# Deciphering key structural elements in the ability of phenolic compounds and plant extracts to alleviate secondary amine N-nitrosation and lipid oxidation in a cured meat model

Charlène Sirvins, Pascale Goupy, Aurélie Promeyrat, Christian Ginies, Claire Dufour

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## To cite this version:

Charlène Sirvins, Pascale Goupy, Aurélie Promeyrat, Christian Ginies, Claire Dufour. Deciphering key structural elements in the ability of phenolic compounds and plant extracts to alleviate secondary amine N-nitrosation and lipid oxidation in a cured meat model. Food Research International, 2025, 221, pp.117387. 10.1016/j.foodres.2025.117387. hal-05236105

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https://hal.inrae.fr/hal-05236105v1

Submitted on 2 Sep 2025

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# Deciphering key structural elements in the ability of phenolic compounds and plant extracts to alleviate secondary amine N-nitrosation and lipid oxidation in a cured meat model

Charlène Sirvins<sup>a,b</sup>, Pascale Goupy<sup>a</sup>, Aurélie Promeyrat<sup>b</sup>, Christian Ginies<sup>a</sup>, Claire Dufour<sup>a,*</sup>

## ARTICLE INFO

### Keywords:

- Flavonoid

- Hydroxycinnamic acid

- N-nitroso-N-acetyltryptophan

- Lipid oxidation

- Grape

- Green tea

- Olive

## ABSTRACT

In cured meats, nitrite provides microbiological safety, lipid oxidation control and typical sensorial properties although it is associated with N-nitrosamine formation, including cytotoxic and genotoxic ones. This work reports a new test to evaluate the antinitrosating capacity of plant extracts or phenolic compounds in an o/w emulsion, in which N-acetyltryptophan is the secondary amine target, and in processing and gastric digestion conditions (pH 5 and 2.5). Extracts from green tea, a fruit mix, grape seed or olive, evaluated at the level of 1 mM gallic acid equiv., were shown to be among the most antinitrosating extracts. Additionally, in the absence of nitrite, they markedly inhibited the metmyoglobin-initiated accumulation of triacylglycerol hydroperoxides highlighting a potent antioxidant capacity for meat preservation. The phenolic composition of the fruit mix, green tea, olive-grape mix extracts was further elucidated allowing the assessment of the antinitrosating capacity of the major phenolic constituents. Finally, this work brings out elements of molecular structure which should be helpful for plant extract selection in the aim of processing newly formulated cured and nitrite-free meat products.

## 1. Introduction

In 2015, the World Health Organization (WHO) classified processed meats as carcinogenic to human while red meats were assessed as probably carcinogenic (Bouvard et al., 2015). Since then, several factors have been investigated to tentatively unravel the underlying mechanisms for the increased colorectal cancer risk associated with the consumption of processed meat products. Most of the studies highlighted the potential role of heme iron, N-nitroso compounds, or aldehydes arising from lipid oxidation, among others (Baradat et al., 2011; Bastide et al., 2015; Keszei et al., 2013; Loh et al., 2011; Seyyedsalehi et al., 2023). In processed meats, nitrate and nitrite are commonly used as preservatives. It is well established that when added to meat, nitrite will give rise to a myriad of nitrosating species (e.g. NO<sup>+</sup>, N<sub>2</sub>O<sub>3</sub>, N<sub>2</sub>O<sub>4</sub>...) likely involved in the N-nitrosation of secondary amines. Since 1978, the WHO has raised concerns about the potential carcinogenicity of some N-nitrosamines (IARC, 1978). Recently, EFSA assessed the risk of N-nitrosamines in the food chain after reviewing the literature available for 32 N-nitrosamines. This report evidenced the carcinogenicity of 23 of them, out of which 10 have actually been detected in food with meat and

meat products being the most important contributors (EFSA, 2023). Apart from food, N-nitrosamines can also form endogenously following the concomitant ingestion of secondary amines and nitrite or nitrate. Secondary amines may originate from proteins and peptides through specific enzymatic activities while nitrite but mostly nitrate is highly brought through water and vegetable consumption. Dietary nitrate can easily be reduced to nitrite by bacterial nitrate-reductases from the commensal flora present in the gastrointestinal tract. For instance, it has been demonstrated that the microbiota from the pig cecum catalyzes the N-nitrosation of several secondary amines and amides in the presence of nitrate (Engemann et al., 2013). Ingested nitrate is also excreted in the saliva through the enterosalivary pathway and reduced to nitrite mainly by oral bacteria. In the stomach, acidic conditions are known to favor N-nitrosamine formation (Tricker, 1997). Taking this into account, the WHO concluded that nitrate or nitrite ingested under conditions that result in endogenous nitrosation is probably carcinogenic to humans (Group 2A) (Grosse et al., 2006).

Since then, solutions to limit the formation of N-nitrosamines have been sought after, especially regarding N-nitrosodimethylamine (NDMA), a volatile N-nitrosamine described as carcinogenic and

<footnote><sup>a</sup> INRAE, Avignon University, UMR408 SQPOV, F-84000 Avignon, France</footnote>

<footnote><sup>b</sup> IFIP, French Pork and Pig Institute, F-35650 Le Rheu, France</footnote>

<footnote>* Corresponding author.</footnote>

<footnote>E-mail address: claire.dufour@inrae.fr (C. Dufour).</footnote>
commonly detected in food (EFSA, 2023). Actually, polyphenol-rich plant extracts from green tea, rosemary, grape seed and other plant sources have already been described to reduce the amount of NDMA and some other volatile N-nitrosamines (VNA) in processed meat products including dry-cured bacon and sausages (Li et al., 2013; Wang et al., 2015). Additionally, processed meats from the Belgian and Danish markets were found to contain 100-times higher amounts of non-volatile N-nitrosamines (NVNA) compared to volatile ones (Herrmann et al., 2015). Process- and digestion-induced decarboxylation of NVNA such as N-nitrososarcosine and N-nitrosoproline could also generate the more toxic VNA counterparts, i.e. NDMA and N-nitrosopyrrolidine (EFSA, 2017).

Therefore, this work aimed at developing an easy-to-handle test to probe the antinitrosating capacity of several plant extracts and phenolic compounds from various classes. An o/w emulsion system was used to take into account lipids in cured and cooked ham. In meat proteins, three aminoacids possess a secondary amine function (histidine, proline and tryptophan). N-acetyltryptophan, presenting a chromophore and retaining the amide bond of covalently linked aminoacids in proteins, was selected as the secondary amine target and its N-nitrosation evaluated in the additional presence of meat heme and non-heme irons. Next, the determination of the phenolic composition for the most efficient plant extracts allowed to further compare the antinitrosating capacity of epigallocatechin gallate, epicatechin, chlorogenic acid, rutin, and hydroxytyrosol. Finally, the capacity of these commercially available plant extracts to inhibit lipid oxidation was also investigated to get a complete overview of their ability to manage the decrease and replacement of nitrite in cured meat products.

