Ellagitannins from Strawberries with Different Degrees of

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Ellagitannins from Strawberry with Different Degree of Polymerization Showed Different Metabolism Through Gastrointestinal Tract of Rats Joanna Milala, Monika Kosmala, El#bieta Karli#ska, Jerzy Juskiewicz, Zenon Zdunczyk, and Bartosz Fotschki J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b04120 • Publication Date (Web): 16 Nov 2017 Downloaded from http://pubs.acs.org on November 19, 2017

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Journal of Agricultural and Food Chemistry

Ellagitannins

from

Polymerization

Strawberry

Showed

with

Different

Different Metabolism

Degree

of

Through

Gastrointestinal Tract of Rats Joanna Milala,a* Monika Kosmala,a Elżbieta Karlińska,a , Jerzy Juśkiewicz,b Zenon Zduńczyk,b Bartosz Fotschkib a

Institute of Food Technology and Analysis, Lodz University of Technology, Stefanowskiego 4/10, 90-924 Lodz, Poland

b

Institute of Animal Reproduction and Food Research of the Polish Academy of Sciences, Division of Food Science, Tuwima 10, 10-748 Olsztyn, Poland

*

Corresponding

author,

(Tel:

+48

426312780;

Fax:

+48

426367488:

E-mail:

[email protected])

ACS Paragon Plus Environment

1


Page 2 of 51

1

Abstract

2

The present paper describes a comparative study of the metabolism of 1) ellagic acid, 2)

3

monomeric ellagitannins – a mixture of α- and β-bis-hexahydroxydiphenoyl-D-glucose, and

4

3) dimeric ellagitannins – mainly agrimoniin with both glucose residues being esterified with

5

hexahydroxydiphenoyl (HHDP), in rats fed polyphenol-rich diets. Their metabolites were

6

identified and quantified in selected parts of the gastrointestinal tract i.e., the stomach, small

7

intestine, and cecum on the 2nd, 4th, and 7th day of the experiment, as well as in the feces,

8

blood serum, and urine of rats. Significant differences between the metabolites of strawberry

9

ellagitannins and ellagic acid were observed in all parts of the gastrointestinal tract. Urolithin

10

A was the predominant polyphenolic metabolite of rats fed a diet supplemented with ellagic

11

acid. On the other hands, in rats fed low DP ellagitannins, the main metabolite was nasutin

12

followed by urolithin A, while ellagitannins with a higher DP led to nasutin only.

13

KEYWORDS: ellagitannins, nasutin, urolithin, rat, agrimoniin, strawberry

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15

Introduction

16

Epidemiological studies have shown that the consumption of fruits and vegetables reduces the

17

risk of lifestyle diseases such as atherosclerosis, cancer, and neurodegenerative disorders. 1,2,3

18

1

19

cholesterol, low-density lipoprotein cholesterol, and triglyceride levels4, which is largely

20

attributable to the activity of polyphenols. Special attention has been attracted by ellagitannins

21

(ETs), which are the most abundant strawberry polyphenols, along with anthocyanins and

22

flavanols 5 In the study of Juśkiewicz et al .6 strawberry ET preparations were shown to lower

23

lipemia and glycaemia.

Berry consumption beneficially modifies the lipid profile by significantly decreasing total

24

Ellagitannins can be defined as hexahydroxydifenoyl esters of carbohydrates of

25

cylitols as well as compounds derived from further oxidative transformations including

26

oligomerization processes.

27

differentiated in terms of the degree of polymerization (DP) and susceptibility to hydrolysis.

28

The elementary building blocks such as ellagic acid and HHDP may exist as free compounds

29

or may be generated as a result of hydrolysis of larger molecules. Ellagic acid is a product of

30

spontaneous lactonization within HHDP acid molecule.

