Analysis of Anthocyanins Extracted from the Acai Fruit ...downloads.hindawi.com/journals/joph/2018/6830835.pdf · clinical trial in 25 humans (unpublished data). e objectives of the

Research ArticleAnalysis of Anthocyanins Extracted from the Acai Fruit(Euterpe oleracea): A Potential Novel VitalDye for Chromovitrectomy

Cristiane S. Peris,1 Rafael R. Caiado,1 Acacio Alves Souza Lima-Filho,1,2

Eduardo B. Rodrigues,1 Michel Eid Farah,1 Mariana Batista Gonçalves,1

Bruno de Queiroz Alves,1 Joao Guilherme Palma Urushima,1 Raul Ragazzi,1,2

and Mauricio Maia 1,3

1Department of Ophthalmology, Federal University of São Paulo, Botucatu Street, 816–Vila Clementino, 04023-062 São Paulo,SP, Brazil2Ophthalmos S/A, Brigadeiro Luıs Antonio Avenue, 4830–Jardim Paulista, 01401-002 São Paulo, SP, Brazil3Brazilian Institute of Fight Against Blindness, Otto Ribeiro Avenue, 901–Jardim Paulista, 19814-470 Assis, SP, Brazil

Correspondence should be addressed to Mauricio Maia; [email protected]

Received 10 March 2018; Accepted 17 May 2018; Published 19 July 2018

Academic Editor: Anat Loewenstein

Copyright © 2018 Cristiane S. Peris et al. 'is is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Purpose. To classify and quantify anthocyanins in a vital dye extracted from the acai fruit (Euterpe oleracea), adjust pH and osmolarity,and perform lyophilization to develop a new chromovitrectomy dye. Methods. 'ree dye concentrations 10%, 25%, and 35%(equivalent to 100, 250, and 350mg of lyophilized acai fruit pulp extract samples) were evaluated when diluted in 1ml of phosphate-buffered solution (pH 7 and 300mOsm). 'e dye was analyzed by mass spectrometry and high-performance liquid chromatography(HPLC) to identify and quantify anthocyanins molecules. Results. 'e pH and osmolarity correction and lyophilization were per-formed without damaging the anthocyaninmolecular structure.Mass spectrometry confirmed the presence of five anthocyanins in thethree concentrations of the dye. Cyanidin-3-O-glucoside was the major anthocyanin found. HPLC showed that the concentration ofanthocyanin was similar, independent of the dye concentration tested. Conclusions. Lyophilization and the correction of pH andosmolarity (7.00 and 300mOsm, resp.) were performed successfully. Five anthocyanins are present in the dye from the acai fruit. 'emajor anthocyanin is cyanidin-3-O-glucoside. Independent of the dye concentration tested, the anthocyanin concentration wassimilar. Standardized chemical characteristics of this new dye may allow its use during chromovitrectomy in humans.

1. Introduction

'eacai fruit (Euterpe oleracea) from the palm tree is native tothe Amazon forest in Brazil. In addition to being one of themain trees that produce palm hearts, the acai palm alsoproduces a fruit with high nutritional properties and eco-nomic value for the food and cosmetic industries. 'e acaifruit has been described as caloric and rich in proteins,polyunsaturated fatty acids, and sugars (Table 1) [1].

Carotenoids and anthocyanins are among the maincomponents of the acai fruit [2–4]. Anthocyanins, derivedfrom the Greek words anthos (flower) and kianos (blue), arepart of a large group of organic components known as

flavonoids that are represented by the basic chemicalstructure C6-C3-C6 (Figure 1). 'e phenolic structure of ananthocyanin provides an antioxidant effect due to electrondonation or transference of hydrogen atoms.

It has been suggested that photosensitizing dyes used inchromovitrectomy could enhance phototoxicity by increasinglevels of free radicals, creating a photoproduct that could beharmful to retinal cells. 'e antioxidant effects of anthocyaninstheoretically could quench the singlet oxygen, which is releasedduring chromovitrectomy [5–7].'e antioxidant property of theacai dye, associated with its high affinity for the internal limitingmembrane (first observed in cadaveric eyes by our researchteam), became the basis for the development of a novel dye [8].

