBLIOGRÁFICAS
Andrés-Lacueva, C., Medina-Remon, A., Llorach, R., Urpi-Sarda, M., Khan, N.,
Chiva-Blanch, G., Zamora-Ros, R., Rochetes-Ribalta, M. & Lamuela-Raventos,
R. M. (2010). Phenolic compounds: chemistry and occurrence in fruits and
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de Moura, F. T., de Melo Silva, S., Schunemann, A. P., & Martins, L. P. (2013).
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diferentes estádios de maturação. Revista Ciência Agronômica, 44, 764.
Giuffrida, D., Menchaca, D., Dugo, P., Donato, P., Cacciola, F., & Murillo, E.,
(2015). Study of the carotenoid composition in membrillo, guanabana toreta,
jobo and mamey fruits. Fruits, 70, 163-172.
Gomes, E. B., Ramalho, S. A., Gualberto, N. C., Miranda, R. C. M., Nigam, N.,
& Narain, N. (2013). A rapid method for determination of some phenolic acids in
Brazilian tropical fruits of mangaba (Hancornia speciose Gomes) and umbu
(Spondias tuberosa Arruda Câmara) by UPLC. Journal of Analytical Sciences,
Methods and Instrumentation, 3, 1-10.
Gouvêa, R. F., Ribeiro, L. O., Souza, É. F., Penha, E. M., Matta, V. M., &
Freitas, S. P. (2017). Effect of enzymatic treatment on the rheological behavior
and vitamin C content of Spondias tuberosa (umbu) pulp. Journal of Food
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scavenging activity of Spondias pinnata. BMC complementary and Alternative
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tuberosa Arruda (Anacardiaceae), a threatened tree of the Brazilian Caatinga?.
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19
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physical-chemical and microbiological characteristics of pasteurized umbu juice.
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CAPITULO II
REVISÃO BIBLIOGRÁFICA
23
REVISÃO BIBLIOGRÁFICA
A B
C D
A B
A B
C D
Figura 2. Fotos dos frutos partidos ao meio, registradas por Gilsandro (2016).
A: cajá (Spondias mombin L.), B: umbu (Spondias tuberosa), C: umbu-cajá
(Spondias spp.) e D: seriguela (Spondias purpurea L.).
2. Carotenoides
Carotenos
all-trans-β-caroteno
all-trans-γ-caroteno
9-cis-β-caroteno
Xantofilas
HO
HO zeinoxantina
Β-criptoxantina
OH
OH
HO
zeaxantina
HO
luteína
OH
3. Compostos fenólicos
B
O
A C
O O
OH O
O O O
O O O
+ O
O
O
OH OH
O
Chalconas Antocianidinas Flavan-3-ol Coumarinas Auronas
OH
O O
COOH
COOH
O
Ácidos Xantonas
Ácidos Acetofenonas
hidroxicinâmicos
hidroxibenzoicos OH
O
Lignanas
Estilbenos Benzofenonas
R1 R1 OH
R2 R2
CH = CH = COOH
COOH CH = CH = COOH
R3 R3 O
Ácido O–cumárico
R4 R4 C
Ácidos cinâmicos O CH
Ácidos benzoicos
R 1 = R2 = R3 = R4 = H → Ácido cinâmico; CH
R1 = OH → Ácido Salicílico; R1 = OH → Ácido o-cumárico;
R1 = R4 = OH → Ácido Gentísico; R 2 = OH → Ácido m-cumárico;
R3 = OH → Ácido p-hidroxibenzóico; R3 = OH → Ácido p-cumárico;
R2 = R3 = OH → Ácido Protocatequínico; R2 = R3 = OH → Ácido Caféico;
R2 = OCH3 ; R3 = OH → Ácido Vanílico; R2 = OCH3 ; R3 = OH → Ácido Ferúlico;
R2 = R3 = R4 = OH → Ácido Gálico; R2 = R4 = OCH3 ; R3 = OH → Ácido Sinápico Cumarina
R2 = R4 = OCH3 ; R3 = OH → Ácido Siríngico
forma livre ou ligados a açúcares e a proteínas (Angelo & Jorge, 2007; Bravo,
1998). Os flavonoides são encontrados na forma de aglicona ou glicosilada,
com o açúcar ligado a um carbono ou a um grupo hidroxila. Os açúcares
ligados às hidroxilas da aglicona encontram-se comumente nas posições C3 ou
C7 e os que estão ligados a carbono apresentam-se normalmente no C6 e C8.
Independentemente da ligação, os monossacarídeos mais comuns são a
ramnose, glicose, galactose e arabinose (Angelo & Jorge, 2007).
5. Bioacessibilidade de carotenoides
através da dieta precisam ser liberadas da matriz alimentar para poderem estar
acessíveis para absorção (Olson, 1999). A bioacessibilidade é então o passo que
antecede a biodisponibilidade, visto que, tanto fatores ligados ao alimento como
aqueles ligados ao organismo influenciam nesse processo (Fernández-García et al.,
2009).
Biodisponibilidade
Bioacessibilidade Bioatividade
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50
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(Spondias tuberosa Arruda Câmara) by UPLC. Journal of Analytical Sciences,
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59
CAPITULO III
a
Department of Food Science, Faculty of Food Engineering, University of Campinas
(UNICAMP), Rua Monteiro Lobato, 80, Campinas, SP, 13083-862, Brazil
ABSTRACT
Carotenoids, free and esterified with fatty acids, were determined in four genus of Spondias
fruits. Non-saponified extracts from freeze-dried edible parts of cajá (Spondias mombin L.),
seriguela (Spondias purpurea), umbu-cajá (Spondias spp.) and umbu (Spondias tuberosa)
were identified and quantified by HPLC-DAD-MS/MS. Despite the high number of
carotenoids quantified, the major carotenoids in all the Spondias evaluated were (all-E)-
lutein, (all-E)-β-cryptoxanthin, (all-E)-β-carotene, (all-E)-β-cryptoxanthin laurate and (all-E)-
β-cryptoxanthin myristate, with 22 % of the (all-E)-β-cryptoxanthin in Spondias mombin L.,
26 % of the (all-E)-lutein in Spondias tuberosa, 18 % of the (all-E)-β-carotene in Spondias
spp. and 18 % of the (all-E)-lutein in Spondias purpurea. Besides those carotenoids, (all-E)-
zeinoxanthin contributed with 11% in Spondias mombin L. and (all-E)-α-carotene with 10 %
in Spondias spp. The highest carotenoid content was found in Spondias mombin L. (192.39
± 8.20 µg.g-1dw), followed by 125.75 ± 4.21 (µg.g-1dw) in Spondias spp., 74.58 ± 1.73 (µg.g-
1
dw) in Spondias purpurea and 28.07 ± 2.04 (µg.g-1dw) in Spondias tuberosa. The levels of
provitamin A value followed the same order given above, 997.6 ± 46.4 µg RAE/100 g in
cajá, 805.41 ± 27.40 µg RAE/100 g in umbu-cajá, 298.89 ± 8.37 µg RAE/100 g in seriguela
and 143.33 ± 9.15 µg RAE/100 g in umbu. The percentage of esters in relation to the total
carotenoids was 36 % in Spondias mombin L., 23 % in Spondias tuberosa, 32 % in
Spondias spp. and 41 % in Spondias purpurea. This fact demonstrates that esters of
carotenoids are important in the composition of such compounds in these fruits.