9

31

agrimoniin,

two

32

hexahydroxydiphenoyl-D-glucose units linked by a C-O-C bond between two gallic acid

33

residues.7,10 Raspberries and blackberries primarily contain sanguiin H-6 and lambertianin C,

34

the monomeric units of which are linked with a sanguisorboyl group.7,11 These structural

35

differences may affect the physiological activity of polyphenols from Rosaceae fruits. 12

a

7,8

GOG-type

ETs constitute a heterogeneous group of compounds

dimer

composed

of

The main strawberry ET is α-1-O-galloyl-2,3:4,6-bis-

36

Polyphenols are known as antioxidants, but have a much wider spectrum of activity,

37

especially considering their low bioavailability as compared to endogenous antioxidants1 like

38

glutathione, alpha-lipoic acid, coenzyme Q, ferritin, uric acid, bilirubin. First of all, it must be

39

remembered that large polyphenolic molecules cannot enter the bloodstream, in contrast to


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some simple polyphenols which are absorbed directly. The majority of ETs are hydrolyzed by

41

colonic microorganisms, first into ellagic acid, and then into even smaller molecules, such as

42

urolithins. The pattern of urolithin formation seems to be as follows: after HHDP acid is

43

released form ETs and converted into poly-hydroxylated dibenzopyranone, and then

44

3,4,8,9,10-pentahydroxy-6H-dibenzo[b,d]pyran-6-one (Uro-M5). Subsequently, sequential

45

dehydroxylation leads to tetrahydroxy- (Uro-M6 and Uro-D), trihydroxy- (Uro-M7 and Uro-

46

C), dihydroxy- (Uro-A and IsoUro-A) and monohydroxy- (Uro-B) dibenzopyranones. These

47

metabolites may be absorbed into the bloodstream and eventually accumulate in the urine in

48

the form of glucuronides and sulfate conjugates; they can be also excreted in the

49

feces.13,14,15,16,17,18 A fully validated methodology for urolithin determination in terms of

50

linearity, sensitivity, precision, recovery, matrix effect, selectivity, stability in different

51

biological samples and quantification was developed by García-Villalba et al.18, even though

52

not all metabolites are commercially available.

53

Depending on the host’s species and individual characteristics, the resulting

54

metabolites may differ.19 20 The significance of polyphenolic metabolites, such as urolithins, is

55

not fully understood. It is known that they have antioxidant properties. Urolithins (especially

56

A and C) were very active towards O2 •− direct scavenging, significantly inhibited neutrophil

57

oxidative burst (stronger than ascorbic acid) 21.and/or activate phase II enzymes, which are

58

involved in antioxidant functions or detoxification (thioredoxin reductase-1 or glutathione

59

peroxidases)1, show anti-inflammatory activity, anti-estrogenic/estrogenic capacity due to

60

structural analogy to estrogens, and anticancer effects.21,22,23,24 On the other hand, non-

61

absorbable large compounds may influence the colonic microbiota with the result of

62

stimulating the activity of some strains while inhibiting others.22 In our previous study, a

63

strawberry polyphenolic extract improved the prebiotic effects of a fructooligosaccharide

64

(FOS) diet, beneficially lowering cecal digesta pH and reducing putrefactive short-chain fatty


4

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65

acid production. On the other hand, the addition of dietary FOS enhanced the metabolism of

66

the examined strawberry extracts in the cecum, thereby increasing the concentrations of

67

polyphenolic metabolites in the cecal digesta and urine.25 Our subsequent study26 showed that

68

the DP influenced the effectiveness of blood glucose reduction by polyphenolics, with

69

monomers more readily mitigating sugar-induced postprandial glycemic loads. Furthermore,

70

it was found that ETs contained in a monomeric ET-rich extract were more prone to intestinal

71

breakdown in the cecum than those present in a dimeric ET-rich extract, and absorption of

72

their metabolites could be increased by dietary FOS; together, they elicited strong

73

antibacterial activity. 27 Interestingly, in previous investigations, nasutin was identified as one

74

of ET metabolites in rats.12,27,28 This compound was previously found in the feces of some

75

wild rodents, including beavers, and termites.13,19,29

76

As molecular structure is important to the physiological function of ETs, the aim of

77

this study was to identify and quantify the metabolites of ellagic acid and strawberry

78

polyphenol preparations with monomeric (EM) and dimeric (ED) ellagitannins (Figure 1) in

79

selected parts of the gastrointestinal tract of rats, i.e., the stomach, small intestine, and cecum,

80

as well as in rat feces, blood serum, and urine.