HindawiJournal of OphthalmologyVolume 2018, Article ID 6830835, 9 pageshttps://doi.org/10.1155/2018/6830835

mailto:[email protected]

http://orcid.org/0000-0002-7034-8091

https://doi.org/10.1155/2018/6830835

In 2017, our research group found that the 10% and 25%concentrations of the acai dye were safe when used ina rabbit model [9]. In this study, rabbits were injectedintravitreously with the acai dye in three concentrations:10%, 25%, and 35%. Control eyes received balanced saltsolution. Functional evaluations were performed usingelectroretinography while morphologic examinations wereperformed by fundus imaging, fluorescein angiography,optical coherence tomography, light microscopy, andtransmission electron microscopy. 'e 10% and 25% dyeconcentrations from the acai fruit did not cause significantfunctional or morphological toxicity. However, the 35%concentration showed evidence of morphologic and func-tional abnormalities suggestive of temporary toxic effects at24 h follow-up.

Hence, the highest concentration of the acai fruit thatwas safe and effective in the rabbit model was 25%. 'isconcentration was used for the sequence of a phase I/IIclinical trial in 25 humans (unpublished data).

'e objectives of the current study were to classify andquantify the anthocyanins of the acai fruit dye in the con-centrations of 10%, 25%, and 35%. 'is information willbecome the basis for further understanding the antioxidantbehavior of the acai dye.

2. Materials and Methods

2.1. Sample Preparation. 'e commercial acai extract wasdivided into flasks of 100, 250, and 350mg of lyophilized

anthocyanin powder to produce the concentrations of 10%,25%, and 35%, respectively.

Ophthalmos SA (São Paulo, Brazil) developed the ly-ophilization process for the final dye preparation that wascomprised mainly of anthocyanins. 'is process was per-formed in a sterile environment, stabilized at −50°C for 24hours, and diluted in 1ml of polyvinyl alcohol in the 10%,25%, and 35% concentrations. 'e pH of the natural extractwas acidic [10]. Hence, both the pH and osmolarity (pH 7.00;300mOsm) were controlled and established using one mil-liliter of phosphate-buffered solution (PBS). 'ese concen-trations also reflect the most suitable values obtained inpreliminary studies in which factors such as staining capacity,pH, osmolarity, solubility, and stability were considered.

'e lyophilized dye solutions were stored in depyro-genized amber flasks based on our previous results thatanthocyanin molecules are highly unstable and light ex-posure might modify the staining capacity of the dye [9].'eanthocyanins were then analytically characterized by massspectrometry.

2.2. Mass Spectrometry. Twenty-five-milligram samples ofthe raw materials and final products were subjected to massspectrometry to compare the specific molecules in lower tohigher quantities. 'is analysis was based on a spectral databank. SGB Chemical Consultant Ltda. (São Paulo, Brazil),which was blinded to the dye components, analyzed thethree lyophilized samples.

2.3. High-Performance Liquid Chromatography (HPLC).'ree microliters of dimethyl sulfoxide (DMSO) (VetecVentiltechnik GmbH) and 7.0mL of the solution from themobile phase were added to the flasks with the dye con-centrations. 'ese were then put in an ultrasonic bath, andthe extraction process continued for 10 minutes. 'e flaskswere removed from the bath, and the solutions were filteredinto 1.5mL vials containing a filter 0.22 micrometer indiameter. Finally, 20 µL was added to the HPLC stabilizedsystem.

'e analysis was performed using the 1260 Infinity IIliquid chromatography system and a diode array detector(Agilent Technologies, Santa Clara, CA). 'e specificmethodology used a 5 µm 150× 4.6mm Agilent EclipseColumn XDB C18 (Agilent Technologies). 'e mobile phasewith an aqueous solution of 0.1% trifluoroacetic acid wasreferred to as phase A and acetonitrile HPLC was referred toas phase B.