1. Introduction
confers stability to the pigment (Fu et al., 2010; Subagio et al., 1999; Mínguez-Mosquera &
Hornero-Méndez, 1994). However, most of the studies on composition of carotenoids applies
a saponification step with consequent hydrolysis of carotenoid esters (de Rosso &
Mercadante, 2007; Hamano & Mercadante, 2001; Tiburski et al., 2011). On the other side,
identification of esterified carotenoids has increased in recent years considering that this is
the most common form of xanthophylls found in most fruits (Delgado-Pelayo et al., 2016;
Schweiggert et al., 2016; Giuffrida et al., 2012; Petry & Mercadante, 2016; Rodrigues et al.,
2016; Mercadante et al., 2017). Moreover, information about the composition of carotenoids
esters can help the elucidation of the arrangement of these molecules into the cells to
understand mechanisms and physical-chemical interactions.
Techniques using HPLC-DAD-MS/MS systems are necessary for the separation and
identification of esters of xanthophylls (Rodrigues et al., 2016; Schlatterer & Breithaupt,
2005; Breithaupt & Bamedi, 2002), thus the present study aims to identify and quantify both
free and esters of carotenoids in samples of four Spondias: cajá, umbu, umbu-cajá and
seriguela.
2.1. Materials
Standard of (all-E)-β-carotene was purchased from Sigma-Aldrich (St. Louis, MO,
USA). Standards of (all-E)-lutein and (all-E)-β-cryptoxanthin were donated by DSM
Nutritional Products (Basel, Switzerland). Standards showed at least 97% of purity for (all-E)-
β-carotene, 92% for (all-E)-β-cryptoxanthin and 90% for (all-E)-lutein, determined by high
performance liquid chromatography-diode array detector (HPLC-DAD).
Methanol (MeOH) and methyl tert-butyl ether (MTBE) of HPLC grade were acquired
from J.T. Baker (Phillipsburg, NJ, USA). The other reagents (petroleum ether, ethyl ether and
acetone) were all of analytical grade from Labsynth (Diadema, Brazil). Water was purified by
a Milli-Q system (Billerica, MA, USA). The samples and solvents were filtered through
Millipore membranes of 0.22 and 0.45 μm, respectively.
2.2. Samples
Fruits of umbu, umbu-caja, caja and seriguela were acquired at the public market
located in the city of João Pessoa, Paraíba State, Brazil, in January 2016. All ripe fruits (5 kg
for each type of Spondias) arrived to the laboratory at UNICAMP (Campinas, São Paulo
State) within two days after purchase. The seeds were manually removed and pulp with peel
was immediately frozen in liquid nitrogen. The frozen pulp was freeze-dried below 40 μHg at
−60 °C until constant weight (Liobrás, São Paulo, Brazil). The freeze-dried pulp was ground
63
into powder, homogenized, vacuum packaged in polyethylene bags, and stored in the dark at
−37 °C.
Twenty fresh fruits of each genus were used for physical-chemical characterization.
Caja and seriguela showed, respectively, similar physical characteristics: 12.90 ± 2.32 g and
12.99 ± 1.24 g of weight, 26.62 ± 1.81 mm and 25.90 ± 0.88 mm of transverse diameter,
35.37 ± 3.21 mm and 33.93 ± 1.48 mm of longitudinal diameter. Umbu and umbu-cajá
showed, respectively, 22.41 ± 3.21 g and 15.85 ± 2.16 g of weight, 13.34 ± 2.14 mm and
9.83 ± 1.85 mm of transverse diameter, and 17.17 ± 1.50 mm and 13.00 ± 1.84 mm of
longitudinal diameter. The percentage of moisture, pH and soluble solids (° Brix) values were
86.60 ± 0.46%, 2.63 ± 0.10 and 11.4 ± 0.5 in cajá; 73.83 ± 0.35%, 3.04 ± 0.04 and 18.4 ± 0.
9 in seriguela; 88.07 ± 0.80%, 2.75 ± 0.02 and 12.2 ± 0.3 in umbu-cajá; and 88.53 ± 0.24%,
2.72 ± 0.07 and 10.7 ± 0.4 in umbu.
remaining in this condition for 5 min, resulting in a 55 min run (Rodrigues et al., 2016). UV-
Vis spectra were acquired from 200 to 600 nm and processed at 270, 348 and 450 nm. The
conditions for the ionization by APCI positive mode and MS conditions were the same as
those previously described by Rodrigues et al. (2016).
Carotenoid identification was carried out through the combined interpretation of all
data obtained by chromatographic elution order, UV-Visible spectra characteristics
(maximum absorption wavelength (λmax), spectral fine structure (% III/II) and peak cis
intensity (% AB/AII)), mass spectra features (protonated molecule ([M+H]+) and MS/MS
fragments) and comparison with literature data (da Costa & Mercadante, 2017; Petry &
Mercadante, 2016; Giuffrida et al., 2015; de Rosso & Mercadante, 2007; Breithaupt et al.,
2002; Hamano & Mercadante, 2001). Quantification was done with the same calibration
curves as described by da Costa & Mercadante (2017). The NAS-IOM (2001) conversion
factor was used to calculate the vitamin A value and the contribution of Z isomers of
provitamin A carotenoids were calculated as other dietary provitamin A carotenoids.
Table 1. Chromatographic, UV-Vis and mass spectroscopic characteristics, obtained by HPLC-DAD-MS/MS, of pigments from fruits from Spondias
genus.