81 82

Methods

83

Preparation of ET-rich strawberry extracts

84

ET extracts were obtained from strawberry fruit pomace, a by-product in the manufacture of

85

concentrated strawberry juice (ALPEX Co., Łęczeszyce, Poland), as described by Jurgoński

86

et al.27 In brief, fresh pomace was dried in an industrial vacuum dryer at 70±2°C and passed

87

through sieves. The seedless fraction was subjected to two-step extraction with 60% aqueous

88

solution of acetone. Next, after partial removal of the solvent via distillation, the resultant

89

solutions were transferred onto a column packed with polymeric resin (Amberlite XAD 16,


5


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90

Sigma-Aldrich, Poznan, Poland). Sugars and other water-soluble compounds present in the

91

solutions were eluted from the column with water. Then, fractions rich in monomeric and

92

dimeric ETs were desorbed with 10% and 40% aqueous solution of ethanol, respectively,

93

concentrated to approx. 15% dry matter, and lyophilized (Table 1).

94

Analytical methods

95

Basic composition. AOAC methods 30 were used to determine dry matter and ash (940.26),

96

protein (920.152), crude fat (930.09), total dietary fiber (985.29), and insoluble dietary fiber

97

(991.42).

98

Polyphenols: HPLC analysis. The concentration of ETs, ellagic acid, anthocyanins, and

99

flavonols was determined in extracts diluted with methanol (1 mg/mL) using a Knauer

100

Smartline HPLC system with a photodiode array detector (Berlin, Germany) coupled with a

101

Gemini C18 column (110 Å, 250×4.60 mm; 5 µm, Phenomenex, Torrance, USA). The

102

detailed separation and detection conditions are described elsewhere Fotschki et al.

103

following standards were used for the identification of polyphenols: ellagic acid, flavonols

104

(quercetin-3-O-glucoside, kaempferol-3-O-glucoside, quercetin, kaempferol, tiliroside),

105

pelargonidin-3-O-glucoside (all from Extrasynthese, Genay, France), p-coumaric acid

106

(Sigma-Aldrich), as well as ETs (bis-hexahydroxydiphenoyl-D-glucose and agrimoniin),

107

obtained by semi-preparative HPLC as described by Sójka et al. 10

25

. The

108

The concentration of proanthocyanidins in the extracts was determined by HPLC

109

following their breakdown in an acidic environment with excess phloroglucinol according to

110

Kennedy and Jones (2001). The obtained products were separated using a Knauer Smartline

111

chromatograph (Berlin, Germany) equipped with a UV–Vis detector (PDA 280, Knauer,

112

Berlin Germany) and a fluorescence detector (Shimadzu RF-10Axl, Kyoto, Japan) coupled

113

with a Gemini C18 column (110 Å, 250×4.60 mm; 5 µm, Phenomenex, Torrance, USA). The

114

separation conditions were as described by Kosmala et al.12 Identification was done at 280 nm


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115

using a UV–Vis detector and the following standards: (–)-epicatechin, (+)-catechin, and (–)-

116

epigallocatechin, and their respective phloroglucinol adducts. Phloroglucinol adducts were

117

previously synthetized and isolated from corresponding standard. Quantification was carried

118

out by measuring peak areas recorded by the fluorescence detector (excitation wavelength:

119

278 nm; emission wavelength: 360 nm). The breakdown products were quantified using

120

standard

121

(–)-epicatechin-phloroglucinol adduct for extender units.

curves

of

(–)-epicatechin

and

(+)-catechin

for

terminal

units

and

122 123

High Performance Liquid Chromatography – Electrospray Ionization – Mass Spectrometry

124

(HPLC-ESI-MS).