## 2. Materials and methods

### 2.1. Material

**Chemicals.** N-acetyl-DL-tryptophan (≥ 98 %), horse heart myoglobin (M1882-type II), iron (II) sulfate heptahydrate (≥ 99 %), ascorbic acid (≥ 99 %), (-)-epicatechin (96 %), rutin (96 %), naringin (97 %), chlorogenic acid (96 %), caffeic acid (99 %), gallic acid (95 %), egg yolk phospholipids, soy phospholipids, 13(S)-hydroxyoctadec-9Z,11E-dienoic acid (13(S)-HODE) and the Folin-Ciocalteu reagent were purchased from Sigma-Aldrich (Saint-Quentin-Fallavier, France). Hydroxytyrosol (≥ 98 %), (-)-epicatechin gallate (ECg ≥ 98 %), (-)-epigallocatechin (EGC ≥ 98 %), and (-)-epigallocatechin-3-O-gallate (EGCg ≥ 98 %) were from Extrasynthèse (Genay, France). Absolute ethanol and sodium nitrite (≥ 99 %) were from VWR (Fontenay-sous-Bois, France). HPLC-MS grade acetonitrile and formic acid were from Carlo Erba Reagents (Val de Reuil, France). Ultrapure water (resistivity 18.2 MΩ·cm⁻¹ at 25 °C) was obtained with a Millipak Express 40 filter (Merck, Darmstadt, Germany).

**Oil.** Sunflower oil was from food store Leclerc (ECO+ brand) and its amount in α-tocopherol was estimated to be 540 ± 12 ppm using UHPLC-DAD. Aliquots (50 mL) were kept frozen at -20 °C under nitrogen until use.

**Plant extracts.** Eleven commercially available and water-soluble plant extracts were obtained from various companies specialized in food applications. Extracts were furnished for free under a material transfer agreement between each company, INRAE and IFIP. Plant origin, plant part and type of product (extract or dehydrated juice) are described in Table 1.

### 2.2. Methods

#### 2.2.1. Total antioxidant capacity and total polyphenol content of plant extracts

The Total Antioxidant Capacity (TAC) of plant extracts was determined by the Folin-Ciocalteu spectrophotometric method adapted to 96-well microplates (Georgé et al., 2005; Singleton & Rossi, 1965). Briefly,

<caption>**Table 1**</caption>

Total Antioxidant Capacity (TAC), Total Phenolic Content (TPC) and TPC/TAC ratio for the selected plant extracts (PE). Mean ± SD (n = 3).

<table><thead><tr><th>Plant matrix (form)</th><th>Total Antioxidant Capacity (TAC)<br>mmol GAE g<sup>-1</sup> PE</th><th>Total Phenolic Content (TPC)<br>mmol GAE g<sup>-1</sup> PE</th><th>TPC<br>TAC<br>%</th></tr></thead><tbody><tr><td>Green tea - leaves<br>(extract)</td><td>4.88 ± 0.29</td><td>4.83 ± 0.35</td><td>99</td></tr><tr><td>Olive - fruits + Grape -<br>seeds (extract)</td><td>4.09 ± 0.35</td><td>3.17 ± 0.30</td><td>78</td></tr><tr><td>Pomegranate - skin<br>(extract)</td><td>3.76 ± 0.06</td><td>3.70 ± 0.06</td><td>98</td></tr><tr><td>Grape - seeds (extract)</td><td>3.49 ± 0.20</td><td>3.20 ± 0.22</td><td>91</td></tr><tr><td>Grape - fruits (extract)</td><td>3.46 ± 0.23</td><td>2.83 ± 0.30</td><td>82</td></tr><tr><td>Fruit mix - fruits (extract)</td><td>3.32 ± 0.19</td><td>3.18 ± 0.21</td><td>96</td></tr><tr><td>Rosemary - leaves<br>(aqueous extract)</td><td>1.87 ± 0.08</td><td>1.65 ± 0.06</td><td>88</td></tr><tr><td>Acerola - fruits<br>(dehydrated juice)</td><td>1.85 ± 0.10</td><td>0.588 ± 0.089</td><td>32</td></tr><tr><td>Olive - fruits (extract)</td><td>0.892 ± 0.035</td><td>0.615 ± 0.032</td><td>69</td></tr><tr><td>Fruit & veg mix<br>(dehydrated juice)</td><td>0.389 ± 0.029</td><td>0.283 ± 0.030</td><td>72</td></tr><tr><td>Broccoli (extract)</td><td>0.044 ± 0.001</td><td>0.010 ± 0.002</td><td>25</td></tr></tbody></table>

to each well were added successively 50 µL of extract solubilized in H₂O (0.1–1 mg mL⁻¹) or calibration solution (10 to 100 µg gallic acid mL⁻¹), 125 µL of 0.2 N Folin-Ciocalteu reagent and 100 µL of 75 g L⁻¹ Na₂CO₃. The plate was then incubated in a spectrophotometer Xenius (SAFAS, Monaco) at room temperature and the absorbance at 730 nm was read after 90 min of reaction. Results were expressed in mmoles of gallic acid equivalent per gram of plant extract (mmol GA eq g⁻¹ PE) using the calibration curve y = 2.359 × 10³ x + 0.011 (R² = 0.999) obtained with 7 solutions of gallic acid varying from 0.059 to 0.59 mM (n = 4).

To distinguish the antioxidant capacity due to phenolic compounds from that from non-phenolic compounds, 100 mg of polyvinylpolypyrrolidone (PVPP) was added to 1 mL of plant extract in water (1 mg mL⁻¹). After 10 min of incubation at room temperature under stirring, the mixture was centrifuged (4 min, 16200 g, 15 °C) and 50 µL of the aqueous phase was transferred to the 96-well microplate and mixed with the Folin-Ciocalteu reagent and the Na₂CO₃ solution as described above. The concentration difference between the native sample and that treated with PVPP represents the Total Polyphenolic Content (TPC) of the extract. Each plant extract was analyzed in triplicate.

