An Anthocyanin-Rich Extract of Acai (Euterpe precatoria Mart

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Article

An anthocyanin-rich extract of Acai (Euterpe precatoria Mart.) increases stress resistance and retards aging related markers in Caenorhabditis elegans. Herbenya Silva Peixoto, Mariana Roxo, Sonja Krstin, Teresa Röhrig, Elke Richling, and Michael Wink J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b05812 • Publication Date (Web): 26 Jan 2016 Downloaded from http://pubs.acs.org on January 26, 2016

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An anthocyanin-rich extract of Acai (Euterpe precatoria Mart.) increases stress resistance and retards aging related markers in Caenorhabditis elegans. Herbenya Peixoto§, Mariana Roxo§, Sonja Krstin§, Teresa Röhrig ¥, Elke Richling¥, Michael Wink§ ⃰ §

Heidelberg University, Institute of Pharmacy and Molecular Biotechnology, INF 364, D-

69120 Heidelberg, Germany ¥

Department of Food Chemistry and Toxicology, Molecular Nutrition, University of

Kaiserslautern, Erwin-Schroedinger-Strasse 52, D-67663 Kaiserslautern, Germany

Corresponding author: Prof. Dr. M. Wink; Email: [email protected]


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1

Abstract

2

Acai fruits (Euterpe precatoria) are rich in antioxidant anthocyanins. Acai consumption is

3

believed to have many health benefits; however, relevant detailed scientific investigations are

4

limited. The current study aimed to investigate an anthocyanin-rich extract from Euterpe

5

precatoria fruits (AE) with regard to its antioxidant and anti-aging properties using the model

6

organism Caenorhabditis elegans. AE can protect the worms against oxidative stress and can

7

ameliorate accumulation of reactive oxygen species (ROS) in vivo. The expression of stress

8

response genes, such as sod-3::GFP was upregulated while hsp-16::GFP was down-regulated

9

after AE treatment. Studies with DAF-16/FOXO mutants indicated that some of the

10

antioxidant effects are mediated by this transcription factor. AE can modulate the

11

development of age related markers such as the pharyngeal pumping. Despite the apparent

12

antioxidant activity, no lifespan prolonging effect was observed.

13

14

Key Words:

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Caenorhabditis elegans, antioxidants, anthocyanins, oxidative stress, aging, Acai

16

17

18

19

20

21



22

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Introduction

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Acai tree (Euterpe spp.) is a large palm found in the Amazon flood plain. Palm hearts

24

and fruits are harvested from this tree. Interest for the fruits, which are traditionally a part of

25

the daily diet of indigenous Amazonian people, has increased recently. Acai fruits are

26

distributed and consumed in all states of Brazil and are currently being exported to several

27

countries. Mixing the fruit’s pulp with iced water produces a drink of a pasty consistency,

28

which is famous and much appreciated in Brazil, especially for its nutritional value.

29

beverage is rich in α-tocopherol, fibers, lipids, polyphenols (including anthocyanins) and

30

mineral ions (such as calcium, magnesium, potassium). 3, 4

1, 2

The

31

The fruits which are being used for preparing the beverage come from two species,

32

Euterpe oleracea Mart., a clustered trunk palm largely distributed in the Amazon estuary,

33

including the northern part of Brazil, French Guyana, Guyana, Suriname, Venezuela and

34

Colombia, and Euterpe precatoria Mart., a single trunk palm found in the north of Brazil,

35

especially Amazonas, Acre and Rondônia as well as in the Bolivian and Peruvian portion of

36

Amazon. 5, 6 The sensorial characteristics of the beverages are similar and they are generally

37

referred to as “Acai”. However, the species bear fruits in alternate periods, the E. precatoria

38

crop period runs from December to June.7

39

Acai has gained the status of “superfruit” together with pomegranate, blueberry, goji

40

berry and others exotic fruits. The term superfruit has no official definition by regulatory

41

agencies; it is a market expression used since the last decade to refer, especially, to fruits with

42

high energetic power, antioxidant capacity, immune boosting and antiaging properties. These

43

bioactivities are mainly attributed to the substantial amounts of phytochemicals in the fruits,

44

such as polyphenols, polysaccharides and fibers. Cosmetic and toiletry industry are also

45

interested in superfruits as a source of natural additives. A large number of products for skin


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and hair care based on superfruit extracts including Acai are available, which claim

47

antioxidant, anti-aging and nourishing properties. 8, 9

48

A number of biological activities have been reported for Acai fruits from E. oleracea

49

including anti-inflammatory, antinociceptive and antioxidant properties. These bioactivities

50

are attributed to their high polyphenol content, especially to anthocyanins and flavones.