Table 1: Reference of nutritional values from 100 grams of acaifruit (E. oleracea) pulp on a diet of 2,500 kcal/day.Protein 13 gIron 26 gFibers 34 gPhosphorus 227 gSodium 56.4 gVitamin C 17mgPotassium 932mgVitamin E 45mgCalcium 286mgLipids 17mgMagnesium 174mgGlycides 36mgCalorie value 349 kcalSource: Federal University of Para State, Brazil.

R2

R1

O

OH

OH∗

OH∗

HO

Figure 1: Basic molecular structure of the anthocyanins.

Table 2: Gradient used for liquid chromatography analysis of fivemajor anthocyanins.

Time Phase A (%) Phase B (%) Flow (mL/minute)0.0 95 5 1.005.0 95 5 1.0020.0 5 95 1.0025.0 95 5 1.0027.0 95 5 1.0035.0 95 5 1.00

2 Journal of Ophthalmology

Retention time (minutes)

120100

80604020

0–20

105 15 20 25 30 35

8

910

11

121314

151617181920 2122 23 242526

1. 2.233

234 65

7

DAD1 B, sig = 265, 4 ref = off (ACAI_FLAVONOIDS\ACAI_1 2015-08-01 10-31-57\AFLAV000009.D)

1

2. 2.8473. 3.0404. 3.1555. 3.2466. 3.4707. 3.9728. 9.873

9. 12.07910. 12.48211. 12.68812. 13.24513. 13.47114. 13.69715. 13.87016. 14.186

17. 14.66418. 15.48019. 15.76320. 16.20121. 20.47322. 20.70223. 22.45724. 24.484

25- 24.64726- 25.142

Are

a (m

illi a

bsor

banc

e uni

t)


302010

–100

–20

105 15 20 25 30 35


1 23

4 65789

1110

12

13

14

1516171819 20

7. 11.7928. 12.0619. 12.346

10. 12.45711. 12.56012. 12.761

13. 13.24614. 13.47615. 13.81916. 14.01317. 14.30318. 14.685

19. 14.87120. 16.208

Are

a (m

illi a

bsor

banc

e uni

t)

Retention time (minutes) 105 15 20 25 30 35

200

150

50

100

0 123 4 5

6

7

1. 3.1112. 3.3173. 3.4804. 11.4465. 12.4926. 12.6027. 13.166

Are

a (m

illi a

bsor

banc

e uni

t)

(a)


DAD1 A, sig = 265, 4 ref = off (ACAI_FLAVONOIDS\ACAI_1 2015-08-01 10-31-57\AFLAV000011.D)

1 2 34 5

80

60

40

20

0

20


17

2 43 56 8

9

1011

12

13

14151617

18

192021222324

7612 453 8

9

10

11

1. 2.2732. 2.8843. 3.0934. 3.1945. 3.3676. 3.5437. 11.799

9. 12.35210. 12.66211. 12.76412. 13.70013. 13.48014. 13.824

17. 14.69018. 14.87719. 15.48220. 15.77021. 16.21222. 16.50523. 17.29224. 17.4898. 12.067

15. 14.21616. 14.368

DAD1 C, sig = 520, 4 ref = off (ACAI_FLAVONOIDS\ACAI_1 2015-08-01 10-31-57\AFLAV000011.D)1. 2.5192. 2.8173. 2.9994. 3.021

9. 12.60710. 12.76411. 13.170

5. 3.1286. 3.3957. 11.4528. 12.496

(mAU

)(m

AU)

500400300200100

0

(mAU

)

250

200

150

100

50

0

5 10 15 20 25 30 35

Retention time (minutes)5 10 15 20 25 30 35

Retention time (minutes)5 10 15 20 25 30 35

6

7 8

9

1011

12131415161718 22 2324 25 26 27 282930212019

1. 2.2402. 2.8863. 3.0874. 3.5375. 3.9796. 9.8807. 12.085

9. 12.69010. 13.25011. 13.47612. 13.70013. 14.00914. 14.190

17. 15.48318. 15.48319. 16.20720. 16.49921. 17.23022. 17.50423. 20.47724. 20.705

25. 21.69326. 22.45827. 23.45628. 24.483

8. 12.48115. 14.66616. 14.878

29. 24.64730. 25.141

(b)

Figure 2: Continued.