7 5’,6’- or 5’,8’-epoxy-β-cryptoxanthin 10.9-12.2 ndd nce 0 569.4 551.3 [M+H-18]+, 533.4 [M+H-18-18]+, 205.1
29 (all-E)-β-cryptoxanthin caprate 29.1-32.1 420,452,478 nce 0 707.6 615.6 [M+H-92]+, 535.4 [M+H-172]+, 443.3 [M+H-172-92]+
30 (all-E)-β-cryptoxanthin laurate 34.1-36.6 428,449,475 nce 0 735.6 643.5 [M+H-92]+, 535.4 [M+H-200]+, 443.4 [M+H-200-92]+
32 (all-E)-β-cryptoxanthin myristate 35.7-37.8 420,445,4478 31 0 763.7 671.6 [M+H-92]+, 535.4 [M+H-228]+, 443.3 [M+H-228-92]+
705.6 [M+H-172]+→ 533.4 [M+H-172-172]+, 441.3 [M+H-
33 (all-E)-lutein dicaprate 40.3-40.7 426,444,472 nce 0 ndg
172-56-92]+, 411.3
733.6 [M+H-228]+, 761.5 [M+H-200]+, 533.4 [M+H-228-
34 (all-E)-lutein-3-O-myristate-3’-O-laurate 41.8-41.9 420,444,474 40 0 ndg
200]+
35 (all-E)-β-cryptoxanthin palmitate 41.2-43.1 425,451,477 29 0 791.7 699.6 [M+H-92]+, 535.4 [M+H-256]+, 443.4 [M+H-92]+*
761.7 [M+H-228]+→ 742.9 [M+H-228-18]+, 705.6 [M+H-228-
36 (all-E)-lutein dimyristate 42.7-43.2 426,446,474 60 0 ndg 56]+, 669.5 [M+H-228-92]+, 533.4 [M+H-228-228]+, 477.3
[M+H-228-228-56]+, 441.3 [M+H-228-228-92]+, 411.3
802.6 [M+H-18]+, 727.8 [M+H-92]+, 535.4 [M+H-284]+, 443.3
37 (all-E)-zeinoxanthin stearate 44.7-45.5 420, 448, 474-5 44 0 819.7
[M+H-284-92]+*
761.7 [M+H-256]+→ 743.8 [M+H-256-18]+, 533.4 [M+H-256-
228]+ 477.4 [M+H-256-228-56]+, 441.3 [M+H-256-228-92]+,
38 (all-E)-lutein-3-O-myristate-3’-O-palmitate 45.6-46.4 420,444,474 nce 0 ndg 411.3; 789.7 [M+H-228]+→ 771.7 [M+H-228-18]+, 533.4
[M+H-228-256]+, 477.4 [M+H-228-256-56]+, 441.3 [M+H-
228-256-92]+, 411.3
789.7 [M+H-256]+→ 533.4 [M+H-256-256]+, 477.4 [M+H-
39 (all-E)-lutein dipalmitate 45.6-46.1 420,446,474 40 0 ndg
256-256-56]+, 441.3 [M+H-256-256-92]+, 411.3
789.7 [M+H-284]+→ 733,7 [M+H-284-56]+, 697.7 [M+H-284-
92]+, 533.4 [M+H-284-256]+, 477.4 [M+H-284-256-56]+,
441.3 [M+H-284-256-92]+, 411.3; 817.7 [M+H-256]+→ 799.7
40 (all-E)-lutein-3-O-stearate 3’-O-palmitate 46.1-46.7 420,444,472 nce 0 ndg
[M+H-256-18]+, 725.7 [M+H-256-92]+, 533.4 [M+H-256-
284]+, 477.4 [M+H-256-284-56]+, 441.3 [M+H-256-284-92]+,
411.3
a
Numbered according to the chromatograms shown in Figures 1A, 1B, 1C e 1D for, respectively, cajá (Spondias mombin L.); umbu (Spondias tuberosa); umbu-cajá (Spondias spp.) and seriguela (Spondias
purpurea L.). bRetention time on C30 column (see Material and Methods for chromatographic conditions). cLinear gradient containing mixture of MeOH, MTBE, and H2O. dUV/vis spectrum was not clearly
detected because of coelution or low concentration. e % III/II was not calculated because of poor definition of the UV/vis spectrum or because it was not detected. f% AB/AII was not calculated because of poor
definition of the UV/vis spectrum or because it was not detected. g[M + H]+ or MS/MS fragments were not detected. Underlined fragments are the most abundant in the MS/MS spectrum. Bold face indicates
MS in-source fragment ions.
67
40
14
12
0
15
Detector response at 450 nm (mAU)
30
-2
15 20 25
time (min)
20
11
5
8
10 22
25 32 37
35
24 30
7 26 36
6 31 38
13
0 33
-10
0 10 20 30 40 50
Time (min)
200
19
150
350 100 17
50
5
300 0
15 20 25 30
Time (min)
Detector response at 450 nm (mAU)
250
17
200
12
150
25
100 20
6 13 21 33 36 37
50 9 10 30
4 19 29 32
8 35
26 38
0 10 20 30 40 50
Time (min)
80
12
Detector response at 450 nm (mAU)
60
40
24
9 22 31
20 8 18
10 25 26 30 35 37 38
7 17
13 32
6 27 28 40
33 36
0
0 10 20 30 40 50
Time (min)
30
5
Detector response at 450 nm (mAU)
20 38
25 32 39
12 23 33
10
2 4
30
11
1 3 40
34
6 16 20
0 10 20 30 40 50
Time (min)
m/z 205 (de Faria et al., 2009). Peaks 9 and 10 were, respectively, identified as (Z)-5,8-
epoxy-β-cryptoxanthin and (all-E)-5,8-epoxy-β-cryptoxanthin, considering their UV-Vis and
elution order, as previously described by de Faria et al. (2009). Although the [M+H]+ and
losses of one and two waters molecules were detected at the same m/z values for all those
three peaks (7, 9 and 10), the location of the epoxy/furanoid group can be assigned at a 3-
hydroxy-β-ring end-group when a MS/MS fragment at m/z 221.1 is detected or at a non-
hydroxylated β-ring in case a fragment at m/z 205 is found (de Faria et al., 2009). Peak 7
was identified only in caja and umbu-caja while peaks 9 and 10 were detected in umbu and
umbu-cajá.
Considering the MS characteristics of peaks 8, 12 and 13, [M+H]+ at m/z 553.4,
MS/MS fragments at m/z 535.4 ([M+H-18]+) and at m/z 461.3 ([M+H-92]+), they were
identified as β-cryptoxanthin isomers. Peaks 8 and 13 were tentatively identified as (13Z)- or
(13’Z)-β-cryptoxanthin and (9Z)-β-cryptoxanthin, respectively, considering their elution order,
% III/II and AB/II values compared to the literature (de Rosso & Mercadante, 2007; de Faria
et al., 2009). The Z isomers (peaks 8 and 13) may be formed by the acids naturally found in
fruits. (all-E)-β-Cryptoxanthin was one of the main carotenoids in all genus Spondias, while
its Z isomers were detected in caja, umbu and umbu-caja.
Peaks 16 and 18 were identified as esters of (all-E)-luteoxanthin acylated,
respectively, with myristic and palmitic acids. The features of UV-Vis were identical to data
reported for (all-E)-luteoxanthin in the literature (Rodrigues et al., 2013). The identification of
(all-E)-luteoxanthin myristate (peak 16) was further based on the [M+H]+ at m/z 811.6 and
MS/MS fragments indicating loss of myristic acid combined with one ([M+H-18-228]+) or two
water molecules ([M+H-18-18-228]+), as can be verified in Table 1. Similarly, peak 18 was
identified as (all-E)-luteoxanthin palmitate considering the [M+H]+ at m/z 839.7 and MS/MS
fragments indicating loss of palmitic acid (molecular mass of 256.4 u) combined with one
water molecule ([M+H-18-256]+). These data are in accordance with those previously
reported by Petry & Mercadante (2016). (all-E)-luteoxanthin myristate was identified in
seriguela while (all-E)-luteoxanthin palmitate was only detected in umbu-cajá.