51

Polysaccharides from Acai have been reported to stimulate T cells, thereby playing a

52

potential immunomodulatory role in the innate immune response.

53

shown to lower the level of cholesterol in the blood in animal models for

54

hypercholesterolemia. 17 Udani et al. 18 demonstrated in a non-controlled pilot study, that the

55

consumption of Acai reduced the levels of metabolic syndrome markers in overweight adults,

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especially the levels of postprandial glucose, insulin and total cholesterol, and slightly of

57

LDL-cholesterol. Stoner et al. 19 showed that the pulp of Acai was able to attenuate tumor

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growth in mice with esophageal cancer. E. precatoria is also rich in polyphenols. The amount

59

of anthocyanins is reported to be 50 % higher than in E. oleracea,5 however, studies about its

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biological properties are limited.

16

10-15

Furthermore, Acai was

61

Reactive oxygen species (ROS) are normal byproducts of the aerobic metabolism of

62

cells responsible for physiological and pathological functions. Among the physiological

63

functions cell signaling and antimicrobial activity in the innate immune response are known.

64

The pathological functions are due to their capacity to negatively interact with biomolecules

65

like proteins, lipids and nucleic acids (causing mutations). ROS imbalance is associated with

66

tissue injuries and vascular dysfunction mediated by inflammation, neurodegenerative

67

diseases, even cancer and aging. 20, 21

68

Considering the dual role of ROS in cells an antioxidant defense system is required to

69

neutralize an overproduction of such reactive compounds. The cellular antioxidant defense



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system is composed of antioxidant enzymes and peptides (such as glutathione) endogenously

71

produced and also vitamins, trace metals, polyunsaturated fatty acids and polyphenolic

72

compounds which are obtained from food sources.

73

for antioxidant defence are gradually impaired throughout age resulting in an inability to deal

74

with the generation of ROS, which contributes to the aging process and the onset of age-

75

related diseases. 24

22, 23

However, the intrinsic mechanisms

76

Polyphenols, including anthocyanins, are widely known by their antioxidant capacity

77

in vitro and in vivo. 25-27 They are found in plants and are incorporated in human diet through

78

consumption of vegetables, fruits and beverages such as tea and juices. There are good

79

evidences that dietary polyphenols can modulate oxidative stress and consequently help in the

80

prevention or retardation of age-related diseases.

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role of dietary Acai in the modulation of ROS production by neutrophils and in the

82

expression of antioxidant genes in the liver of rats. 30

25, 28, 29

Previous works reported a positive

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In the current study a methanol/water extract from E. precatoria fruits was

84

investigated with regard to its antioxidant and anti-aging properties using the nematode

85

Caenorhabditis elegans, a model organism widely used in this context.

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antioxidant activity the capacity of the extract to protect the nematodes against acute

87

oxidative stress was investigated, as well as the influence on reactive oxygen species (ROS)

88

accumulation in vivo. The expression of stress response genes, such as sod-3::GFP and hsp-

89

16.2::GFP was also assessed as well as the participation of the DAF-16/FOXO transcription

90

factor. In order to study a potential anti-aging activity, aging markers such as the

91

autofluorescent pigment in C. elegans gut granules and the pharyngeal pumping rate were

92

analyzed. Fertility rate, body length and lifespan of the nematodes were also assessed.

93


31-33

With regard to

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Methods

96

Extraction and Plant Material

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A hydroalcoholic extract was obtained from ripe E. precatoria fruits. The fruits, with

98

origin in Codajás-AM (Brazil), were purchased in a local market in Manaus-AM (Brazil) in

99

February, during the crop period for this species. The whole fruits were submitted to

100

hydroalcoholic extraction (80 % (v/v, methanol/water) acidified with citric acid 0.15 %) and

101

then concentrated in vacuum at 37 ºC using a rotavapor. Finally, the extract was submitted to

102

a spray-drying (180 °C inlet temperature and 100 °C outlet temperature). The final product

103

was stored at room temperature (25 ºC) in amber glasses.

