# Lipid oxidation and its inhibition by apple polyphenols in static and dynamic in vitro digestion systems

Gaëtan Boléa, Pascale Goupy, Christian Ginies, Claire Dufour

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Gaëtan Boléa, Pascale Goupy, Christian Ginies, Claire Dufour. Lipid oxidation and its inhibition by apple polyphenols in static and dynamic in vitro digestion systems. 5th International Conference on Food Digestion, Apr 2017, Rennes, France. 2017. [hal-01605063](https://hal.science/hal-01605063)

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https://hal.science/hal-01605063

Submitted on 2 Jun 2020

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INRA
SCIENCE & IMPACT

# Lipid oxidation and its inhibition by apple polyphenols in
static and dynamic in vitro digestion systems

Geëtan Boléa, Pascale Goupy, Christian Ginies, Claire Dufour*

INRA, Avignon University UMR408 Safety and Quality of Plant Products, F-84914 Avignon, France.

Claire.dufour@inra.fr

UNIVERSITÉ
D'AVIGNON

## Introduction

Atherosclerosis is frequently associated with ischemic pathologies and one of the main risk factors is the Western diet. This diet is rich in ω-6 polyunsaturated fatty acids (ω-6 PUFA) which are sensitive to oxidation and lead to the formation of genotoxic and cytotoxic aldehydes. Indeed, the modification of low density lipoproteins by coupling with 4-hydroxy-2-nonenal (4-HNE), a specific lipid oxidation product of ω-6 PUFA, is a key step in the development of atherosclerosis [1,2]. By contrast, the consumption of either fruit and vegetables or flavonoids, the main class of dietary polyphenols (PP), is inversely associated with the development of coronary artery disease [3,4].

**Objectives** : This study aims at assessing lipid oxidation during the digestion of a Western type meal and the antioxidant capacity of apple polyphenols in different matrix forms. Two in vitro gastrointestinal digestion systems are compared: a static one with conditions derived from the COST Infogest [5] and the dynamic system DIDGI® developed at INRA.

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## Methodology

### Static in Vitro Digestion Model (COST Infogest)

### Dynamic in Vitro Digestion Model (INRA DIDGI®, Storm Software)

#### Gastric Phase

**Sunflower oil-in-water (10%) emulsion**
stabilized by egg yolk phospholipids in
Simulated Gastric Fluid pH 5

+ Pepsin (1000 U/ml) / CaCl₂ (0,75 mM)

± Epicatechin or apple puree / extract
(100 µM epicatechin equiv.)

+ Myoglobin (20 µM) → T0

Adjustment at pH 5 (1 M HCl)
1 h, 37 °C, 280 rpm (sampling 0, 30 60 min)

Adjustment at pH 3 (1 M HCl)
1 h, 37 °C, 280 rpm (sampling 90, 120 min)

#### Intestinal Phase

Gastric phase: 1:1 dilution with
Simulated Intestinal Fluid pH 7

+ Pancreatin (100 U trypsin/ml)

+ Bile (10 mM) / CaCl₂ (0,675 mM)

Adjustment pH 6.5 (1 M HCl)
2 h, 37 °C, 280 rpm

Sampling 0, 30, 60, 90, 120 min

**Evaluation of lipid oxidation**

Sample dilution with iPrOH then
centrifugation

✓ Conjugated dienes (Abs 234 nm)

✓ 4-hydroxy-2-nonenal (4-HNE) after
coupling with DNPH (UPLC-MS)

<figure><img src="image_4.png" /></figure>

**Composition (w/w)**

6% epicatechin
77% oligomeric
procyanidins

19% chlorogenic
acid

1.4% flavonols

2.6% phloretin
glycosides

9% sunflower oil-in-water emulsion (200 g)
stabilized by egg yolk phospholipids
or 10% emulsion (180 g) + apple puree (20 g)
+ Myoglobin

<figure><img src="image_5.png" /></figure>

**STEPS**

**Gastric step**

Pepsin (Sigma P6887) 3 g/L - 0,5 mL/min

Myoglobin (MbFe¹¹¹) 62,5 µM initial

HCl 0,1 M

beta 1

T 1/2 60 min

pH 5 → 2 over 120 min

**Duodenal Step**

Pancreatin (Sigma P7545) 4 g/L - 0,25 mL/min

Bile extract (Sigma B8631) 55g/L - 0,4 mL/min

NaHCO3 0,5 M

Volume max. 50 mL

pH 6.5

**Lipid oxidation / phenolic compounds**

Sampling in stomach and duodenum

✓ Conjugated dienes (Abs 234 nm)

✓ 4-hydroxy-2-nonenal / phenolic compounds
(UPLC/DAD/MS)

## Results & Discussion

### Static in Vitro Digestion

#### Gastric step

✓ Metmyoglobin-initiated lipid oxidation is evidenced during both steps of *in vitro* gastrointestinal digestion through the accumulation of lipid-derived conjugated dienes (CD) and 4-HNE.

<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

✓ Epicatechin and apple puree or extract totally inhibited the accumulation of CD and 4-HNE at a dietary level (100 µM epicatechin equivalents).

✓ Opposite results are observed in the duodenal step suggesting that apple products could produce compounds absorbing at 234 nm during proteolysis or lipolysis (to be checked).

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#### Duodenal step

n = 3-4, mean ± SD, p < 0,05

### Dynamic in Vitro Digestion - Gastric Step

✓ Metmyoglobin-initiated lipid oxidation is also evidenced during dynamic gastric digestion. Apple puree (1,2 mM total PP) appears to slow down the formation of CD and 4-HNE in a similar way during the first 60 min, although not statistically owing to the heterogeneity of digesta (more replicates needed).

<figure><img src="image_9.png" /><figcaption>Conjugated dienes</figcaption></figure>

<figure><img src="image_10.png" /><figcaption>4-HNE</figcaption></figure>

✓ Polyphenol bioaccessibility is found to be *ca.* total for chlorogenic acid and increasing but partial for quercetin and phloretin glycosides. Recovery of procyanidins B1 and B2 as well as epicatechin is low to null owing to binding to proteins and low solubility.

<figure><img src="image_11.png" /></figure>

$$ \text{Bioaccessibility} = \frac{\text{Content in PP in the aq. phase of the digesta}}{\text{Content in PP initially brought by apple}} $$

n = 3, mean ± SD

### CONCLUSION

MbFe¹¹¹-initiated lipid oxidation of emulsions stabilized by phospholipids has been demonstrated to occur during gastric digestion in static and dynamic systems as well as in *in vivo* in minipigs of a Western type diet [5]. Polyphenols brought by apple proved to be protective, limiting the formation of toxic lipid-oxidation products.

### References

[1] Uchida K., Free Radicals Biol. Med., 2000, 28, 1685.

[2] Ursini F. and Sevanian A., Biol. Chem., 2002, 383, 599.

[3] Dauchet L. et al., J. Nutr. 2006, 136, 2588.

[4] Arts I. C. W. and Hollman P. C. H. Am. J. Clin. Nutr. 2005, 81, 3175.

[5] Minekus M. et al., Food & Function, 2014,5, 1113.

[6] Gobert M. et al., Food & Function, 2014, 5, 2166.

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