Peak 21 was identified as (all-E)-ζ-carotene considering its UV-Vis with λmax at 380,
400, 426 nm, [M+H]+ at m/z 541.4, and MS/MS fragments at m/z 471.4 ([M+H-69]+) indicating
a cleavage between C-3 and C-4 and at m/z 337.2, as previously reported (de Rosso &
Mercadante, 2007). This carotene was only identified in umbu.
Besides free lutein, 7 monoesters and 6 diesters of lutein were identified in different
genus of Spondias. (all-E)-Lutein-3’-O-laurate (peak 20), (all-E)-lutein-3-O-laurate (peak 23),
(all-E)-lutein-3’-O-myristate (peak 24), (all-E)-lutein-3-O-myristate (peak 26), (all-E)-lutein-3-
O-palmitate (peak 27), (all-E)-lutein-3’-O-oleate (peak 28) and (all-E)-lutein-3-O-stearate
(peak 31) were the lutein monoesters identified and (all-E)-lutein dicaprate (peak 33), (all-E)-
71
when compared to the caja, umbu and umbu-caja (Santos & Oliveira, 2008; de Souza
Almeida et al., 2007).
Table 2 shows the quantification of 24 peaks in umbu-caja (Spondias spp.), 21 in caja
(Spondias mombin L.), 20 in umbu (Spondias tuberosa) and 19 in seriguela (Spondias
purpurea L.). Despite the high number of quantified carotenoids, the major carotenoids in all
Spondias evaluated were (all-E)-lutein, (all-E)-β-cryptoxanthin, (all-E)-β-carotene, (all-E)-β-
cryptoxanthin laurate and (all-E)-β-cryptoxanthin myristate, with their sum representing 51 %,
67 %, 54 % and 52 % of the total carotenoids in cajá, umbu, umbu-cajá and seriguela,
respectively. Besides those carotenoids, (all-E)-zeinoxanthin contributed with 11% in caja
(Spondias mombin L.) and (all-E)-α-carotene with 10 % in umbu-cajá (Spondias spp.).
Similar result was reported for pulp of caja (Spondias mombin L.), harvested in
Panama, with (all-E)-lutein, (all-E)-β-crytoxanthin, (all-E)-β-carotene, (all-E)-β-cryptoxanthin
myristate and (all-E)-β-cryptoxanthin palmitate as the major carotenoids (Giuffrida et al.,
2015). High amounts of (all-E)-zeinoxanthin, a not so commonly found carotenoid, was
previously reported in caja as 15-17% of the total carotenoid content (Hamano &
Mercadante, 2001; da Costa & Mercadante, 2017).
Among the major carotenoids presented in Table 2, we highlight (all-E)-lutein with
14.89 ± 0.93 (µg.g-1dw) and 13.92 ± 0.73 (µg.g-1dw) in umbu-cajá and seriguela, (all-E)-β-
cryptoxanthin with 42.70 ± 2.32 (µg.g-1dw) and 19.12 ± 0.67 (µg.g-1dw) in cajá and umbu-
cajá, (all-E)-β-carotene with 22.98 ± 0.85 (µg.g-1dw) and 13.83 ± 0.60 (µg.g-1dw) in umbu-
cajá and cajá, (all-E)-β-cryptoxanthin myristate with14.73 ± 0.79 (µg.g-1dw) in umbu-cajá and
(all-E)-zeinoxanthin stearate with 10.63 ± 0.58 (µg.g-1dw) in cajá.
Murillo et al. (2010), reported the amounts of carotenoids in fresh weight, 8.6 ± 0.7
µg.g of (all-E)-lutein and 1.2 ± 0.2 µg.g-1 (all-E)-zeaxanthin in cajá, 6.3 ± 0.5 µg.g-1 (all-E)-
-1
lutein and 0.8 ± 0.1 µg.g-1 (all-E)-zeaxanthin in seriguela, values higher than those obtained
in this study, also in fresh weight, 1.6 ± 0.1 µg.g -1 (all-E)-lutein and 0.3 ± 0.0 µg.g-1 (all-E)-
zeaxanthin values in caja and 3.6 ± 0.2 µg.g-1 (all-E)-lutein and 0.7 ± 0.02 µg.g-1 (all-E)-
zeaxanthin in seriguela.
The highest total carotenoid content was found in caja (Spondias mombin L.) (192.39
± 8.20 µg.g-1dw), followed by 125.75 ± 4.21 (µg.g-1dw) in umbu-caja (Spondias spp.), 74.58 ±
1.73 (µg.g-1dw) in seriguela (Spondias purpurea L.) and 28.07 ± 2.04 (µg.g-1dw) in umbu
(Spondias tuberosa) (Table 2). The levels of provitamin A value followed the same order
given above.
74
Table 2. Pigment composition and vitamin A value of pulp from different fruits of Spondias genus.
Only results for carotenoid contents from caja (Spondias mombin L.) were found in the
literature. Total carotenoid 25.78 ± 1.1 µg.g-1 fw in cajá fruit found in the present study was
similar to 22.86 ± 3.76 µg/g fw in frozen pulp of cajá (Hamano & Mercadante, 2001) and to
23.9 ± 1.2 µg/g fw in frozen pulp of caja (da Costa & Mercadante, 2017). On the other hand,
the contents were lower than those from cajá fruits harvested in Panama, 45.8 ± 3.1 µg.g -1fw
(Giuffrida et al., 2015) and in Aracaju state of Brazil, 48.69 ± 1.57 µg.g -1fw (Tiburski et al.,
2011).
The percentage of esters in relation to the total carotenoids was 36 % in cajá, 22 % in
umbu, 29 % in umbu-cajá and 37 % in seriguela. This fact demonstrates that esters of
carotenoids play an important role in the carotenoid composition of fruits despite that the
percentage in Spondias is lower that in other fruits. For example, in murici 66 % of the total
carotenoids corresponded to carotenoids esters (Rodrigues et al., 2016), in red bell peppers
96 % of all existing carotenoids are carotenoids esters (Gregory et al., 1987) and in fruit
espinheiro-maritimo (Hippophae rhamnoides L., ssp. Carpatica) cv. Victoria, 65% of the total
content of carotenoids corresponded to carotenoid esters (Pop et al., 2014).