104

105

Characterization of Anthocyanins

106

The anthocyanin content of the extract was characterized and quantified by HPLC-

107

UV/VIS. An aliquot of the sample was diluted in solvent A (87 % water / 3 % acetonitrile /

108

10 % formic acid (v/v/v)), mixed (3 min), centrifuged (5.000 rpm, 20 min, 20 °C) and filtered

109

using Millipore membrane (0.45 µm PVDF). The internal standard (IS), delphinidin-3 5-

110

diglucoside (del-3.5-di-glc) 100 µg/ml, was added to the aliquots which were analysed in

111

duplicate. A calibration curve was established using cyanidin-3-glucoside (cy-3-glc) 0.1-10

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µg/ml. Briefly, the analyses were conducted with a Jasco HPLC-UV system equipped with a

113

Jasco PU-2080 intelligent HPLC pump, a Jasco LG-2080–02 ternary gradient unit, a Jasco

114

UV-2075 plus intelligent UV/VIS detector, a Jasco DG-2080–53 3-line-degasser, and an AS-

115

2055 plus intelligent sampler (Jasco, Gross-Umstadt, Germany). The samples were eluted

116

according to the following elution gradients with solvent A (see above) and solvent B (40 %



117

water / 50 % acetonitrile / 10 % formic acid): between 0 to 1 min, 2 % B; between 1 and 20

118

min, 2 %–14 % B; 20–40 min held at 14 % B; 40–50 min increased to 15 % B; 50–55 min

119

increased to 19 % B; 55–65 min raised to 20 % B, 65 – 65.1 min, from 20-99 % B; 65.1 – 70

120

min, constant 99 % B; 70.1–110 min re-equilibrated with 2 % solvent B. 34 The flow rate was

121

0.5 mL/min, the detection wavelength was 520 nm, and the injection volume was 20 µl at

122

room temperature.

123

124

DPPH

125

The antioxidant activity of Acai extract (AE) was assessed by measuring the decrease

126

in the absorbance of the stable free radical DPPH (2,2-diphenyl-1-picrylhydrazyl) (Sigma-

127

Aldrich GmbH, Steinheim, Germany) as described by Blois

128

microplate analysis. Briefly, 100 µl of DPPH 200 µM methanol solution were added to 100

129

µl of sample or standard. The standard, epigallocatechin gallate (EGCG) (Sigma-Aldrich

130

GmbH, Steinheim, Germany), and the sample were both diluted in methanol. The reaction

131

ran protected from light at room temperature (25 ºC) for 30 min. The absorbance was

132

measured at 517 nm in a microplate reader (Tecan Group Ltd., Männedorf, Switzerland). The

133

ability to scavenge the DPPH radicals was calculated using the following equation:

35

, adapted to a 96-wells

134

DPPH scavenging effect (%) = [(A0 –A1)/A0]×100

135

Where A0 means the absorbance of the control reaction and A1 is the absorbance in

136

the presence of AE. All measurements were performed in triplicate. The EC50 value was

137

estimated by sigmoid non-linear regression using GraphPad Prism version 6.00 for Windows

138

(GraphPad Software, La Jolla, CA, USA) adequate software and is presented in µg/ml.

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141

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SPF in vitro The sun protection factor (SPF) was determined spectrophotometrically following the 36

143

method described by Mansur as cited in Mishra.

Briefly, AE 0.01 % hydroalcoholic

144

solution (ethanol 40 %) was scanned with a Jasco V-630 UV-VIS Spectrophotometer (Jasco,

145

Gross-Umstadt, Germany) between 290-320 nm, at intervals of 5 mm using a HELL 6040-

146

UV quartz cuvette (Hellma, Müllheim, Germany). The SPF was calculated using the

147

following equation:

= . .

148

Where CF = correction factor (10), EE (λ) = erythmogenic effect of radiation with

149

wavelength λ, Abs (λ) = spectrophotometric absorbance values at wavelength λ. The values

150

of EE (λ) x I are constants previously determined by Sayre. 37

151

152

Total Phenolic Content

153

The assay was carried out according to the Folin-Ciocalteu method with minor modifications

154

to 96-well microplate analysis. Briefly, 100 µl of Folin-Ciocalteu reagent (Merck, Darmstadt,

155

Germany) were added to 20 µl of sample; 5 min later 80 µl of sodium carbonate (7.5 %

156

solution) were also added. Gallic acid was used as standard. The reaction ran in dark, at room

157

temperature (25 ºC) for 2 h. Then the absorbance was measured at 750 nm. The sample and

158

standard were dissolved in methanol. All measurements were carried out in triplicate and at

159

least three times. The phenolic content is expressed as gallic acid equivalents (GAE/mg of

160

sample).