#### 2.2.2. Antinitrosating capacity of plant extracts and phenolic compounds in a cured and cooked meat model

Cooked ham was modeled by a 5 % sunflower oil-in-water emulsion (lipid rate as in the (CIQUAL food composition table, 2020)) containing 30 µM free iron and 50 µM heme iron as in cured and/or cooked pork meat (Bou et al., 2024; Lombardi-Boccia et al., 2002; Min et al., 2008). The level in sodium nitrite was 120 ppm, the upper rate allowed by the French Code of Practice in 2020. The content in tryptophan was found to be 1.4 g per 100 g pork proteins (Mahan & Shields, 1998). Taking a rate of proteins of 20.8 % for cured and cooked ham (CIQUAL food composition table, 2020), the content in Trp can be calculated to be 14.0 mmol kg⁻¹ ham. However, due to solubility limits, the level in AcTrp was fixed at 3 mM. The N-nitrosation of AcTrp was evaluated at pH 5 to simulate meat product formulation and the early stage of gastric digestion as well as at pH 2.5 to take into account the final stage of gastric digestion. The corresponding buffers were obtained by mixing adequate volumes of 20 mM aqueous citric acid and 40 mM aq. disodium hydrogen phosphate. Emulsions were prepared by adapting the protocol described by Tarone et al. (2021). In a 60 mL short-necked glass bottle, 53 mg of soy phospholipids were dispersed in 38 g of 3.3 mM AcTrp in buffer at either pH 2.5 or pH 5 using a rotor stator homogenizer (Polytron PT 2500 E, Kinematica) at 24000 rpm for 1.5 min. Then, 2 g of sunflower oil were added to the mixture and both aqueous and lipid phases were
homogenized at 24000 rpm for 2 min. The emulsion was next sonicated on ice for 4 periods of 1 min with rest periods of 30 s and an amplitude of 60 % (Q700 Misonix, QSonica, 20 kHz). Horse heart myoglobin (35.2 mg) was added as powder into the emulsion which was placed under magnetic stirring (350 rpm) until total dissolution (ca. 30 min). Then, 50 µL of 25 mM FeSO₄ in 20 mM aq. citric acid were added. To test the antinitrosating capacity of pure molecules, 1 mL of a 41.74 mM solution in phenolic compounds or ascorbic acid was introduced into the medium. Chlorogenic acid, caffeic acid, (-)-epicatechin and hydroxytyrosol were prepared in ethanol while (-)-epallocatechin-3-O-gallate (EGCg), rutin, naringin and ascorbic acid were dissolved in DMSO/ ethanol (1:9). Plant extracts were tested at the final level of 1 mM GAE, as evaluated in the TAC test, and added directly as powders into the emulsion in amounts reported in Table S5. To keep with the same total volume, 1 mL of water was added. As ethanol and DMSO/EtOH (1:9) exhibited a slight similar antinitrosating capacity in this model, control conditions were obtained by adding either 1 mL of ethanol for pure molecules or 1 mL of H₂O for plant extracts. For all the emulsions, 1 mL was sampled at T0 prior to the addition of 500 µL of a 135 mM NaNO₂ solution in the adapted buffer to initiate AcTrp N-nitrosation. Final concentrations were 50 µM in myoglobin, 30 µM in FeSO₄, 3 mM in AcTrp, 1.7 mM in NaNO₂, and either 1 mM in phenolic compounds or 1 mM in ascorbic acid or 1 mM GA equiv. in plant extract. The reaction medium was incubated at 37 °C under magnetic stirring (350 rpm) and 1 mL was sampled after 30 min and 60 min of reaction. Samples were centrifuged for 10 min (16,200 g, 4 °C) and supernatants were injected directly into UHPLC-DAD. A more comprehensive extract screening was initiated at pH 5 with 23 extracts (n = 2). The eleven most active extracts were further evaluated at pH 2.5 (n = 2). Pure phenolic compounds were evaluated at least in triplicates.

### 2.2.3. Analysis of AcTrp N-Nitrosation by UHPLC-DAD

The analysis of AcTrp and N-nitroso-N-acetyltryptophan (NO-AcTrp) was conducted as described in Sirvins et al. (2024). Briefly, a Waters Acquity UPLC chromatograph (Milford, MA) coupled to an UV-vis diode-array detector was used to carry out the separation using an Acquity BEH C18 column (100 mm × 2.4 mm i.d., 1.7 µm; Waters) at 35 °C. A binary solvent system with water/formic acid (99.95/0.05, v/v, solvent A) and acetonitrile (solvent B) constituted the mobile phase. Flow rate was 0.4 mL min⁻¹ and injection volume was 4 µL. The elution gradient (Table S1) separates AcTrp (Rt 15.8 min) from NO-AcTrp (Rt 20.9 min). The antinitrosating capacity of plant extracts and phenolic compounds was expressed as the inhibition rate and calculated as follows: (relative extent of N-nitrosation in control – relative extent of N-nitrosation in test condition) / relative extent of N-nitrosation in control. The relative extent of N-nitrosation at a given time was defined as the ratio between the areas at 280 nm for the peaks of NO-AcTrp and AcTrp. Exact extents of AcTrp N-nitrosation are not required for this calculation but can be obtained after calibrations with AcTrp and a synthesized standard of NO-AcTrp as in Sirvins et al. (2024).

### 2.2.4. Phenolic profile of the selected plant extracts by UHPLC-DAD-MSn

The identification and quantification of the major phenolic compounds in the most efficient plant extracts were performed by UHPLC-DAD-MSn using an Acquity UPLC system (Waters Corp., Milford, MA, USA) coupled with an UV-Vis diode array detector and a Bruker Daltonics HCT Ultra Ion Trap MS equipped with an electrospray ion source. Separations were carried out in the same conditions as for AcTrp and NO-AcTrp for the Green tea extract and with a slightly modified gradient for the Fruit mix and Olive and grape extracts (Tables S1 and S2). Negative electrospray ionization mode was used for mass detection for m/z ranging from 100 to 1000. Conditions were as follows: capillary voltage of ±3 kV, nitrogen flow rate at 9 L min⁻¹, desolvation temperature at 365 °C and nebulization pressure at 50 psi. Acquisition and data processing were performed with Compass and DataAnalysis softwares from Bruker (Bruker Daltonics, Bremen, Germany). Chlorogenic acid

was quantified at 325 nm while gallic acid and hydroxytyrosol were quantified at 280 nm with respective standards. Flavonols were quantified at 360 nm using isoquercitrin as standard. Flavanols, which were mostly (epi)catechin derivatives, were quantified at 280 nm, using the most adequate standard between (-)-epicatechin, (-)-epallocatechin, (-)-epallocatechin-3-O-gallate and (-)-epicatechin-3-O-gallate.