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81
CAPITULO IV
IDENTIFICAÇÃO E QUANTIFICAÇÃO POR HPLC-DAD-MS/MS DE COMPOSTOS
FENÓLICOS EM ESPÉCIES DE SPONDIAS
RESUMO
O objetivo do trabalho foi identificar e quantificar os compostos fenólicos em
frutos do gênero Spondias. Os extratos hidrofílicos obtidos com metanol/água dos
frutos liofilizados de cajá (Spondias mombin L.), seriguela (Spondias purpurea L.),
umbu-cajá (Spondias spp.) e umbu (Spondias tuberosa) foram analisados por
cromatografia líquida de alta eficiência acoplada aos detectores de arranjo de diodos
e de espectrometria de massas (HPLC-DAD-MS/MS) e quantificados por DAD com
curva de calibração analítica. De uma forma geral, as espécies de Spondias
estudadas apresentaram perfis cromatográficos semelhantes, com exceção da
seriguela. Em coluna C18, foram separados 26 compostos, 19 compostos fenólicos
foram identificados nos quatro frutos estudados do gênero Spondias. As maiores
quantidades de compostos fenólicos totais foram encontrados em cajá e seriguela,
405,51 ± 10,26 µg.g-1 base seca (b.s.) e 202,64 ± 3,79 µg.g-1 b.s., respectivamente. A
rutina foi o composto fenólico majoritário em cajá, umbu-cajá e umbu, enquanto o
galoil-HHDP-hexosídeo foi o majoritário em seriguela. O maior número de ácidos
fenólicos foi identificado em cajá, já o maior número de flavonóis derivados de
quercetina foi identificado em seriguela. Os frutos de cajá, umbu, umbu-cajá e
seriguela apresentaram os seguintes teores de rutina, respectivamente, 193,33 ±
7,40 µg.g-1 b.s., 137,24 ± 5,67 µg.g-1 b.s., 90,46 ± 0,53 µg.g-1 b.s. e 30,74 ± 1,77 µg.g-
1 b.s.. Em seriguela foram tentativamente identificados dois compostos como galoil-
HHDP-hexosideo, apresentando 78,93 ± 3,18 µg.g-1 b.s. e 43,37 ± 1,81 µg.g-1 b.s..
De forma geral, foram encontrados onze ácidos fenólicos e seus derivados em cajá,
dois em umbu, quatro em umbu-cajá e cinco em seriguela. Já os flavonóis
identificados foram um em cajá, um em umbu, um em umbu-cajá e dois em seriguela.
INTRODUÇÃO
et al., 2012; Silva et al., 2012; de Sousa Araújo et al., 2012; Tiburski et al., 2011). No
caso da casca de seriguela, foram identificados ácidos fenólicos e flavonoides, ácido
3-cafeoilquinico, quercetina 3-O-pentosilhexosideo, quercetina 3-O-
pentosilglicopiranose e ramentin hexosilpentosideo (Engels et al., 2012). Outros
compostos fenólicos, como os ácidos: clorogênico, p-cumárico e ferúlico foram
identificados em polpa de umbu, porém, a identidade foi confirmada somente pela
comparação dos espectros e tempo de retenção com padrões (de Barros Gomes et
al., 2013).
Conhecer a composição de compostos fenólicos em espécies de Spondias
disponibilizará não só conhecimento científico sobre essas espécies, mas também
chamarão a atenção para a riqueza da biodiversidade existente na região tropical do
Brasil. Assim esse trabalho tem como objetivo identificar e quantificar compostos
fenólicos em cajá, umbu, umbu-cajá e seriguela, através do uso do sistema HPLC-
DAD-MSn.
MATERIAL E MÉTODOS
PADRÕES E REAGENTES
Os padrões de ácido cafeico, ácido gálico, ácido quínico, ácido elágico, ácido
hidroxibenzoico, ácido 4-cumárico, ácido ferúlico, catequina, epicatequina, miricetina,
kaempeferol e quercetina foram adquiridos da Sigma-Aldrich (St. Louis, MO, EUA).
Os padrões apresentaram cerca de 93% de pureza, determinada por HPLC-DAD. O
metanol e a acetonitrila de grau HPLC foram adquiridos da J. T. Baker (Phillipsburg,
NJ, EUA). A água ultrapura foi obtida por meio do sistema Milli-Q (Billerica, MA,
EUA). O reagente de Folin-Ciocalteu foi adquirido da Dinâmica (São Paulo, Brasil).
As amostras e os solventes foram filtradas em membrana Millipore de 0,22 µm e 0,45
µm, respectivamente.
AMOSTRAS DE FRUTOS
Hydro RP80A (4 µm, 250 x 4,6 mm i.d.) (Phenomenex, Torrance, CA, EUA) mantida
a 29 °C, utilizando como fase móvel água (A) e acetonitrila (B), ambos contendo
0,5 % de ácido fórmico (v/v), em um gradiente linear (v/v) de A:B 99:1 para 50:50
em 50 min, depois para 1:99 em 5 min, mantendo esta proporção por 5 min, voltando
para 99:1 em 5 min e mantendo esta proporção por 10 min para re-equilíbrio da
coluna, a uma vazão de 0,9 mL/min (Chisté & Mercadante, 2012).
Para análise no MS, o fluxo proveniente do DAD foi dividido, permitindo a
entrada de somente 0,15 mL/min na fonte ESI. O MS foi configurado com os
seguintes parâmetros: modo de ionização positivo e negativo; voltagem no capilar:
2000 V; voltagem na saída do capilar: -110 V; skimmer 1: 10 V, skimmer 2: 5 V; end
plate offset: -500 V; temperatura do gás de secagem (N2): 310 °C; vazão: 5 L/min;
nebulizador: 30 psi. A energia de fragmentação para obtenção do MS/MS foi de 1,4
V, e energia de fragmentação para obtenção do MS 3 foi de 1,8 V (Chisté &
Mercadante, 2012).
Os espectros UV-visível foram obtidos entre 200 e 600 nm e os
cromatogramas foram processados em 280 e 360 nm. A faixa de varredura para os
espectros de massas foi de m/z 100 a 1000. Os compostos fenólicos foram
identificados a partir dos seguintes parâmetros: ordem de eluição e tempo de
retenção em coluna C18, características dos espectros UV-visível e de massas
comparados a padrões nas mesmas condições e a informações na literatura
(Bystrom et al., 2008; Chisté & Mercadante, 2012; Clifford et al., 2003; Engels et al.,
2012; Fischer et al., 2011; Mariutti et al., 2014; Rodrigues et al., 2015; Sentandreu et
al., 2015; Silva et al., 2014; Cuyckens & Claeys (2004); Singh et al., 2009; Tao et al.,
2014; Weisz et al., 2009). A quantificação dos compostos foi realizada a partir de
curva analítica externa de seis pontos (em triplicata) para ácido gálico (1,0 – 20,0
µg/mL), rutina (1,0 – 20,0 µg/mL) e quercetina (1,0 – 20,0 µg/mL). Para todos os
compostos, r2 ≥ 0,997, com limite de detecção de 1,4, 0,6 e 0,8 µg/mL, e limite de
quantificação de 4,2, 1,8 e 2,3 µg/mL, de ácido gálico, rutina e quercetina,
respectivamente (Mariutti et al., 2014). Estes limites foram calculados usando-se
parâmetros das curvas analíticas segundo o ICH (2005).