161

162

C. elegans – Strains and Maintenance

163

C. elegans strains (N2 (wt), TK-22 (mev-1(kn1)III), TJ375 (gpIs1[hsp-16-2::GFP]),

164

CF1553 (muls84[pAD76(sod-3::GFP)]), TJ356 (zIs356 [daf-16p::daf-16a/b::GFP+rol-6]),

165

CF1038 (daf-16(mu86)I), GR1307 (daf-16(mgDf50)I), BA17 (fem-1(hc17)IV) were

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purchased from the Caenorhabditis Genetics Center (CGC, University of Minnesota,

167

Minneapolis, USA) and cultured in NGM agar medium, seeded with E. coli OP50 as food

168

source (CGC, University of Minnesota, Minneapolis, USA). The worms were incubated at

169

20°C, except when mentioned. For assays that demand liquid medium the S-medium was

170

employed which was complemented with E. coli OP50 (DO600=1.0). Synchronous

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populations were obtained by treating the gravid worms during 5 min with 5 M NaOH and

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5% NaOCl in the ratio 3:1, for lysis and decontamination. The lysate was pelleted by

173

centrifugation (1200 rpm, 1 min). The eggs were separated from the pellet by density gradient

174

using sucrose solution (60 % w/v) and water in the ratio 1:1, followed by centrifugation

175

(1200 rpm, 4 min). The upper layer, in which the eggs float, was collected and transferred to

176

a fresh tube with sterile water (1:3) then, submitted to a last centrifugation step (1200 rpm, 1

177

min) to pellet the eggs and wash out the sucrose. The eggs were allowed to hatch in M9

178

buffer. 38

179

180

Survival Assay Under Oxidative Stress

181

Synchronized wild type (N2 grown in S-medium) and transgenic worms (CF1038,

182

GR1307 grown in S-medium) at L1 larval stage were sorted into populations and treated with

183

AE for 48 h, except the control group. Each group contained 75 individuals. Thereafter, all


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the groups were exposed to the pro-oxidant juglone (5-hydroxy-1,4-naphthoquinone) (Sigma-

185

Aldrich GmbH, Steinheim, Germany) 80 µM for 24 h. Dead and live worms were counted

186

afterwards. The worms were considered dead when they did not respond to a gentle touch

187

with a platinum wire on their bodies.39 The survival rate is presented as mean of three

188

independent runs (mean ± SEM) and compared by one-way ANOVA followed by Bonferroni

189

(post-hoc) using Graphpad Prism for windows, Version 6.01.

190

191

Intracellular ROS Accumulation

192

Synchronized worms (N2 grown in S-medium) at L1 larval stage were sorted into

193

populations and treated with AE for 48 h, except the control group. After treatment, the

194

worms were washed with M9 buffer and pelleted by centrifugation. The pellet was

195

reconstituted in 1 ml of 50 µM CM-H2DCFDA solution (Fluka Chemie GmbH, Buchs,

196

Switzerland) and incubated, protected from the light, for 1 h at 20 °C. To remove an excess

197

of the dye, the worms were washed once more with M9 buffer and then mounted on a glass

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slide with a drop of 10 mM sodium azide (AppliChem GmbH, Darmstadt, Germany) for

199

paralysis. Using the BIOREVO BZ-9000 fluorescence microscope (Keyence Deutschland

200

GmbH, Neu-Isenburg, Germany) equipped with a mercury lamp, the slides were analyzed

201

(λEx 480/20 nm; λEm 510/38 nm) and photographed. Images were taken from at least 30

202

worms at constant exposure time using a 10X objective lens. The relative fluorescence of the

203

whole body was determined densitometrically using Image J version 1.48 (National Institutes

204

of Health, MD, USA) software. The results are presented as mean fluorescence intensity of

205

three independent runs (mean ± SEM) and compared by one-way ANOVA followed by

206

Bonferroni (post-hoc).

207



208

Quantification of hsp-16.2::GFP Expression

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Synchronized worms (transgenic strain TJ375 grown in S-medium) at larval stage L4

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were sorted into populations and treated with AE for 48 h, except the control group. Then all

211

the groups were exposed to 20 µM juglone for 24 h. After juglone incubation, the worms

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were analysed as described before.