### 2.2.5. Inhibition of the metmyoglobin-initiated lipid oxidation by plant extracts

The inhibition of lipid oxidation by plant extracts was evaluated in a 12.5 % oil-in-water emulsion devoid of nitrite and free iron. Emulsions were prepared according to Tarone et al. (2021). Briefly, 100 mg of egg yolk L-α-phosphatidylcholine were dispersed into 35 mL of a pH 5 buffer, obtained by mixing aq. citric acid (20 mM) and aq. disodium hydrogen phosphate (40 mM), using a rotor stator homogenizer (Polytron PT 2500 E, Kinematica) at 24000 rpm for 2 min. Five g of sunflower oil were added and both aqueous and lipid phases were homogenized and sonicated as described above for the cured and cooked meat model. Then, 12 mL of this emulsion were transferred into a 25 mL beaker, followed by the addition of 500 µL of an aq. solution of plant extract at 7.5 mM GA equiv. and then 2.5 mL of 300 µM myoglobin prepared in the pH 5 buffer. Final concentrations are 10 % in lipids, 50 µM in myoglobin and 250 µM GAE in plant extracts. The medium was incubated at 37 °C under magnetic stirring (350 rpm). Every hour, 200 µL of emulsion were sampled and diluted in 1600 µL of isopropanol before centrifugation (10 min, 16200 g, 15 °C) and injection of the supernatant in UHPLC-DAD-MSn. The identification and quantification of triacylglycerols and their hydroperoxydes were carried out using a Waters Acquity UPLC chromatograph (Milford, MA) coupled to an UV-vis diode-array detector and a Bruker Daltonics HCT Ultra Ion Trap MS equipped with an electrospray ion source. The separation was conducted using an HSST3 C18 column (50 mm × 2.1 mm i.d., 1.8 µm; Waters) at 40 °C. The mobile phase was a binary solvent system with acetonitrile/10 mM aq. ammonium formate (80/20, v/v, solvent A) and isopropanol (solvent B). The flow rate was 0.4 mL min⁻¹ and the injection volume was 2 µL. Mass detection was conducted in the alternate mode for negative and positive electrospray ionizations from m/z 250 to 1200. Other MS conditions were as above. The elution gradient (Table S3) separates native triacylglycerols (LLL, LLO, LOO) from their mono- and dihydroperoxide forms (LLL-OOH, LLO-OOH, LOO-OOH, LLL-diOOH, LLO-diOOH, LOO-diOOH) whose structures has been assessed by MS as detailed in Table S4. Hydroperoxides were quantified using 6-point calibration curves at 234 nm with 13(S)-HODE. The concentration of the initial 13(S)-HODE in EtOH was evaluated by UV spectroscopy at 234 nm using an epsilon of 27,000 M⁻¹ cm⁻¹. Lipid oxidation was expressed as the sum of the six hydroperoxides above (TAGox). Each plant extract was analyzed in triplicate.

### 2.3. Statistics

Results are expressed as mean ± standard deviation (SD) on at least three replicates except for the antinitrosating capacity of plant extracts that has been evaluated in duplicate. One way analysis of variance (ANOVA) was performed to test the effect of the structure of phenolic compounds at each pH (antinitrosating test). The Tukey-Kramer post-hoc test was applied to identify groups among phenolic compounds (*p* < 0.05). Differences in TAGox levels were compared using a repeated measures ANOVA and a pair-wise comparison with Tukey's HSD method at each time (*p* < 0.05). Analyses were performed with XLStat software (Addinsoft, Paris, France).

## 3. Results

### 3.1. Inhibition by plant extracts of N-acetyltryptophan N-nitrosation in a cured and cooked meat model

The eleven plant extracts selected for this study presented a high
variability in the nature and contents of their bioactive compounds owing to different commercial forms (i.e., extracts, dried juices, with or without carriers, etc). One needs to standardize them using a common and relevant parameter before the assessment of their antinitrosating and antioxidant capacities. The Total Antioxidant Capacity (TAC) and the Total Phenolic Content (TPC) were thus evaluated to differentiate total reducing substances from total phenolic compounds. Both can be obtained by differential spectrophotometry using the Folin-Ciocalteu method, respectively in the absence and presence of PVPP (Table 1). As a matter of fact, *Broccoli* displayed a TAC per weight of dry matter that is 100-fold lower compared to that of *Green tea*. Additionally, the ratio between TPC and TAC indicates that phenolic compounds contribute for 25 % to the TAC of *Broccoli* and 99 % to that of *Green tea*. In this study, plant extracts were further evaluated for their antinitrosating capacity at equal TAC to take into consideration their large molecular diversity. All the plant extracts limited *N*-AcTrp *N*-nitrosation at pH 5 although in varying extents, while only part of them exhibited this ability at pH 2.5 (Fig. 1 and Table S5). As a matter of fact, the *Broccoli*, *Acerola* and *Fruit & Veg mix* extracts showed a markedly high antinitrosating capacity at pH 5, inhibiting between 56 and 82 % the formation of *NO*-AcTrp after 30 min of reaction. By contrast, they proved to have no effect at pH 2.5. It is worth noting that phenolic compounds explain less than 32 % of the antioxidant capacity of the *Broccoli* and *Acerola* extracts. As a matter of fact, broccoli and acerola have been described as excellent vitamin C providers with ascorbic acid ranging between 13 and 110 mg per 100 g of fresh broccoli florets and between 425 and 2625 mg per 100 g of fresh acerola pulp (Farinelli et al., 2021; Koh et al., 2009). Zhang and Hamauzu (2004) additionally estimated that the broccoli content in ascorbic acid was three-times higher compared to the TPC (respectively 103 and 34.5 mg per 100 g of fresh florets) while Koh et al. (2009) reported similar ascorbic acid content and TPC (Koh et al., 2009; Zhang & Hamauzu, 2004; Koh et al., 2009). Ascorbic acid being well-known as a strong reducing agent, most of the antioxidant capacities of the *Broccoli* and *Acerola* extracts must largely be due to this non-phenolic compound.

On the contrary, for most of the other plant extracts tested, phenolic compounds represented more than 69 % of the TAC. The *Green tea*, *Fruit mix*, *Olive and Grape*, and *Olive* extracts exerted high and rather similar

*N*-nitrosation inhibition at pH 5 (41–49 %) while being slightly less active at pH 2.5 (21–44 %). They also appear rather more efficient than the *Grape seeds* extract at pH 5 although the latter presented a TPC/TAC ratio of 92 %. This may suggest that phenolic compounds typical of green tea, grape seed and olive fruit play a major role in the antinitrosating capacities of these extracts. Although highly rich in phenolic compounds (TPC/TAC = 82 %), the *Grape fruits* extract largely differs in its antinitrosating capacity from the *Grape seeds* extract in particular at pH 5. As to the *Pomegranate* extract, it presented a moderate antinitrosating capacity, being twice less efficient than the best extracts, while presenting one of the highest TPC/TAC ratios (98 %). Free phenolic compounds in pomegranate skin are mainly ellagitannins, in the form of hexahydroxydiphenic acid and gallic acid derivatives, followed by flavonols (Ambigaipalan et al., 2016). Surprisingly, the *Rosemary* extract, which also exhibited a high TPC/TAC ratio (88 %), proved to be markedly efficient at pH 5 while being strongly less antinitrosating at pH 2.5. Rosmarinic acid is usually the most abundant phenolic compound in rosemary aqueous extracts (Pizani et al., 2024). Regarding the *Fruit & veg mix* extract, it is a mixed extract containing among others rosemary and acerola. It is thus not surprising that its behavior is related to that of both the *Acerola* and *Rosemary* extracts.