RESULTADOS E DISCUSSÕES
50 A 100
30 18
60
17
3
16
5
20 7 40
1 14 21
13 24
6 19
10 20
8
26
0 0
10 20 30 40 50 0 10 20 30 40 50
Tempo (min) Tempo (min)
40
B
80
Resposta do detector em 280 nm (mAU)
20
30 9 60
50
20 40
2
30
4
10 20
10
0 0
10 20 30 40 50 0 10 20 30 40 50
Tempo (min) Tempo (min)
50 C
Resposta do detector em 280 nm (mAU)
9 20
Resposta do detector em 360 nm (mAU)
40
40
30
30
20 20
20
2 10
5 11
10 4
26
10 24
0
0
10 20 30 40 50 0 10 20 30 40 50
Tempo (min) Tempo (min)
14
D
Resposta do detector em 360 nm (mAU)
20
60
Resposta do detector em 280 nm (mAU)
19 12
50 10
40 8
17
22
6
30 19
23
4 17
15
20
6 2 24
5 12
10
0
0
-2
10 20 30 40 50 0 10 20 30 40 50
Tempo (min) Tempo (min)
Figura 1. Cromatogramas obtidos por HPLC-DAD de compostos fenólicos em Spondias mombin L. (A), Spondias tuberosa (B),
Spondias spp. (C) e Spondias purpurea (D), processados em 280 nm e 320 nm. Condições cromatográficas: ver texto.
Caracterização dos picos é dada na Tabela 1.
90
não forneceu a quebra da massa 169, no entanto, a massa 125 que corresponde a
quebra do 169 apareceu no MS2. As características deste pico foram similares às da
literatura (Chisté & Mercadante, 2012). O pico 6 foi encontrado em cajá e seriguela.
O pico 8 foi tentativamente identificado como um derivado do ácido gálico,
devido ao λmax de 276 nm e [M-H]- de 411 u e fragmentos de íons (m/z) em MS2 de
331, 241, 169 e 125. O fragmento de íon (m/z) em MS2 de 169 corresponde ao ácido
gálico (massa molecular 170 Da) que aparece em MS2 como sendo fragmento da
massa 411, ainda em MS2 o fragmento a m/z 125 corresponde à perda de 44 u (CO2)
do fragmento 169. A massa 169 foi submetida à quebra em MS 3, no entanto, não se
obteve resultado. A confirmação foi feita com a comparação em literatura (Chisté &
Mercadante, 2012). Este composto foi somente encontrado em cajá.
O pico 10 foi tentativamente identificado como ácido p-cumárico hexosídeo,
apesar de não ter apresentado comprimento de onda característico nítido que
permitisse ser utilizado na sua identificação. O espectro de massas mostrou a [M-H]-
a m/z de 325 e fragmentos de íons MS2 a m/z 265, 187, 163, 145 e 119. O fragmento
de íon 163 é correspondente ao ácido p-cumárico (massa molar 164 Da) e o
fragmento 119 u indica a quebra do fragmento 163 u. Este pico apresentou
características similares às da literatura (Bystrom et al., 2008). O pico 10 foi
identificado apenas umbu-cajá.
O pico 11 foi tentativamente identificado como ácido 5 ou 3-O-cafeoilquínico. A
identificação das ligações no carbono na posição 5 ou 3 da estrutura da molécula
torna-se complicada por tratar-se apenas de arranjo dimensional da estrutura que
possui mesma fórmula molecular C16H18O9. O pico 11 não apresentou comprimento
de onda nítido que permitisse a comparação para ajudar na identificação, mas
apresentou espectro de massas em modo negativo [M-H]- (m/z) de 353 e fragmento
de íons (m/z) em MS2 de 191. O fragmento (m/z) em MS2 de 191 corresponde ao
ácido quínico (massa molecular 192 Da). Foram feitas inúmeras tentativas, mas não
ocorreu a quebra da massa 191 para confirmação no MS 3. No entanto, tempo de
retenção e espectro de massas estão de acordo com a literatura (Rodrigues et al.,
2015; Engels et al., 2012). O pico 11 foi identificado apenas em amostras de umbu-
cajá.
O pico 12 apresentou comprimento de onda com λmax de 277 nm, [M-H]- (m/z)
de 483 e fragmentos de íons (m/z) MS2 de 423, 405, 331, 271 e 193. Este pico foi
tentativamente identificado como um derivado do monogaloil hexosídeo,
94
O pico 25 foi denominado não identificado 7, apresentou λmax de 312 nm, [M-
H]- (m/z) 677 em MS e fragmentos de íons (m/z) de 659, 645, 617 e 585 em MS2. É
interessante salientar que o λmax e espectro de massas e tempo de retenção do pico
25 foram idênticos ao descrito em literatura como não identificado em extratos de
frutas de murici (Mariutti et al., 2014). O pico 25 foi somente encontrado em amostras
de umbu-cajá.
Os picos foram quantificados em equivalentes de ácido gálico1 (LOD 1,40 µg/mL, LOQ 4,24 µg/mL), rutina2 (LOD 0,60 µg/mL, LOQ 1,81 µg/mL) e quercetina
(LOD 0,75 µg/mL, LOQ 2,27 µg/mL).
99
CONCLUSÃO
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Capitulo V
In vitro bioaccessibility of free and esterified carotenoids in cajá frozen pulp-
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DISCUSSÃO GERAL
116
CONCLUSÃO GERAL
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146
ANEXOS
147
0.75
1+
0.50 583.42 1+
1+
685.56
1+ 551.41
1+ 1+ 1+ 1+ 1+
0.25 540.58 1+ 1+
499.44 511.41 663.48 673.54 779.79
635.48 705.55 747.58 794.59 807.68
0.00