213

214

Quantification of sod-3::GFP Expression

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Synchronized cultures of transgenic C. elegans strain CF1553 (L1 larvae, grown in S

216

media) carrying a sod-3::GFP fusion protein as reporter were treated with AE for 72 h, except

217

the control group, and then submitted to fluorescence microscopy as described above.

218

219

Quantification of DAF-16::GFP Expression

220

Synchronized worms (transgenic strain TJ356 grown in S-medium) at larval stage L1

221

were sorted into populations and treated with AE for 72 h, except the control group. The

222

analysis by fluorescence microcopy was carried out as described above.

223

224

Quantification of Autofluorescent Pigment in C. elegans

225

Synchronized wild-type worms (N2 grown in S-medium) were sorted into

226

populations at day 1 of adulthood and treated during 5 days with AE, except the control

227

group. The analysis by fluorescence microcopy (λEx 360/20 nm; λEm 460/38 nm) was carried

228

out as described above.

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Brood Size and Body Length

231

Synchronized N2 worms from larval stage L4 were sorted and placed one by one on

232

individual NGM agar plates. E. coli OP50 lawn was provided as food source. In the treatment

233

group the bacterial lawn was supplemented with AE. The adult worms were transferred daily

234

to fresh medium to separate them from their progeny. The eggs were counted every day for 5

235

days using a dissecting microscope.

236

compared by two-way ANOVA followed by Bonferroni (post-hoc). To measure the body

237

length, the worms (at day 1 of adulthood) were mounted on a glass slide, paralyzed with a

238

drop of 10 mM sodium azide and analyzed by bright field microscopy. Images were taken

239

from at least 30 worms using a 10X objective lens. The body length was measured using the

240

software BZ-II Analyzer (Keyence Corporation). The results are presented as body length in

241

µm (mean ± SEM) and compared by one-way ANOVA followed by Bonferroni (post-hoc).

33

The result is presented as mean brood size and

242

243

Pharyngeal Pumping Rate

244

Age synchronized worms from N2 (wt) strain, at day 1 of adulthood, were sorted into

245

populations and placed on NGM agar plates, containing E. coli OP50 lawn as a food source.

246

In the treatment group the bacterial lawn was supplemented with AE. The adult worms,

247

during the reproductive period, were transferred every second day to fresh medium to

248

separate them from their progeny. After egg laying had ceased the worms were transferred to

249

fresh medium, always on the day before the analyses of pumping activities. On day 5, 10, 12

250

and 15 of adulthood the pumping frequency of the terminal pharyngeal bulb was counted

251

using a dissection microscope. Each single worm was observed for 60 sec and the pumping

252

frequency was recorded. Each group had a minimum of 10 worms. The pumping frequency

253

was scored when the worms were crawling on the bacterial lawn. 33, 40 The result is presented



254

as pumps min-1 (mean ± SEM) and compared by two-way ANOVA followed by Bonferroni

255

(post-hoc).

256

257

258

Longevity Assay

259

Synchronized worms (N2 and TK-22, grown in S-medium) at day 1 of adulthood were

260

sorted into populations of 90 individuals each and treated with AE, except the control group.

261

During the reproductive period they were daily transferred to fresh medium to separate the

262

adults under study from their progeny, thereafter the transfer was carried out every second

263

day. The worms were counted during the transfer to fresh medium. The worms with internally

264

hatched progeny or extruded gonads were discarded from the assay. Worms that no longer

265

responded to a gentle touch with the platinum wire were scored as dead and excluded from

266

the plates. The results are presented as percentage of survival and the statistical significance

267

determined by Log-rank (Mantel-Cox) tests followed by Gehan-Breslow-Wilcoxon Test.

268

269

Results and Discussion

270

An HPLC-UV/VIS analysis revealed the presence of three anthocyanins in the

271

methanol/water extract from E. precatoria fruits, which were identified as cyanidin-3-

272

rutinoside (corresponding to 89.44 % of the anthocyanins) and traces of cyanidin-3-glucoside

273

and peonidin-3-rutinoside (Table 01). Our analysis thus agrees with literature data indicating

274

that cyanidin-3-rutinoside is the main anthocyanin of E. precatoria fruits. 5

275

The polyphenol rich AE exhibits powerful antioxidant activity in vitro. When tested in

276

the DPPH assay, AE effectively scavenged the radical (EC50= 3.8 µg AE ml-1). These values


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are in a similar range as of known antioxidants, such as EGCG or ascorbic acid (Table 02). In

278

accordance with the antioxidant activities, a high phenolic content of 205 GAE mg-1 was

279

determined by the Folin-Ciocalteu method.