Journal of Ophthalmology 3

'e reagents used water (produced with purified waterfrom a permutation technique), trifluoroacetic acid (VetecVentiltechnik GmbH, Speyer-am-Rhein, Germany), DMSO,and acetonitrile HPLC (Sigma-Aldrich, St. Louis, MO). 'isanalysis used the gradients listed in Table 2, thus enablingquantification of all anthocyanins in the same chromatogram.

For the anthocyanin quantification using HPLC, it wasfirst necessary to obtain the anthocyanin profiles using massspectrometry. 'is information was used to determine theanalytical standards of the anthocyanins which were thenobtained from ChromaDex Inc. (Irvine, CA).

Five 25-milligram samples of the same major isolatedanthocyanin molecules were used as comparative patternsfrom higher to lower quantities: cyanidin-3-O-glucoside,homoorientin, orientin, taxifolin, and isovitexin. 'e sameamount (2.0± 0.20mg) of the anthocyanin chemical stan-dards was weighed in separate 10mL volumetric flasks of10ml each. Subsequently, 3mL of DMSO was added to eachflask and subjected to high-frequency ultrasound for 5minutes until the mobile phase.'e patterns weremixed anddiluted with the mobile phase.

'e anthocyanin concentrations were quantified byHPLC using an electrochemical detector. 'e 350-nano-meter wavelength was identified by the detector showing1.00mL/min flow by maintaining the column with the aid ofa 40°C oven. 'e five molecules were used in the mobilephase of the solution in 10ml/vial containing water and

30% DMSO without being retained in the filter. Such datawere used to obtain the anthocyanin concentration for eachconcentration of the dye.

3. Results

3.1. pH and Osmolarity. Correction of pH and osmolarityand the lyophilization process were performed successfully.

3.2.Mass Spectrometry. 'is analysis showed the presence offive anthocyanins in the vital dye of the acai fruit from lowerto higher quantities: taxifolin, orientin, isovitexin, homo-orientin, and cyanidin-3-O-glucoside. Mass spectrometryanalysis of the 10%, 25%, and 35% concentrations of the vitaldye are shown in Figure 2.

3.3. HPLC. 'is analysis quantified the five major antho-cyanins in the vital dye of the acai fruit in the three testedconcentrations based on the retention time by area shown inthe chromatographs (Figures 3(a)–3(c)). Results showed thatindependent of the concentration of the acai dye, the an-thocyanin concentration was similar (Table 3).

Chromatography results were compared with those ofthe isolated patterns, which facilitated confirmation of thepresence of the anthocyanin molecules through separationof the flavonoids (Figures 4(a)–4(e)).

120100

60

20

–20

0100200300400500600700

123 4 5

6

8

7

0

40

80

1 2345

678

109

11

12

13

1416

15171819202122232425 26

400

300

200

100

0 1 26534 7

8910

11

121314151617181920

2122232425 2627 28 29 30 313233

1. 2.2502. 2.9333. 3.1824. 3.5735. 3.7366. 3.9827. 9.8828. 12.087

9. 12.47910. 12.61211. 12.76512. 13.25413. 13.48214. 13.76515. 13.89116. 14.016

17. 14.19818. 14.67219. 14.88420. 15.49121. 15.77522. 16.21623. 16.50624. 17.237

25. 17.50926. 20.47927. 20.70528. 21.69429. 22.45930. 23.45931. 24.48532. 24.64833. 25.142

1. 2.2712. 2.8253. 3.0574. 3.3535. 3.1186. 11.803

9. 12.46410. 12.50811. 12.76512. 13.25413. 13.255

14. 13.83115. 14.11716. 14.31417. 14.52818. 14.69619. 14.86320. 15.490

23. 16.51224. 17.30025. 17.49026. 19.807

7. 12.071

8. 12.355 21. 15.77822. 16.220

1. 3.1172. 3.2073. 3.4394. 11.4555. 12.4986. 12.6407. 12.7658. 13.175





105 15 20 25 30 35


105 15 20 25 30 35


105 15 20 25 30 35

Are

a (m

illi a

bsor

banc

e uni

t)A

rea (

mill

i abs

orba

nce u

nit)

Are

a (m

illi a

bsor

banc

e uni

t)

(c)

Figure 2: (a) A mass spectrometry graph of the 10% concentration of the vital dye extracted from the acai fruit. (b) A mass spectrometrygraph of the 25% concentration of the vital dye extracted from the acai fruit. (c) A mass spectrometry graph of the 35% concentration of thevital dye extracted from the acai fruit.