In a general manner, the dual efficiency of some plant extracts at pH 5 and 2.5 indicates that they may interfere with the formation of *N*-nitrosamines in cured meat processing but also during gastric digestion. Our results are supported by scarce data in in vitro systems and real cured matrices. As a matter of fact, green tea and its molecular constituents were found to inhibit the formation of volatile *N*-nitrosamines such as NDMA or NMOR at pH ranging between 2 and 4 (Masuda et al., 2006; Tanaka et al., 1998). Furthermore, green tea and grape seed extracts decreased final levels in NDMA and NPYR in dry-cured bacon and sausages (Li et al., 2013; Wang et al., 2015). Additionally, green tea consumption has been associated with a decrease of the urinary excretion of NDMA in humans (Choi et al., 2002; Vermeer et al., 1999). Grape seed and green tea are established sources of flavonols which may also be found as oligomers in the case of grape seed (Labarbe et al., 1999; Zheng et al., 2018). A deeper insight into the phenolic composition of the most active extracts is thus required to highlight the main structural features underlying the antinitrosating capacity.

<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Inhibition by selected plant extracts (PE, 1 mM GA equiv.) of the *N*-nitrosation of *N*-acetyltryptophan (3 mM) in the presence of NaNO₂ (1.7 mM), myoglobin (50 µM) and FeSO₄ (30 µM) in a 5 % o/w emulsion at pH 5 and 2.5 after 30 min of reaction at 37 °C. Mean ± SD (n = 2).</figcaption></figure>
## 3.2. Phenolic composition of the three most antinitrosating plant extracts

The major phenolic compounds of the *Green tea*, *Fruit mix* and *Olive and grape* extracts were identified (Fig. S1, Tables S6-S8) and quantified by UHPLC-DAD-MS<sup>n</sup> (Table 2). Regarding *Green tea*, most of the phenolic compounds were flavanols. (–)-Epigallocatechin-3-O-gallate (EGCg) was the main compound representing more than half of the flavanol pool with 487 mg g<sup>-1</sup> extract while (–)-epicatechin-3-O-gallate (81 mg g<sup>-1</sup>), (–)-epicatechin (74 mg g<sup>-1</sup>), and (–)-epigallocatechin (61 mg g<sup>-1</sup>) were present in 6- to 8-fold lower amounts. This composition and the flavanol distribution is well supported by literature (Zheng et al., 2018). The sum of phenolic compounds, as assessed by UHPLC, amounted to 758 mg g<sup>-1</sup> extract, in total agreement with the TPC evaluated at 822 mg g<sup>-1</sup> extract.

For the *Fruit mix* extract, (+)-catechin and (–)-epicatechin were the most important contributors with 103 mg g<sup>-1</sup> and 77 mg g<sup>-1</sup> extract. Several B-type procyanidin dimers and trimers were quantified. Various minor compounds were not quantitatively assessed, possibly explaining the discrepancy between the sum of phenolic compounds (405 mg g<sup>-1</sup> extract) and TPC (541 mg g<sup>-1</sup> extract). Chlorogenic acid was also present although in a 10-times lower amount compared to (+)-catechin. Additionally, anthocyanins were identified in the positive ESI mode and found to contribute for 8 mg g<sup>-1</sup> extract (in malvidin-3-O-glucoside equivalent, data not shown). Finally, gallic acid (5.5 mg g<sup>-1</sup> extract) and flavonols (2.6 mg g<sup>-1</sup> extract), namely quercetin hexoside and rutin, contributed to this fruit mix.

<caption>**Table 2**</caption>

Contents in the major phenolic compounds identified in the *Green tea*, *Fruit mix* and *Olive and grape* extracts by UHPLC-DAD-MS<sup>n</sup>.

<table><thead><tr><th></th><th>Compound</th><th>mg g<sup>-1</sup> PE<sup>a</sup></th></tr></thead><tbody><tr><td rowspan="12"><strong>Green tea</strong></td><td>Gallic acid</td><td>8.3 ± 0.7</td></tr><tr><td>(–)-Epigallocatechin</td><td>60.5 ± 5.4</td></tr><tr><td>(+)-Catechin</td><td>17.1 ± 0.7</td></tr><tr><td>(–)-Epicatechin</td><td>73.9 ± 1.8</td></tr><tr><td>(–)-Epigallocatechin-3-gallate</td><td>487 ± 13</td></tr><tr><td>Gallocatechin gallate</td><td>16.1 ± 0.3</td></tr><tr><td>Trigalloyl hexoside</td><td>3.7 ± 0.4</td></tr><tr><td>Methyl-epigallocatechin gallate</td><td>7.0 ± 0.4</td></tr><tr><td>(–)-Epicatechin-3-gallate</td><td>80.9 ± 1.8</td></tr><tr><td>Kaempferol hexoside</td><td>2.3 ± 0.1</td></tr><tr><td>(Epi)afzelechin-3-gallate</td><td>1.35 ± 0.02</td></tr><tr><td><strong>Sum of phenolic compounds</strong></td><td><strong>758 ± 22</strong></td></tr><tr><td rowspan="12"><strong>Fruit mix</strong></td><td>Gallic acid</td><td>5.5 ± 0.7</td></tr><tr><td>B-type procyanidin dimer</td><td>67.2 ± 6.9</td></tr><tr><td>(+)-Catechin</td><td>103 ± 14</td></tr><tr><td>Chlorogenic acid</td><td>10.8 ± 1.4</td></tr><tr><td>B-type procyanidin dimer</td><td>35.1 ± 3.5</td></tr><tr><td>(–)-Epicatechin</td><td>77.2 ± 6.8</td></tr><tr><td>B-type procyanidin trimer</td><td>26.5 ± 5.3</td></tr><tr><td>Mix of procyanidin dimers<sup>b</sup></td><td>73.7 ± 6.2</td></tr><tr><td>(–)-Epicatechin-3-gallate</td><td>3.7 ± 0.5</td></tr><tr><td>Quercetin hexoside</td><td>1.7 ± 0.4</td></tr><tr><td>Rutin</td><td>0.9 ± 0.2</td></tr><tr><td><strong>Sum of phenolic compounds</strong></td><td><strong>405 ± 42</strong></td></tr><tr><td rowspan="8"><strong>Olive and grape</strong></td><td>Gallic acid</td><td>1.6 ± 0.1</td></tr><tr><td>Hydroxytyrosol</td><td>41.5 ± 0.6</td></tr><tr><td>B-type procyanidin dimer</td><td>35.0 ± 1.8</td></tr><tr><td>(+)-Catechin</td><td>12.3 ± 0.9</td></tr><tr><td>Two B-type procyanidin trimers</td><td>30.2 ± 1.4</td></tr><tr><td>B-type procyanidin dimer</td><td>33.9 ± 1.0</td></tr><tr><td>(–)-Epicatechin</td><td>12.2 ± 0.1</td></tr><tr><td>B-type procyanidin trimer</td><td>25.8 ± 0.8</td></tr><tr><td><strong>Sum of phenolic compounds</strong></td><td><strong>195 ± 4</strong></td></tr></tbody></table>

Regarding the *Olive and grape* extract, the phenolic profile was first typical of olive with hydroxytyrosol as the major compound (41.5 mg g<sup>-1</sup> extract). Other abundant compounds were mostly B-type procyanidin dimers and trimers, (+)-catechin, and (–)-epicatechin which are typical of grape. The large discrepancy between the sum of phenolic compounds (195 mg g<sup>-1</sup> extract) and TPC (540 mg g<sup>-1</sup> extract) can be assessed to the presence of a diversity of unquantified flavanol oligomers with higher degrees of polymerization. These compounds appeared as a hump at the end of the chromatogram at 280 nm (Fig. S1).