125

Ellagitannins were confirmed by HPLC-ESI-MS using a Dionex UltiMate 3000 UHPLC and

126

a Thermo Scientific Q Exactive series quadrupole ion trap mass spectrometer (Jurgoński et

127

al., 2015). A Phenomenex Luna 5 µm C18 (250 × 4.6 mm) column was used with a binary

128

gradient of 1% formic acid as mobile phase A and water/acetonitrile (1:4 v/v) as mobile phase

129

B at a flow rate of 1.0 mL/min. The gradient program was 5% of mobile phase B for the first

130

6.5 min, 5–15% B from 6.5 to 12.5 min, 15–45% B from 12.5 to 44 min, 45–75% B from 44

131

to 45 min, isocratic conditions at 75% B from 45 to 50 min, 75–5% B from 50 to 52 min, and

132

column equilibration at 5% B from 52 to 65 min. Mass spectrometry analysis was performed

133

in negative ion mode under the following conditions: capillary voltage +4 kV, sheath gas

134

pressure 75 au (arbitrary units), auxiliary gas at 17 au, and scan range from 200 to 2000 m/z.

135 136

In vivo experiments

137

Animals and diets. Experiments were conducted on 30 male Wistar rats aged approx. 4 weeks,

138

randomly assigned to one of three groups of ten rats each. The animals were maintained

139

individually in metabolic cages at a stable temperature (21–22°C), a 12 h light:12 h dark


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140

cycle, and a ventilation rate of 20 air changes per hour. The rats were used in compliance with

141

the European guidelines for the care and use of laboratory animals (EU Directive

142

2010/63/EU), and the animal protocol was approved by the Local Institutional Animal Care

143

and Use Committee (permission number 32/2012). Individual body weight and food intake

144

data were recorded. Samples of intestinal digesta, feces, urine, and blood were collected after

145

2, 4 and 7 days of experimental feeding, from 3 rats each time. All urine and feces for 24 h

146

were collected to measure how much of ET and ellagic acid metabolites was produced during

147

1 day. The rats had free access to tap water and semipurified diets, which were prepared and

148

then stored at 4°C in sealed containers until the end of the experiment. The diets were

149

modifications of a casein diet for laboratory rodents recommended by the American Institute

150

of Nutrition

151

achieve a final concentration 0.1% of ellagitannins or ellagic acid. The composition of

152

experimental diets is summarized in Table 3. All diets contained 3% FOS to mitigate any

153

potential negative effect of polyphenols on the fermentative function of the large gut

154

microbiota 32. Three individuals from each experimental group fed polyphenol-rich diets were

155

anesthetized after 2 days, another 3 after 4 days, and the rest after 7 days in order to collect

156

intestinal digesta and blood samples. Three rats fed a control diet without polyphenols were

157

sacrificed after 7 days of the experiment. Urine was collected after 1, 2, 4, and 7 days, and

158

feces were collected after 1, 4, and 7 days. Upon the termination of the experiment, the rats

159

were weighed and anesthetized with 50 mg sodium pentobarbital/kg body weight (they were

160

not deprived of feed prior to anesthesia). Following laparotomy, blood samples were collected

161

from the caudal vein and serum was prepared by solidification and low-speed centrifugation

162

(350×g, 10 min, 4°C). Serum samples were kept frozen at -70°C until assayed. Selected

163

intestinal segments (stomach, small intestine, cecum) were removed, and samples of digesta

164

were collected.

31

.Polyphenolic preparations were added to the diets in such proportions as to


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165

Free ellagic acid and ET metabolites in stomach, small intestinal, and cecal digesta and

166

feces. Extraction of free ellagic acid and ET metabolites was carried out using 100% and 70%

167

acetone. To 500 mg of digesta or feces 2 mL of 100% acetone was added, mixed, sonicated

168

for 10 min, and centrifuged (800 rad/s). The procedure was repeated twice with 1.5 mL of

169

70% acetone and the extracts were combined, evaporated to dryness under vacuum, dissolved

170

in 1 mL of 100% methanol and analyzed with HPLC. For HPLC parameters see Polyphenols:

171

HPLC analysis. Detection was performed at 360 nm. Standards used were: Ellagic acid,

172

Urolithin A, Urolithin B (Sigma Aldrich, Steinheim, Germany). Urolithin A glucuronide was

173

prepared as described by Cerdá et al. (2004).18 The raw material for the isolation of urolithin

174

A glucuronide was two liters of urine from three healthy volunteers (25−45 years old) who

175

consumed 400 g of strawberry per day, as described in Fotschki et al. (2014).Nasutin was

176

isolated from rats fed ellagitannin preparation faeces in the same manner. Identification of

177

metabolites was confirmed by HPLC-MS and compared with literature.