280

Also in vivo data confirm the antioxidant activity of AE as it can effectively protect C.

281

elegans against oxidative stress and reduce endogenous ROS levels. The extract can attenuate

282

the deleterious effects of juglone, a secondary metabolite of Juglans regia, which can

283

increase the formation of free radicals in vivo. After 80 µM juglone exposure, the survival

284

rate of the worms pre-treated with 200 µg.ml-1 of AE was significantly higher (up to 86.34 ±

285

3.08 %) when compared to the untreated control group (38.50 ± 1.50 %), (adjusted P-value of

286

0.0007). (Figure 1a).

287

To assess the effect of AE on ROS accumulation the cell permeant reagent H2DCF-

288

DA (2’,7’- dichlorofluorescin diacetate) was employed. This compound becomes

289

deacetylated by intracellular esterases and remains inside the cells. Inside cells, H2DCF-DA is

290

subjected to oxidation in the presence of intracellular ROS. The oxidized compound emits

291

fluorescence, whose intensity correlates with intracellular ROS levels.

292

fluorescence intensities were observed among nematodes treated with AE in relation to the

293

untreated control group (Figure 1b). The treatment with 200 µg.ml-1 AE decreased by 58.1%

294

the intracellular ROS accumulation (adjusted P-value < 0.0001). This effect showed to be

295

concentration-dependent, indicating that the antioxidants have been absorbed by the worms

296

and are thus bioavailable.

41

Significant lower

297

More evidence that AE can attenuate oxidative stress was obtained by quantifying the

298

expression of heat shock proteins (HSP) in the mutant strain TJ375, which has a hsp-16.2

299

promoter fused with a GFP reporter. HSPs represent a family of proteins found in nearly all

300

living organisms that exhibits among others chaperone activity. HSP expression is induced

301

mainly by heat shock or oxidative stress.

42, 43

In C. elegans the HSP16.2, categorized as



302

small HSP, is not expressed unless the organism is facing pernicious environmental

303

conditions. These conditions can be high temperature or the presence of oxidants like juglone

304

that are directly associated with protein damage.

305

with 20 µM juglone the hsp-16.2 is expressed as seen through the increased fluorescence

306

intensity. Among the groups pre-treated with AE the fluorescence intensity is significantly

307

lower compared to the untreated control group under the same conditions (Figure 2a). Thus,

308

as lower fluorescence intensity correlates with lower expression of hsp-16.2, this data

309

indicates that AE is able to attenuate oxidative stress in C. elegans. The most pronounced

310

effect was observed at a concentration of 200 µg.ml-1 AE that decreased by 59.06 % the

311

fluorescence intensity in comparison to the untreated control group (adjusted P-value

312

0.0001). The ability to suppress the expression of hsp-16.2 under induced oxidative stress

313

was also described for a green tea extract and its main constituent EGCG. 45, 46

44

After incubation of the TJ375 mutants

314

Antioxidant balance inside the cells also requires the participation of enzymes with

315

the ability to neutralize ROS. SOD-3 is a mitochondrial enzyme with O2˙-scavenging activity.

316

SOD-3 is important for C. elegans to maintain a balance of ROS; its expression can be

317

influenced by antioxidants. 47, 48 Using the mutant strain CF1553, which has a sod-3 promoter

318

fused with a GFP reporter, a higher expression was detected among AE treated worms in

319

comparison with untreated control worms (Figure 2b). 200 µg.ml-1 AE increased by 67.65 %

320

the expression of sod-3::GFP which correlates with the measured fluorescence intensity

321

(adjusted P-value < 0.0001). EGCG was also shown to up-regulate sod-3::GFP expression. 49

322

These findings indicate that AE performs its antioxidant activity not only by

323

scavenging radicals but additionally by modulation of the expression of stress response genes

324

such as sod-3 and hsp-16.2.

325

DAF-16/FOXO is the main transcription factor involved in the regulation of stress

326

response genes. Under normal conditions, DAF-16/FOXO remains inactive in the cytosol.


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However, environmental conditions and stress or certain ligands can stimulate its

328

translocation to the nucleus, where it influences the expression of a variety of genes involved

329

in stress response, metabolism and longevity.