## 3.3. Inhibition of the N-nitrosation of N-acetyltryptophan by pure phenolic compounds

The major phenolic compounds identified in the extracts, e.g. (–)-epicatechin, EGCg, hydroxytyrosol, rutin, and chlorogenic acid, or presenting complementary structural features, e.g. naringin and caffeic acid, were compared to ascorbate for their ability to inhibit N-AcTrp N-nitrosation in the same emulsion system as for plant extracts, at both pH 5 and 2.5 (Fig. 2). Ascorbic acid was found to decrease the formation of NO-AcTrp by 81 % after 30 min at pH 5 while being totally inefficient in more acidic conditions. Interestingly, pure ascorbic acid behaved similarly to the ascorbic-rich *Broccoli* and *Acerola* extracts that were tested at similar concentrations (1 mM GAE). In aqueous conditions, the antinitrosating efficiency of ascorbic acid at pH 5 was found to be time-limited, the formation of NO-AcTrp probably being delayed until ascorbic acid was totally consumed (within approx. 180 min) (Sirvins et al., 2024). Ascorbic acid (E300), but also sodium and calcium ascorbates (E301 and E302), erythorbic acid (E315) or sodium erythorbate (E316) that are traditionally used in cured meat production, are likely able to manage N-nitrosamine formation during processing and storage. Indeed, most of cured meats exhibit a pH between 4 and 6. Nevertheless, this result also suggests the inefficiency of ascorbic acid to limit N-nitrosation in the last stage of the gastric digestion.

By contrast, all the selected phenolic compounds, except naringin, exhibited a rather different behavior as they proved to be efficient at both pH. The extent of their antinitrosating activity appears to depend on both the phenolic structure and pH. Interestingly, (–)-epicatechin, EGCg, and caffeic acid were the three most efficient phenolic compounds at both pH. At the level of 1 mM, caffeic acid reduced N-AcTrp N-nitrosation by 70 % at pH 5 and 92 % at pH 2.5 after 30 min. As to the two flavanols, EGCg and (–)-epicatechin, they exhibited very similar inhibition rates of 57 % and 52 % at pH 5 and 94 % and 89 % at pH 2.5, respectively. This was rather unexpected as they markedly differ in their B-ring (1,2,3-trihydroxyphenyl moiety for EGCg and 1,2-dihydroxyphenyl moiety for (–)-epicatechin) with EGCg being further acylated by a galloyl group at the 3-OH. However, they were both similarly consumed while exerting their antinitrosating role (Fig. 3). By contrast, half of caffeic acid was recovered at pH 2.5 suggesting different mechanisms of action. Additionally, a less active group encompasses hydroxytyrosol, rutin and chlorogenic acid which differs statistically from the first group (Table S9). Inhibition rates were of 44 %, 40 % and 33 % at pH 5, while being of 21 %, 26 % and 27 % at pH 2.5, respectively. Although hydroxytyrosol, rutin and chlorogenic acid were less efficient than EGCg and epicatechin at both pH, they were also rapidly consumed at pH 2.5 suggesting degradation pathways not interfering with AcTrp N-nitrosation. Last, naringin, a flavanone but also the only monophenol in this series, was unable to prevent the formation of NO-AcTrp. This highlights the key role played by the *ortho*-diphenol moiety in the antinitrosating capacity of flavonoids as described previously (Hirota & Takahama, 2014; Lee et al., 2006; Sirvins et al., 2024).

In general, the inhibition rates were found relatively similar or slightly increasing between 30 and 60 min at pH 5. As a matter of fact, kinetic studies conducted in related aqueous conditions showed that NO-AcTrp remained stable once formed at this pH when, in the presence of (–)-epicatechin, rutin or quercetin, NO-AcTrp slowly decayed (Sirvins et al., 2024). This result supports the relatively steady inhibition rates

<footnote><sup>a</sup> PE: plant extract; <sup>b</sup>Procyanidin dimer monogallate (66 %) + B-type procyanidin dimer (34 %).</footnote>
<figure><img src="image_5.png" /><figcaption>Fig. 2. Inhibition of AcTrp N-nitrosation in the presence of selected phenolic compounds (1 mM) in a 5 % o/w emulsion containing N-acetyltryptophan (3 mM), myoglobin (50 µM), NaNO₂ (1.7 mM) and FeSO₄ (30 µM) at pH 5 and 2.5 after 30 min and 60 min of reaction at 37 °C. Mean ± SD (n ≥ 3).</figcaption></figure>

<figure><img src="image_6.png" /><figcaption>Fig. 3. Recovery of the evaluated phenolic compounds (1 mM) in the inhibition of AcTrp N-nitrosation in a 5 % o/w emulsion containing N-acetyltryptophan (3 mM), myoglobin (50 µM), NaNO₂ (1.7 mM) and FeSO₄ (30 µM) at pH 5 and 2.5 after 30 and 60 min of reaction at 37 °C. Mean ± SD (n ≥ 3).</figcaption></figure>

observed here. By contrast at pH 2.5, these rates were usually higher after 60 min compared to 30 min. In the previous kinetic study, NO-AcTrp peaked at 60 min and then rapidly disappeared over 5 h in the control experiment. The presence of (–)-epicatechin, rutin or quercetin was found to drastically accelerate this decay. As a consequence, inhibition rates increased upon time at pH 2.5.

(+)-Catechin, (–)-epicatechin, dimer B2, EGCg, EGC, ECg, and ascorbic acid were all found to limit the formation of NDMA, NMOR or N-nitrosomethylaniline in aqueous medium for pH ranging from 2 to 4 (Lee et al., 2006; Tanaka et al., 1998). As to caffeic acid, it appeared to be a better inhibitor than chlorogenic acid, its esterified form, in the N-nitrosation of the primary amine 2,3-diaminonaphtalene at pH 4 and that of the secondary amine morpholine at pH 1.5 (Combet et al., 2010; Kono et al., 1995). Our results are thus in good agreement with the scarce literature existing for some volatile and non-volatile N-nitrosamines.

### 3.4. Capacity of selected plant extracts to inhibit the lipid oxidation initiated by heme iron in the emulsion system

The best antinitrosating plant extracts were evaluated for their ability to inhibit lipid oxidation initiated by heme iron in a 10 % o/w emulsion at pH 5. Lipid oxidation products were monohydroperoxide derivatives of triacylglycerols (TAG<sub>ox</sub>) containing linoleic acid followed by minor dihydroperoxides as assessed by mass spectrometry (Table S4).