178

the ellagitannin metabolites was presented in Table 2.

179

Urine samples. Urine (2–4 mL) was acidified with 0.25 mL of 1 mol/L H3PO4 and was

180

applied to a 60 mg SPE Strata X-33 column conditioned according to the manufacturer’s

181

instructions (Phenomenex, Torrance, USA). The column was washed with 3 mL of water, 3

182

mL of 0.1 mol/L acetate buffer, and 6 mL of water, and dried. The analyte was eluted with

183

100% methanol (2 × 0.5 mL) and analyzed by UHPLC-MS.

184

The metabolites were determined by HPLC-ESI-MS using a Dionex UltiMate 3000 UHPLC

185

and a Thermo Scientific Q Exactive series quadrupole ion trap mass spectrometer. The

186

column was a Kinetex 2.6 µ C 18 100 Å; 150×2.1 mm (Phenomenex, Torrance, CA, USA)

187

kept at 35°C. A binary gradient of 0.1% formic acid as mobile phase A and 0.1% formic acid

188

in acetonitrile as mobile phase B was used at a flow rate of 0.5 mL/min. The gradient program

189

was 5% of mobile phase B for the first 1.44 min, 5–15% B from 1.44 to 2.98 min, 15–40% B


13,18,19,27

Recovery of

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190

from 2.98 to 10.1 min, 40–73% B from 10.1 to 11.5 min, isocratic conditions at 73% B from

191

11.55 to 12.7 min, 73–5% B from 12.7 to 13.28 min, and column equilibration at 5% B from

192

13.28 to 18 min. Mass spectrometry analysis was performed in negative ion mode under the

193

following conditions: capillary voltage +4 kV, sheath gas pressure 60 au (arbitrary units),

194

auxiliary gas 10 au, and scan range from 120 to 1200 m/z.

195

Blood serum samples. To 0.5 mL serum 1 mL 100% acetone was added, mixed, sonicated for

196

10 min, and centrifuged (900 rad/s). The procedure was repeated with 1.0 mL of 100%

197

acetone and the extracts were combined and evaporated to dryness under vacuum in a

198

ScanSpeed 40 low speed centifuge (Labogene, Denmark), lyophilized in an Alpha 1-2 LD

199

plus freeze dryer (Germany), solubilized in 0.2 mL of 100% methanol, and then analyzed by

200

UHPLC-MS (parameters as for urine).

201

Statistical analysis. The results are expressed as means and pooled SEM. Two-way analysis

202

of variance (ANOVA) was used to evaluate the effects of polyphenolic supplementation (S;

203

extracts rich in monomeric or dimeric ETs, and ellagic acid), feeding duration (D; 2, 4, 7 days

204

for gastrointestinal digesta and feces and blood samples and 1, 2, 4, 7 days for urine samples),

205

and interactions between these two factors (S × D). If analysis revealed a significant

206

interaction (P≤0.05), differences between the respective study groups were determined with

207

Duncan’s post hoc test at P≤0.05. Statistical analysis was performed using STATISTICA

208

software, version 10.0 (StatSoft Corp., Cracow, Poland).

209 210

Results

211 212

The preparations used in the experiment were characterized by a high content of polyphenols,

213

more than 95%, mostly ellagitannins. Both preparations, EM and ED, contained ellagitannins

214

typical for strawberry fruits.5,6,7,10,12 EM was characterized by a high quantity of monomeric


10

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215

ellagitannins, 67%, while ED was characterized by a high quantity of dimeric ellagitannins,

216

mostly agriimonin. Structures are presented in Figure 1. The remaining polyphenols were

217

proanthocyanidins, 27 and 11% respectively, which are difficult to separate. Both

218

preparations were deprived of other groups of polyphenols like anthocyanins or flavonols

219

(Table 1).

220

The highest concentration of ellagic acid in stomach digesta was found in rats fed a diet

221

supplemented with ellagic acid (EA treatment) with the difference being statistically

222

significant (P

Ellagitannins from Strawberries with Different Degrees of

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