30

20

10

0

0 2 4 6 8 10 12 14

30

20

10

0

2

4

5

11.177 11.300 11.672 (+) -taxifolin 11.793

1

Are

a (m

illi a

bsor

banc

e uni

t)

Wavelength = 350 nm


3 I

9.813 9.987 cyanidin-3-O-glucoside 10.380 homoorientin 10.587 orientin 10.893 isovitexin 11.033

67

8 910

DAD: signal D, 350.0 nm/Bw: 4.0 nmSample 80-2

1.2.3.4.5.6.7.8.9.

10.

(a)

60

40

20

0

0 2 4 6 8 10 12 14

5

611

Wavelength = 350 nm


I

60

40

20

0

I

7891012A

rea (

mill

i abs

orba

nce u

nit)

11.040 11.220 11.333 11.457

9.813 9.980 cyanidin-3-O-glucoside 10.227 10.380 homoorientin 10.593 orientin 10.893 isovitexin

1

234 11.667 (+) -taxifolin 11.787


1.2.3.4.5.6.7.8.9.

10.11.12.

(b)

30

20

10

0

0 2 4 6 8 10 12 14

30

20

10

0

2

3

4

56 7 89

1

Are

a (m

illi a

bsor

banc

e uni

t)

Wavelength = 350 nm


I

11.300 11.672 (+) -taxifolin 11.793 11.457

9.813 9.987 cyanidin-3-O-glucoside 10.380 homoorientin 10.587 orientin 10.893 isovitexin 11.033

11.667 (+) -taxifolin 11.787

1.2.3.4.5.6.7.8.9.

10.11.12.


(c)

Figure 3: (a) A chromatograph of the 80-2 vital dye extracted from the acai fruit in a 10% concentration (equivalent to 100mg ofa lyophilized sample extracted from the acai pulp diluted in 1ml PBS (pH 7.00; 300mOsm). (b) A chromatograph of 81-2 vital dye extractedfrom the acai fruit in a 25% concentration (equivalent to 250mg of a lyophilized sample extracted from the acai pulp diluted in 1ml PBS (pH7.00; 300mOsm). (c) A chromatograph of 82-2 vital dye extracted from the acai fruit in a 35% concentration (equivalent to 350mg oflyophilized extracted from the acai pulp diluted in 1ml PBS (pH 7.00; 300mOsm)).


Table 3: Quantification of the anthocyanins based on the retention time by area of HPLC results at the three concentrations.

Anthocyanins 10% concentration 25% concentration 35% concentrationCyanidin-3-O-glucosideRetention time (minutes) 9.987 9.980 9.973Area (milli absorbance unit) 23.909 24.410 23.886HomoorientinRetention time (minutes) 10.380 10.380 10.380Area (milli absorbance unit) 38.511 36.950 40.106OrientinRetention time (minutes) 10.587 10.593 10.580Area (milli absorbance unit) 22.945 22.784 23.988TaxifolinRetention time (minutes) 11.673 11.667 11.667Area (milli absorbance unit) 0.966 0.894 0.998IsovitexinRetention time 10.893 10.893 10.880Area (milli absorbance unit) 6.755 6.737 6.194

HPLC chromatogram of cyanidin-3-O-glucoside (CDXA-13-3273)


2

1 4

D EG

–400

–200

0

200

400

600

Are

a (m

illi a

bsor

banc

e uni

t)

A

DAD1 C, sig = 210,8 ref = 600,100 (E2013\E2013_22.D)

653

B C

Area: 30.2248 Area: 30.1798

F

Area: 8.94004

10.582 10.891

Area: 2933.85

11.854 11.927 12.174 13.649

Area: 33.6881 Area: 30.0238

Area: 17.9114

0 5 10 15 20 25

A.B.C.D.E.F.G.