In the control condition, the sum of these oxidized triacylglycerols was found to almost linearly accumulate during the 6 h of reaction (Fig. 4). The six plant extracts were evaluated at 250 µM GAE, rather

<figure><img src="image_7.png" /><figcaption>Fig. 4. Inhibition of the metmyoglobin-initiated lipid oxidation by plant extracts (0.25 mM GAE) in a 10 % o/w emulsion at pH 5 and 37 °C. Metmyoglobin concentration was 50 µM. Mean ± SD (n ≥ 3).</figcaption></figure>
than at 1 mM GAE as previously, as this concentration was sufficient to inhibit lipid oxidation and discriminate plant extracts. Most of them led to a decay of the initially present lipid-derived hydroperoxides in the early phase of the reaction. This suggests a degradation of these TAG<sub>ox</sub>, likely through their reaction with metmyoglobin to produce the hypervalent ferrylmyoglobin form (MbFe<sup>IV</sup>=O) that is the true initiating species in lipid oxidation. In a general manner, all six extracts limited the lipid oxidation initiated by metmyoglobin in this emulsified system (Fig. 4). TAG<sub>ox</sub> concentrations remained statistically lower than that of control during the whole kinetics, except for *Grape seeds* and *Olive and grape* at 6 h (Table S10). Two extracts were remarkably efficient. The *Green tea* extract exhibited the highest antioxidant capacity as lipid oxidation remained totally inhibited throughout the reaction while the *Pomegranate* extract led to a relative accumulation rate of 18 % after 5 h, a time that appears to be the most representative for extract comparison. Although not statistically discriminated, extracts from *Grape seeds*, *Olive* and *Fruit mix* were also highly inhibitory, leading to relative TAG<sub>ox</sub> accumulation rates of 26 %, 36 % and 38 %, respectively, after 5 h of reaction. Regarding the *Olive and grape* extract, a faster lipid oxidation rate was evidenced and the relative TAG<sub>ox</sub> rate was 60 % after 5 h.

The phenolic profiles of the three extracts analyzed for their composition clearly highlight their richness in galloylated monomeric flavanols (Green tea) or monomeric flavanols and oligomeric proanthocyanidins (Olive and grape, Fruit mix). Additionally, the Olive and grape extract was rich in hydroxytyrosol. For the others, the literature indicates that grape seed is an abundant source of monomeric and oligomeric flavanols which are partially galloylated while the olive extract should be rich in hydroxytyrosol, tyrosol and caffeic acid derivatives resulting from the hydrolysis of major compounds such as oleuropein, ligstroside and verbascoside (Labarbe et al., 1999; Malapert et al., 2018). By contrast, gallotannins and ellagitannins are the main constituents of pomegranate skin (Ambigaipalan et al., 2016). All these molecules, except tyrosol, display the typical *ortho*-diphenolic structure that is critical for the antioxidant activity. Further hydroxylation of the aromatic nucleus, as in (–)-epigallocatechin (E7 = 0.43 V), is known to lower the redox potential and thus to increase the antioxidant activity when compared to (+)-catechin (E7 = 0.57 V) (Jovanovic et al., 1996).

These results are in good agreement with the already well-established capacity of phenolic compounds, among which are (+)-catechin and (–)-epicatechin, to protect against the metmyoglobin-initiated lipid oxidation in model systems (Boléa et al., 2019; Lorrain, Dangles, Genot and Dufour, 2010). Additionally, an apple polyphenol extract containing 70 % oligomeric procyanidins was found to be only slightly less inhibitory than (–)-epicatechin, at equivalent unit contents, in a phospholipid-stabilized emulsion at pH 5 (Boléa et al., 2019).

# 4. Discussion

## 4.1. The antinitrosating activity of plant extracts explained by their phenolic composition

The *Green tea* extract (758 mg of phenolic compounds g⁻¹ extract, UHPLC data) is mostly composed of EGCg (487 mg g⁻¹ extract) which proves to be highly antinitrosating in the cured and cooked meat model. As no difference could be observed between the capacities of EGCg and (–)-epicatechin, one can attribute the remarkable inhibitory effect of the *Green tea* extract to its elevated content in flavanols (EGCg, ECg, (–)-epicatechin, EGC, (+)-catechin, gallocatechin gallate in decreasing levels).

For the *Fruit mix* extract (405 mg g⁻¹ extract), major compounds are (+)-catechin, (–)-epicatechin, B-type procyanidin dimers as well as oligomers of higher degrees of polymerization that were only partly quantified. Flavanols again explain the superior antinitrosating activity of the *Fruit mix* extract although the capacity of oligomeric forms remains to be evaluated in this model. Interestingly, Lee et al. showed that the procyanidin dimer B2 could inhibit by a two-fold factor the

formation of NDMA at pH 3 when compared to epicatechin, suggesting that both flavanol units contributed additively (Lee et al., 2006).

In the *Olive and grape* extract, procyanidin dimers and trimers are the major contributors along with hydroxytyrosol which represents one fifth of the phenolic pool (195 mg g⁻¹ extract) but probably less due to the expected presence of highly oligomerized procyanidins in this extract. Hydroxytyrosol is similarly efficient than (–)-epicatechin at pH 5, although less at pH 2.5, and this difference can also be observed in the contrasted capacities of the *Olive and grape* extract at these pHs. On the opposite, the inhibition rates for the *Olive* extract are closely related at both pHs suggesting the additional presence of caffeic acid derivatives.

Finally, extracts from *Pomegranate* and *Grape fruits* were overall less antinitrosating in this model although presenting elevated TPC/TAC ratios of 98 % and 82 %, respectively. This suggests that their phenolic compounds (ellagitannins and flavonols for pomegranate; flavanols, flavonols and esterified hydroxycinnamic acids for grape fruit) could display lower antinitrosating activities as observed for two molecules of these classes, e.g. rutin and chlorogenic acid (Ambigaipalan et al., 2016; Ehrhardt et al., 2014). In the case of the aqueous *Rosemary* extract, rosmarinic acid can be seen as a chlorogenic acid analog with dihydroxyphenyllactic acid replacing quinic acid. The good antinitrosating capacity of the aqueous *Rosemary* extract at pH 5 is in agreement with that of chlorogenic acid and hydroxytyrosol, this last compound being structurally related to dihydroxyphenyllactic acid. However, the low capacity of the *Rosemary* extract at pH 2.5 seems difficult to explain by the sole contribution of rosmarinic acid.