330

antioxidant effects via the DAF-16/FOXO pathway, survival assays were carried out with a

331

daf-16 loss-of-function mutant (CF1038) and a daf-16 null mutant (GR1307). In both strains,

332

AE failed to increase the survival rate (Figure 3), in contrast to results obtained with N2 (wt)

333

worms (Figure 1a). When AE was tested in the mutant strain TJ356, which has a daf-16 fused

334

with a GFP reporter, a higher percentage of worms exhibited a nuclear location pattern of

335

DAF-16::GFP than the untreated control group. The worms treated with 200 µg.ml-1 AE

336

showed a highly significant fraction of 64.00 ± 4 % DAF-16 nuclear localisation compared

337

with 8,5 ± 4,5 % observed among the untreated worms (adjusted P-value < 0.0001). These

338

findings strongly suggest that AE enhances stress resistance in C. elegans via the DAF-

339

16/FOXO pathway. An anthocyanin-rich extract of purple wheat was also shown to activate

340

DAF-16/FOXO in C. elegans. 48

50, 51

To assess whether AE also exerts its

341

C. elegans is a widely used model to analyze longevity as its lifespan is short, usually

342

around 15 d. Using N2 (wt) and TK-22 nematodes we have analyzed whether AE can

343

influence longevity as observed with other polyphenols.

344

performance regarding its antioxidant capacity in vivo, AE did not show any lifespan

345

prolonging effects (Table 3).

346

46, 52, 53

Despite of the good

In mammals, aging is accompanied by a decline in muscle function, accumulation of 33, 54

347

age pigments (lipofuscin) and impaired proteostasis.

These processes are also active in

348

C. elegans which can thus be used as model organism in aging studies. We employed the

349

wildtype N2 worms to study the influence of AE on aging. Regarding the autofluorescent

350

pigment, worms treated with AE showed a lower level of the autofluorescent pigment when

351

measured at the day 5 of adulthood (Figure 4a). Compared to the untreated control group, the



352

treatment with 200 µg.ml-1 AE decreased the level of the autofluorescent pigment by 51.09

353

%. Coburn55 demonstrated that the inhibition of the pathway that generates the

354

autofluorescent pigment can protect animals against stress-induced death. The capacity of

355

polyphenolic compounds to decrease the autofluorescent pigment level had already been

356

reported for EGCG, quercetin, caffeic acid and kaempferol. 45, 56, 57

357

Muscle activity was estimated by measuring the pharyngeal pumping rate at different

358

intervals of adulthood. AE treated worms showed a better performance when compared with

359

untreated control group throughout the entire period (Figure 4b). At the day 15, the

360

pharyngeal pumping rate of the worms treated with 300 µg.ml-1 AE was 173 % higher than

361

the rate observed in the untreated control group (adjusted P-value < 0.0001). This data

362

implies that a dietary restriction (DR) effect can be ruled out; hence the capacity of the

363

worms to feed on E. coli OP50 was not compromised.

364

Brood size and body length were not influenced by AE treatment (Table 4) indicating

365

that AE did not impair the fertility rate nor body development (e.g., via DR). These

366

parameters are mentioned in the literature as toxicity markers. 58, 59

367

In summary, an anthocyanin-rich extract of Euterpe precatoria (Acai) enhanced

368

oxidative stress resistance in C. elegans by promoting DAF-16/FOXO translocation, thus

369

modulating the expression of stress response genes. Worms under treatment showed lower

370

ROS accumulation as well as a higher survival rate when challenged by the pro-oxidant

371

juglone inducing oxidative stress. Our data indicate that Acai also has beneficial effect on the

372

aging process, evidenced by its capacity to ameliorate the age-related decrease in pharyngeal

373

pumping rate, a marker that correlates with the human age-related sarcopenia. The level of

374

the autofluorescent pigment was also attenuated by Acai treatment. However, a lifespan

375

prolonging effect was not noticed. Additionally, the Acai extract exhibits UV protective

376

properties, a SPF of 8 was demonstrated for a solution 0.01 %; this feature might correlate


Page 18 of 31

Page 19 of 31


377

with the high polyphenolic content of AE. These solar protective properties might support the

378

use of Acai in cosmetic formulations for hair and skin care. In conclusion, the Acai extract

379

demonstrated antioxidant and anti-aging activities in C. elegans which might support certain

380

nutraceutical claims. Further studies are needed to clarify to what extent the results can be

381

exploited in human health and wellness, including in vivo tests with more complex model

382

organisms.