1.2.3.4.5.6.

(a)

DAD1 B, sig = 205,8 ref = 600,100 (J1413\J1413_10D)HPLC Chromatogram of (+) -taxifolin (CDXA-13-5818)

–600

–400

0

200

400

600

10Retention time (minutes)

B

A

2

–200

15 20 25

1

B. Area: 2.51301A. Area: 4.049.22

2. 12.1281. 12.008

Are

a (m

illi a

bsor

banc

e uni

t)

0 5

(b)




HPLC chromatogram of orientin (CDXA-12-7714)

5

A

F

E

CB

D

13 6

78

DAD1 A, sig = 250,80 ref = 600,100 (X:\YOUNG\DATA\2012\K0712\K0712_04.D)

100

200

300

400

0

11.684

Area: 1731.89

Area: 3.16356 Area: 3.16122

Area: 3.16052 Area: 0.33088

Area: 0.34587

11.507

24

12.044 12.045 13.167

11.544 11.544 11.567

Are

a (m

illi a

bsor

banc

e uni

t)

0 5 10 15 20 25

1.2.3.4.5.6.7.8.

A.B.C.D.E.F.

(c)


D

3

A

1

HPLC chromatogram of isovitexin (CDXA-13-4304)

B C

2

4

-400

-200

200

0

600

800 Area: 7.87127

12.900

Area: 4064.58 12.176

DAD1 C, sig = 210,8 ref = 600,100 (GO113\GO113_05.D)

600

Area: 60.3881 Area: 13.5539

12.717

13.018

Are

a (m

illi a

bsor

banc

e uni

t)

0 5 10 15 20 25

A.B.C.D.

1.2.3.4.

(d)



4. Discussion

'e acai fruit is a multistemmed palm widely distributed inthe northern South America. 'e fruit varies a great dealin the appearance of the plant, flowers, and fruiting prod-ucts, which can be affected by genetic and environmentalcomponents. Other factors can affect the anthocyanincomposition of fruits and their pulp, such as the harvest, typeof soil, water quality, and spatial localization [11].

'e current study aimed to characterize the physical-chemical composition of the anthocyanins present in theacai extract used in this research. 'e current data will beused to produce a standardized acai dye for use in chro-movitrectomy that was first tested in cadaveric eyes [8] andrabbits [9] and nowwill be used in a phase I/II clinical trial inhumans (unpublished data).

Mass spectrometry and HPLC identified and quantifiedthe presence of five anthocyanins in the vital dye of the acaifruit: cyanidin-3-O-glucoside, homoorientin, orientin, iso-vitexin, and taxifolin. Data showed that cyanidin-3-O-glu-coside is the major anthocyanin. 'ese findings agreed withthose of previous studies that also found that cyanidin-3-O-glucoside was the major anthocyanin found in the acaiextracts [12, 13]. Results also showed that independent of theconcentration of the acai dye, the anthocyanin concentrationwas similar (Table 3), and this result is probably because weemployed the same prime material.

We hypothesized previously [8] that the anthocyanins ofthe acai fruit might have low potential for causing retinal andRPE toxicity in humans. Spectrophotometry analysis ofdifferent dye concentrations from the acai fruit had a peaklight absorption around 1,000 nm; it is a fact that the lower

the wavelength of the light source, the higher the energyemission and consequently the more toxic the light source isto the retina [7]. 'e high absorption peak of light from theacai solution suggests that this dye might have a safer toxicityprofile regarding light absorption than other currentlyavailable dyes. A study that evaluated the absorbance spectraof nine vital dyes for chromovitrectomy found that most hadtwo absorbance peaks, the first below 400 nm and the otherin the visible light ranging from 400 to 700 nm [6].

In conclusion, the pharmacologic correction of the pH to7.00 and osmolarity to 300mOsm and the lyophilizationprocess were successfully performed. 'e five major an-thocyanins are present in the vital dye extracted from theacai, the major one being cyanidin-3-O-glucoside. In-dependent of the concentration of the acai dye, the an-thocyanin concentration was similar. 'ese standardizedchemical characteristics of this new dye may allow its useduring chromovitrectomy in humans. Further basic andclinical studies are needed to fully understand the role of acaidye anthocyanins and their antioxidant capacity.