500 550 600 650 700 750 800 m/z
1.0 1+
491.37
0.5 1+ 1+
1+ 1+ 1+ 2+
1+ 221.08 265.21 299.15 317.17 1+ 2+ 1+ 1+ 1+
377.25 393.23 509.33 565.39
209.07 235.13 279.01 335.18 427.34 464.28 525.42 543.52
0.0
200 250 300 350 400 450 500 550 600 m/z
5’,6’ou 5’,8’-epoxy-β-cryptoxanthin
2.0
1.5
1+
409.32
1.0 1+
569.44
1+ 1+
0.5 391.25 1+ 1+ 1+ 1+ 663.58
427.35 485.37 551.41 577.53 605.46 623.50 632.57
441.32 453.40 529.30 538.46 561.43 593.40
0.0
400 450 500 550 600 650 m/z
2 1+
1+ 1+ 1+ 1+ 1+ 1+
1+ 1+ 285.17 351.20 363.22 377.26 1+ 477.34
1 1+ 205.10 1+ 1+ 1+ 459.29 1+
241.11 259.12 271.18 319.15 415.34
165.11175.13 193.16 221.13 338.13 395.27 441.30 499.13 511.33 533.39
0
150 200 250 300 350 400 450 500 550 m/z
(Z)-5,8-epoxy-β-cryptoxanthin
1.00 1+
565.56
0.75
1+
0.50 664.66
1+
1+ 1+ 1+ 1+ 1+
553.45
0.25 529.37 1+ 1+
583.41 617.48 1+ 1+ 1+
643.57 682.69
523.39 539.25 543.30 559.51 577.53 593.57 601.43 609.44 621.49 630.58634.50 671.51
0.00
520 540 560 580 600 620 640 660 680 m/z
4
1+ 1+ 1+
1+ 361.18 1+ 477.34
1+ 221.08 1+ 335.17 2+ 1+
2 203.12 277.03 1+ 411.24 431.25 1+
1+ 1+ 1+ 379.23 2+ 1+ 1+
177.17 1+ 310.29 343.17 367.17
459.35 495.40 533.36
227.19 247.10 283.31 321.18 397.27
300.15
0
200 250 300 350 400 450 500 550 m/z
(all-E)-5,8-epoxy-β-cryptoxanthin
1.0
2+ 1+ 2+ 1+
1+ 310.29 379.23 1+ 1+
0.5 221.09 1+ 282.32 333.21 1+
209.11 459.32
2+ 1+
267.14
1+ 403.13 413.22 1+ 477.46 1+
195.13 251.03 297.12 361.22 387.30 1+ 441.29 1+ 535.43
349.13 427.31 511.27
489.33 498.38
0.0
200 250 300 350 400 450 500 550 m/z
149
(all-E)-zeinoxanthin
0.8
0.6
2+
0.4 397.28
1+ 1+ 1+ 1+
0.2
461.33 535.38 595.44 663.42 873.66
0.0
100 200 300 400 500 600 700 800 900 1000 1100 m/z
2.0
1.5
3+ 1+
1+ 1+ 321.11 401.20 1+
1.0 1+ 1+ 255.08 1+ 1+ 1+ 1+
1+176.94 1+ 220.92 279.06 1+ 429.15
196.95 267.08 1+ 335.17 1+ 1+ 416.18 1+ 473.28 1+
170.95 2+ 210.97 345.08 1+ 1+
1+ 291.05 307.02 359.08 387.15 455.24 483.18 497.25
0.5 182.89 228.96 374.16 443.36 1+ 1+
511.30 525.25
0.0
200 250 300 350 400 450 500 m/z
(all-E)-β-cryptoxanthin
2
1+ 1+
1 397.32 1+ 1+ 660.62
409.32 473.35 535.42 573.35 609.51 646.71 678.69
0
350 400 450 500 550 600 650 m/z
phytofluene
0.00
200 250 300 350 400 450 500 550 m/z
(all-E)-luteoxanthin myristate
4 1+ 1+
619.53 811.64
1+ 1+ 1+
1+ 1+ 1+ 1+ 1+
2 1+ 541.48 1+ 749.59 793.65
573.43 593.59 1+ 661.46 692.69 705.54 1+
529.38 674.67 1+ 1+ 1+ 1+
509.43 521.43 652.54 719.56 737.65 769.63 779.72 840.70 867.67 902.84
0
500 550 600 650 700 750 800 850 900 m/z
1.00
0.75
0.50 1+
583.41 1+
1+ 1+
0.25 565.35 701.56
1+ 1+ 1+ 1+ 719.54 1+
511.41 527.29 547.31 603.49 775.58
0.00
500 550 600 650 700 750 800 850 900 m/z
150
Pheophytin a
1 2+
615.41 812.56 839.46
0
600 650 700 750 800 850 m/z
3 1+
533.21
2
1
839.46
0
200 400 600 800 1000 m/z
1.0 1+
533.23
0.5 1+
615.51
0.0
150 200 250 300 350 400 450 500 550 600 m/z
(all-E)-lutein-3’-0-laurate
1.5
1+
1.0 575.52
1+
599.48
0.5 1+ 1+ 1+
1+ 1+ 1+
533.39 617.37 661.46 705.48 815.71
541.44 638.65 649.58 749.53 762.66 779.59 789.63
0.0
550 600 650 700 750 800 m/z
0.6 1+
411.24
1+ 1+ 1+
0.4 265.12 345.18 1+ 1+
175.11 1+ 1+
1+ 271.01 1+ 1+ 429.30 495.33
197.11 1+ 359.21 1+ 1+
1+ 1+ 219.06 291.15 333.13 385.19 2+
253.07 309.01 319.23 1+ 441.28 459.28 471.29
0.2 185.14 209.13 229.15 399.33
374.25 520.38
504.13
0.0
200 250 300 350 400 450 500 550 m/z
(all-E)-α-carotene
0.8
0.6
0.4
1+
537.43 1+
0.2 1+ 1+ 1+ 663.41
337.17 521.39 549.44 575.47 627.49
327.00 495.37 561.50
0.0
300 350 400 450 500 550 600 650 m/z
1+
413.22
4
1+ 2+ 1+
3+ 1+ 3+ 321.14 347.12 1+ 399.15
2 1+ 1+ 1+ 1+ 268.97 1+ 1+ 1+ 1+
1+ 299.05 1+ 313.21 359.19 1+
196.98 210.90 243.01 254.98 333.17 429.32 1+ 1+
174.97 184.89 285.01
307.11
388.21 1+
457.30 467.20 481.31 518.49
0
200 250 300 350 400 450 500 m/z
151
(all-E)-lutein-3-0-laurate
0.8 1+
2+ 411.27
297.15 478.38
0.6
(all-E)-β-cryptoxanthin laurate
1+
1 443.37
1+ 1+ 1+ 1+ 1+
1+ 1+ 1+ 1+ 1+ 2+ 1+
267.11 643.50
223.07 237.25 291.10 307.16 335.24347.30359.28 375.40 399.24 413.28 465.22 479.37 611.44 628.51 679.48 718.74
0
250 300 350 400 450 500 550 600 650 700 m/z
(all-E)-β-cryptoxanthin myristate
1
1+ 1+ 1+ 1+ 1+ 1+
1+ 1+ 1+ 1+ 1+ 1+ 1+ 1+
263.14 443.34 671.59
221.02 247.13 289.11 317.09 333.20 371.25 397.20 413.18 494.63 508.57 573.51 599.39 626.59 648.63 743.70
0
300 400 500 600 700 m/z
(all-E)-lutein-3-O-myristate-3’-O-laurate
1+
2 601.56
1+ 1+
1+ 577.56 1+ 1+ 1+
1 645.59 733.64 1+
1+ 533.41 1+ 633.61647.64 1+ 1+ 1+ 885.84
493.41 551.52 699.61 761.61 813.70
473.45 777.58 853.74
0
450 500 550 600 650 700 750 800 850 900 m/z
1.0 1+ 1+
1+ 397.20 477.21
303.05 1+ 357.12 1+
1+ 1+ 1+ 1+
0.5 186.99 1+ 248.95 319.08 1+ 1+