383

384

Acknowledgements. HP gratefully acknowledge CNPq (National Council of Scientific and

385

Technological Development, Brazil) for the PhD scholarship.

386

387

Conflict of interest: “The authors declare no competing financial interest.”

388 389 390 391 392 393 394 395 396 397 398 399 400



401 402 403 404 405 406

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566


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567

Tabular Results

Table 01: Anthocyanin composition and content of Euterpe precatoria (Acai) Identity

[ ] µg.mg-1

Cyanidin-3-Rutinoside

0.5

Cyanidin-3-Glucoside

Traces

Peonidin-3-Rutinoside

Traces

Table 02: DPPH assay. In vitro evaluation of antioxidant effect. Sample

EC50 (µg.mg-1)

Euterpe precatoria (Acai)

3.83 ± 0.04

Vitamin C

2.12 ± 0.04

EGCG

1.03 ± 0.06



Page 26 of 31

Table 03: Effect of Euterpe precatoria (Acai) on lifespan in C. elegans Mean Lifespan (days)* Strain Genotype

Control

AE 300 µg.ml-1

BA-17

fem-1(hc17)

16.0 ± 3.5

16.0 ± 5.1

TK-22

mev-1(kn1)

15.0 ± 3.5

15.0 ± 4.7

* Mean ± SD

Table 04: Effect of Euterpe precatoria (Acai) on markers of aging and development in C. elegans Body Length*

Brood Size**

Strain

N2 (wt)

Control

AE 300 µg.ml-1

Control

AE 300 µg.ml-1

1261 ± 116

1339 ± 99

183 ± 1

160 ± 7

Results are mean ± SEM. * mean length (µm) and ** mean egg lay 568 569 570 571 572 573 574 575


Page 27 of 31


Figure 1: Euterpe precatoria extract (AE) protects against oxidative stress in wild-type C. elegans. (a) Effect of AE on stress resistance under a lethal dose of juglone. Survival rate of N2 (wild type) worms was significantly enhanced after AE treatment. (b) Quantification of intracellular ROS levels in N2 worms using DCFDA. Worms treated with AE showed lower levels of ROS compared to the control group. Data are presented as mean ± SEM (n=40, replicated 3 times). ** p< 0.01 and *** p< 0.001, compared to the untreated control by oneway ANOVA followed by Bonferroni (post-hoc).



Figure 2: Influence of Euterpe precatoria extract (AE) on the expression of stress response genes in C. elegans. (a) AE treatment decreases hsp-16.2 expression in mutant TJ375 worms [hsp-16.2::GFP(gplsI)] treated with pro-oxidant juglone. (b) AE increases sod-3 expression in mutants CF1553 [(pAD76) sod-3p::GFP + rol-6]. Data are presented as mean pixel intensity (mean ± SEM, n=40, replicated 3 times). * p < 0.05, ** p< 0.01 and *** p < 0.001 related to the control, analysed by one-way ANOVA followed by Bonferroni (post-hoc).


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576

577

Figure 3: Antioxidant effects of Euterpe precatoria extract (AE) depend on DAF-16/FOXO. (a) AE induces a significant translocation of DAF-16::GFP in mutant TJ356 worms [daf16p::daf-16a/b::GFP + rol-6]. The results are presented as percentage of worms exhibiting a DAF-16 sub-cellular localisation pattern, namely cytosolic, intermediate and nuclear. (b) no significant difference was observed in the survival rate after juglone induced oxidative stress between AE treated and untreated DAF-16 mutant worms (GR1307 [daf-16(mgDf50) I] and CF1038 [daf-16(mu86) I]). Data are presented as mean ± SEM (n=40, replicated 3 times). *** p < 0.001 compared to the untreated control by one-way ANOVA followed by Bonferroni (post-hoc).



Figure 4: Effect of Euterpe precatoria extract (AE) on aging-related markers. (a) AE attenuates the autofluorescent pigment in N2 (Wild type) worms. Autofluorescent granules were monitored under blue wavelength band. Data are presented as mean ± SEM (n=40, replicated 3 times). (b) AE influences the pharyngeal pumping rate throughout C. elegans aging process. The age-associated decline in muscle function was attenuated by AE treatment in wild-type worms. Data are presented as mean ± SEM (n=30, replicated 3 times). * p < 0.05, ** p< 0.01 and *** p

An Anthocyanin-Rich Extract of Acai (Euterpe precatoria Mart

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