Data Availability

'e data used to support the findings of this study areavailable from the corresponding author upon request.

Disclosure

'e authors hold a patent related to the intraocular use ofanthocyanins from acai fruit (Euterpe oleracea). 'e patentnumber (P11333244-8A2) is registered at the National In-stitute of Intellectual Property, Rio de Janeiro, Brazil.

C

4

123

Area: 4.83387

GFDBA5


0

200

600

400

1000

- DAD1 A, sig = 250,80 ref = 600,100 (C:\HPCHEM\ZEEMAN\DATA\2011\H2511\H2511_05.D)HPLC chromatogram of homoorientin (CDXA-11-4337)

800

H

Area: 4.53078

Area: 3909.84

Area: 4.38157 Area: 4.03112

Area: 3.90411 Area: 3.65581

11.837

10.873 11.136 11.598

12.070 Area: 4.38157

E

Are

a (m

illi a

bsor

banc

e uni

t)

0 5 10 15 20 25

1.2.3.4.5.

A.B.C.D.E.F.G.H.

(e)

Figure 4: (a) A chromatograph of isolated anthocyanin molecules of cyanidin-3-O-glucoside 25mg. (b) A chromatograph of isolatedanthocyanin molecules of taxifolin 25mg. (c) A chromatograph of isolated anthocyanin molecules of orientin 25mg. (d) A chromatographof isolated anthocyanin molecules of isovitexin 25mg. (e) A chromatograph of isolated anthocyanin molecules of homoorientin 25mg.


Conflicts of Interest

'e authors declare that they have no conflicts of interest.

Acknowledgments

'is work was supported by the Conselho Nacional deDesenvolvimento Cientıfico e Tecnologico, Brasılia, Brazil;Fundação de Amparo a Pesquisa do Estado de São Paulo,São Paulo, Brazil; and Pan-American Association ofOphthalmology/Pan-American Ophthalmological Founda-tion, Paul Kayser Global Award, Arlington, Texas. 'eauthors are grateful to Rubens Belfort Jr., M.D., Ph.D., fordonating supplies for the development of this study and toPaulo Schor, M.D., Ph.D., for support regarding applicationfor financial support at Conselho Nacional de Desenvolvi-mento Cientıfico e Tecnologico, Brasılia, Brazil.

References

[1] H. Rogez, Açaı: Preparo, Composição e Melhoramento daConservação, Universidade Federal do Para, Belem, Brazil,2000.

[2] S. Darnet, J. L. Serra, A. C. Rodrigues et al., “A high-performance liquid chromatography method to measure to-copherols in assai pulp (Euterpe oleracea),” Food ResearchInternational, vol. 44, no. 7, pp. 2107–2111, 2011.

[3] D. Del Pozo-Insfran, C. H. Brenes, and S. T. Talcott, “Phy-tochemical composition and pigment stability of acai (Euterpeoleracea Mart.),” Journal of Agricultural and Food Chemistry,vol. 52, no. 6, pp. 1539–1545, 2004.

[4] M. S. M. Rufino, J. Perez-Jimenez, S. Arranz et al., “Açaı(Euterpe oleraceae) ‘BRS Para’: a tropical fruit source of an-tioxidant dietary fiber and high antioxidant capacity oil,” FoodResearch International, vol. 44, no. 7, pp. 2100–2106, 2011.

[5] C. S. Peris, E. Badaro, M. A. Ferreira et al., “Color variationassay of the anthocyanins from acai fruit (Euterpe oleracea):a potential new dye for vitreoretinal surgery,” Journal ofOcular Pharmacology and ?erapeutics, vol. 29, no. 8,pp. 746–753, 2013.

[6] P. Costa Ede, E. B. Rodrigues, M. E. Farah et al., “Vital dyesand light sources for chromovitrectomy: comparative as-sessment of osmolarity, pH, and spectrophotometry,” In-vestigative Opthalmology and Visual Science, vol. 50, no. 1,pp. 385–391, 2009.