170.91 208.92
239.01
272.90 291.09 341.13 373.15385.13 441.36
463.26 514.39
1+
551.46
0.0
200 250 300 350 400 450 500 550 m/z
152
(all-E)-zeinoxanthin stearate
1.0
1+
747.69
0.5
1+ 1+
705.48 727.70 739.71
0.0
700 720 740 760 780 800 820 840 860 m/z
1.0
0.5
1+
1+ 1+ 1+ 1+ 443.33 1+ 1+
1+ 345.15 1+ 2+
269.14 289.10 319.18 377.35 413.30 467.30 683.50 727.83
247.02 493.48 564.38 577.51 617.66 661.51
0.0
300 400 500 600 700 m/z
(all-E)-lutein-3-O- myristoleic-3’-O-palmitate
1+
4 759.68 1+
789.68
1+ 1+ 1+
395.34 1+ 1+ 617.43 1+
2 1+ 573.42 661.46 1+ 749.51
1+ 1+ 529.37 551.52 705.51
1+ 1+ 1+ 1+ 1+ 1+
383.32 485.36 1+ 1+ 1+
423.36 441.29 677.73
467.36 509.40 593.56 607.56 635.63 650.70 734.69
775.69 817.67
491.42
0
400 450 500 550 600 650 700 750 800 m/z
2
533.39
1 1+
1+ 2+ 1+ 2+
742.72
382.51 397.02 425.31 477.25 493.51 508.54 521.09 587.38 635.64 681.52 706.38
0
400 450 500 550 600 650 700 750 800 m/z
1.00
0.75
0.50
1+ 1+
0.25 1+ 1+ 411.26 1+ 1+ 1+ 1+ 1+ 1+
441.32 477.34
385.31 397.20 427.33 629.56 667.57 697.54 755.65 772.74
0.00
400 450 500 550 600 650 700 750 800 m/z
(all-E)-lutein dipalmitate
2 1+
607.53
1 1+ 1+ 1+
1+
533.46 551.52 577.50 635.56
631.55 699.62
565.44 621.64 649.62 687.61 759.72 789.67
0
525 550 575 600 625 650 675 700 725 750 775 m/z
1+
321.22 1+ 2+
2 373.03 1+ 477.19
3+ 1+ 411.22 1+
1+ 252.97 277.05 2+ 491.27 1+
158.90 1+ 1+
212.90 1+ 1+ 395.11 506.43
1 187.02 236.91 293.23 329.03 345.11 1+
268.08 441.14
174.95 462.38 1+
551.58
0
150 200 250 300 350 400 450 500 550 m/z
153
(all-E)-lutein-3-O-stearate 3’-O-palmitate
1.0
1+
817.71
0.5 1+
1+ 1+ 1+ 1+ 1+ 789.68
1+ 1+ 1+ 617.44 1+
533.39 555.52 661.45 705.46
441.34 485.31 573.43 678.71689.64 725.68 736.70 749.54
391.32 597.49 635.53 766.87 804.73 837.60
0.0
400 450 500 550 600 650 700 750 800 m/z
1.0
0.5 1+ 1+
1+ 411.30 1+ 1+ 1+ 799.72
1+ 1+ 1+ 1+
397.25 427.21 441.34 453.35 477.35 781.63
519.41 645.63 725.65 761.67
0.0
400 450 500 550 600 650 700 750 800 m/z
1 1+
1+
411.31 1+ 1+ 1+ 2+
1+ 1+ 1+ 1+ 772.85
441.33 1+ 549.43 697.51 758.92 1+
387.22 425.26 465.17 477.36 491.33 505.26 564.51575.52 617.76 644.76 667.44 721.60 733.74 818.71
0
400 450 500 550 600 650 700 750 800 m/z
Monogaloil hexosídeo
MS
Intens. 1- -MS, 8.5min #749
1-
x104 330.96
339.16
4 1- 1-
353.15 397.23
3 1-
1- 666.04
2 197.66 1- 1-
3-
311.10 418.03 451.27
1- 1- 1- 3- 1- 2- 1-
1 125.80 2- 1- 559.77 770.12
162.72 249.01 495.89 603.72 633.13 693.99 734.08
219.72 286.66 538.92
0
100 200 300 400 500 600 700 m/z
MS2
Intens. -MS2(330.99), 8.6min #758
1-
x104
168.80
1.0
1-
0.8 270.85
0.6 1-
210.85
0.4
1-
0.2 124.94 192.83 330.91
0.0
100 200 300 400 500 600 700 m/z
MS
Intens. 1- -MS, 10.2min #889
x104 339.15
1-
397.20
1- 1-
3
416.04 536.16
1- 1-
2 197.60 353.11 666.07 2-
1- 780.50
1- 1- 455.33
367.13 1- 2- 1- 1-
1 160.68 300.96 1-
249.03 280.75 325.14 505.02 557.26576.76 632.38 715.75 743.38
125.77
0
100 200 300 400 500 600 700 m/z
154
MS2
Intens. -MS2(397.20), 10.2min #891
1-
x104
337.07
0.8
0.6 1-
162.96
0.4 1-
220.93
0.2 1- 1-
178.90 268.63 360.52 395.08
0.0
100 200 300 400 500 600 700 m/z
MS
Intens. 1- -MS, 11.4min #985
x105 410.98
0.8
1-
339.17
0.6 1-
397.28
0.4
1- 1-
1-
197.65 353.15 1- 666.04 1-
0.2 451.28
125.73 160.69 248.99 495.14 550.22 610.56 688.74 725.61 754.09
284.54
0.0
100 200 300 400 500 600 700 m/z
MS2
Intens. -MS2(410.98), 11.4min #986
1-
x104
240.77
3
1 1- 1-
168.81 330.90 1- 1-
124.86 366.91 409.17
0
100 200 300 400 500 600 700 m/z
Galoil–HHDP-hexosídeo
MS
Intens. 1- -MS, 20.2min #1705
x104 633.13
1-
339.19 1-
4 397.29
1- 1-
3 353.16 416.06 1-
197.65 1- 455.29 2-
325.11 1-
2 1- 666.05
467.01
311.07 2- 600.50 3- 2- 2-
125.76 162.65 683.89
1 215.73 297.06 532.70 555.13 717.14 752.95 787.25
244.90 276.94
0
100 200 300 400 500 600 700 m/z
MS2
Intens. -MS2(633.13), 20.2min #1708
1-
300.82
4000
3000
2000
1- 1-
274.82 1-
1000 462.94 596.63
418.95 558.52
256.87 356.88 516.44
228.83
0
100 200 300 400 500 600 700 m/z
155
Rutina
MS
Intens. 1- -MS, 24.6min #3771
x106 609.16
1.0
0.8
0.6
0.4 1-
677.14
0.2 1- 1-
645.11 723.09 745.09
0.0
100 200 300 400 500 600 700 m/z
MS2
Intens. -MS2(609.16), 24.6min #3772
1-
x105
300.82
1
1- 1- 1-
178.81 270.83 342.88
0
100 200 300 400 500 600 700 m/z
MS
Intens. 1- -MS, 25.8min #2165
x104 339.17
1- 1-
4 397.22 433.01
3 1-
325.10 1- 1-
2 1- 1- 353.16
1- 451.30 1- 1- 1-
197.64 1- 311.08 2-
1- 2- 666.05 686.38
1 125.78
162.71 255.65 521.28 612.28 724.35 747.74
219.69 284.00 493.11 551.16
0
100 200 300 400 500 600 700 m/z
MS2
Intens. -MS2(433.01), 25.8min #2168
1-
x104
300.80
1.5
1.0
0.5
1-
162.66 374.63 431.92
0.0
100 200 300 400 500 600 700 m/z