[7] Y. Yanagi, Y. Inoue, W. D. Jang, and K. Kadonosono, “A2emediated phototoxic effects of endoilluminators,” BritishJournal of Ophthalmology, vol. 90, no. 2, pp. 229–232, 2006.

[8] J. Chen, M. A. Ferreira, M. E. Farah et al., “Posterior hyaloiddetachment and internal limiting membrane peeling assistedby anthocyanins from acai fruit (Euterpe oleracea) and 10other natural vital dyes: experimental study in cadaveric eyes,”Retina, vol. 33, no. 11, pp. 89–96, 2013.

[9] R. R. Caiado, C. S. Peris, A. A. S. Lima-Filho et al., “Retinaltoxicity of acai fruit (Euterpe oleracea) dye concentrations inrabbits: basic principles of a new dye for chromovitrectomy inhumans,” Current Eye Research, vol. 42, no. 8, pp. 1185–1193,2017.

[10] S. Gallori, A. R. Bilia, M. Bergonzi et al., “Polyphenolicconstituents of fruit pulp of Euterpe oleracea Mart (Acaipalm),” Chromatographia, vol. 59, no. 11-12, pp. 739–743,2004.

[11] G. C. Cretu and G. E. Morlock, “Analysis of anthocyanins inpowdered berry extracts by planar chromatography linkedwith bioassay and mass spectrometry,” Food Chemistry,vol. 146, pp. 104–112, 2014.

[12] A. V. Carvalho, “Chemical composition and antioxidant ca-pacity of acai (Euterpe oleracea) genotypes and commercialpulps,” Journal of the Science of Food and Agriculture, vol. 97,no. 5, pp. 1467–1474, 2017.

[13] K. K. Yamaguchi, L. F. Pereira, C. V. Lamarao, E. S. Lima, andV. F. da Veiga-Junior, “Amazon acai: chemistry and biologicalactivities: a review,” Food Chemistry, vol. 179, pp. 137–151,2015.


Stem Cells International

Hindawiwww.hindawi.com Volume 2018


MEDIATORSINFLAMMATION

of

EndocrinologyInternational Journal of



Disease Markers


BioMed Research International

OncologyJournal of



Oxidative Medicine and Cellular Longevity


PPAR Research

Hindawi Publishing Corporation http://www.hindawi.com Volume 2013Hindawiwww.hindawi.com

The Scientific World Journal

Volume 2018

Immunology ResearchHindawiwww.hindawi.com Volume 2018

Journal of

ObesityJournal of



Computational and Mathematical Methods in Medicine


Behavioural Neurology

OphthalmologyJournal of


Diabetes ResearchJournal of



Research and TreatmentAIDS


Gastroenterology Research and Practice


Parkinson’s Disease

Evidence-Based Complementary andAlternative Medicine

Volume 2018Hindawiwww.hindawi.com

Submit your manuscripts atwww.hindawi.com

https://www.hindawi.com/journals/sci/

https://www.hindawi.com/journals/mi/

https://www.hindawi.com/journals/ije/

https://www.hindawi.com/journals/dm/

https://www.hindawi.com/journals/bmri/

https://www.hindawi.com/journals/jo/

https://www.hindawi.com/journals/omcl/

https://www.hindawi.com/journals/ppar/

https://www.hindawi.com/journals/tswj/

https://www.hindawi.com/journals/jir/

https://www.hindawi.com/journals/jobe/

https://www.hindawi.com/journals/cmmm/

https://www.hindawi.com/journals/bn/

https://www.hindawi.com/journals/joph/

https://www.hindawi.com/journals/jdr/

https://www.hindawi.com/journals/art/

https://www.hindawi.com/journals/grp/

https://www.hindawi.com/journals/pd/

https://www.hindawi.com/journals/ecam/

https://www.hindawi.com/

https://www.hindawi.com/

Documents

Analysis of Anthocyanins Extracted from the Acai Fruit ...downloads.hindawi.com/journals/joph/2018/6830835.pdf · clinical trial in 25 humans (unpublished data). e objectives of the