## 4.2. Mechanisms underlying the antinitrosating capacity of phenolic compounds

As described in previous works, the antinitrosating capacity of flavonoids possessing an *ortho*-diphenolic B-nucleus first relies on their consumption of two nitrite ions through the B-ring oxidation. Nitrite is thus reduced into nitric oxide as demonstrated in vitro at pH 1.5–2 (Combet et al., 2010; Peri et al., 2005). Additionally, NO° was recovered in the air expelled from the stomach of subjects consuming lettuce, a source of nitrate, and red wine, apple, onions and black tea, all sources of flavonoids (Rocha et al., 2009). In the case of (–)-epicatechin, (+)-catechin, EGCg, rutin and quercetin, the resulting *ortho*-quinone can add nucleophilic species directly present in the environment (i.e. AcTrp, H₂O, thiocyanate, nitrite, *ortho*-phenylenediamine) (Panzella et al., 2005; Sirvins et al., 2024; Takahama et al., 2009; Takahama et al., 2014). With (–)-epicatechin, dimerization is an additional pathway where the electron-rich B-ring of a native molecule plays the role of a nucleophile leading to (–)-epicatechin dehydrodimers (Sirvins et al., 2024). EGCg may offer an additional pathway with the preferential oxidation of the acylating galloyl group. But most of all, flavanols such (+)-catechin, (–)-epicatechin, and EGCg undergo the C-nitrosation of their *meta*-trihydroxylated A-ring (Lee et al., 2006; Panzella et al., 2005; Sirvins et al., 2024; Takahama et al., 2014). The antinitrosating mechanisms proposed for (–)-epicatechin and EGCg likely operate for other flavanols such as ECg and EGC. This additional consumption of two nitrite ions per molecule likely explains the superior antinitrosating activity of EGCg and (–)-epicatechin, as well as that of the three best extracts at pH 5, e.g. *Green tea*, *Fruit mix* and *Olive and grape*. By contrast, the inability of naringin to react with nitrite can be attributed, on one hand, to the absence of an *ortho*-diphenolic B-ring, and on the other hand, to a low electron density on the A-ring in relation with the presence of two electron-withdrawing groups (carbonyl at C4 and glycosyl at O7).

Regarding hydroxycinnamic acids, the contrasted antinitrosating capacities of caffeic acid and its esterified form, chlorogenic acid, can be first assessed to largely differing reactivity pathways. Neoformed products issued from the reaction between chlorogenic acid and nitrite include nitrochlorogenic acid, nitrochlorogenic acid dimer (Cotelle & Vezin, 2001; Napolitano & d'Ischia, 2002) as well as 2-, 2,5-di-, and
2,5,6-tri-S-glutathionylchlorogenic acids when glutathione is present in the environment (Panzella et al., 2003). These results support the preferential oxidation of chlorogenic acid by nitrite with the production of NO°. Interestingly, rosmarinic acid, which is also an esterified form of caffeic acid, yielded the two corresponding nitro and dinitro derivatives in the presence of nitrite (Dubois et al., 2008). With non-esterified hydroxycinnamic acids such as caffeic acid, structurally different products are formed. First, caffeic acid undergoes C-nitrosation at the C2 of the propenoic acid chain forming a highly electrophilic nitrosated cation. Nucleophilic addition of nitrite, water or glutathione onto the C3 of the propenoic chain yields nitroso carboxylic acid derivatives which are prompt to decarboxylation. Furoxan derivatives, 3,4-dihydroxyphenylacetaldehyde oxime derivatives and 3,4-dihydroxybenzaldehyde are thus produced (Cotelle & Vezin, 2001; Napolitano & d'Ischia, 2002; Panzella et al., 2002). Interestingly, our study revealed that AcTrp had the ability to add onto the 3-position of the propenoic system affording isomeric 2-AcTrp-(3',4'-dihydroxyphenyl)acetaldehyde oximes while favoring the corresponding N-nitroso forms at pH 2.5 (unpublished data). Finally, oxidation by nitrite of the catechol nucleus of the oxime Z-isomers was shown to afford the corresponding benzoxazines.

As to hydroxytyrosol, the only product identified so far is 6-nitrohydroxytyrosol (d'Ischia et al., 2011). Yet, in our parallel work, di- and trimerization of hydroxytyrosol were observed, along with the nitration of these derivatives (unpublished data).

If one evaluates the number of nitrite ions scavenged per molecule of phenolic compound to generate the above described neoeformed structures, it can be inferred that 2 to 5 nitrite ions can react with each (-)-epicatechin or EGCg molecule, 3 nitrite ions with rutin, 3 to 5 with chlorogenic acid, and 2 to 7 with hydroxytyrosol. This scavenging potential compares well with the inhibition rates evaluated in this work at pH 5 (Fig. 2). A major discrepancy is however observed for caffeic acid which can scavenge only 1 to 4 nitrite ions. Besides, this scavenging is always accompanied by coupling with one AcTrp molecule, leading to a decrease in the measurable marker level. The main consequence is likely an overestimation of the inhibition rate as observed for caffeic acid in this study. Because non-esterified hydroxycinnamic acids are seldom found in traditional plants, the evaluation of the antinitrosating capacity of most plant extracts using this newly developed test can be safely conducted.

At pH 2.5, additional compounds resulting from covalent coupling with NO-AcTrp have been evidenced for flavonoids, (-)-epicatechin and rutin (Sirvins et al., 2024) as well as for chlorogenic acid, caffeic acid and hydroxytyrosol (unpublished results) although in a very minor extent. This structure-dependent bias should be better quantified to refine the antinitrosating test at pH 2.5.

### 4.3. The potential of plant extracts to modulate secondary amine N-nitrosation and lipid oxidation in cured and cooked meat processing and digestion

The higher antinitrosating capacity of the *Green tea*, *Olive and grape* and *Fruit mix* extracts is well supported by data in dry-cured bacon and sausages in which green tea and grape seed extracts were found to limit the formation of volatile *N*-nitrosamines (Li, Shao, Zhu, Zhou and Xu, 2013; Wang et al., 2015). Although a large use of ascorbic acid will limit *N*-nitrosamine formation in processed meat products cooked at low temperature, overcooking as for cured ham on pizza or digestion may generate secondary amine *N*-nitrosation. As a matter of fact, the consumption of processed red meat products enriched in phytochemicals from green tea, oregano, grape, rosemary and acerola, either reduced or not in nitrite, led to a significant reduction in the fecal excretion of apparent total *N*-nitroso compounds (ATNC), as compared to traditionally processed red meat products (Van Breda et al., 2021). In addition, Bastide et al. demonstrated that red wine and pomegranate extracts added to cured ham suppressed the promotion of mucin-depleted foci in carcinogen-initiated rats while these extracts and a carnosic acid-rich

rosemary extract also decreased a urinary lipid oxidation marker and fecal ATNC (Bastide et al., 2017). These data and our study clearly highlight the potential of polyphenol-rich plant extracts in health issues such as colorectal cancer.

On the other hand, nitrite removal is known to increase lipid oxidation in meat products as nitrosylmyoglobin, an inactive form of the prooxidant, is no longer formed. The potency of plant extracts to inhibit lipid oxidation has been extensively investigated in real meat matrices. As a matter of fact, 1 % and 2 % w/w of a pomegranate peel extract were shown to replace 150 ppm of nitrite during the drying of uncured sausages, similarly inhibiting the formation of TBARS (Cava & Ladero, 2023). With a phenolic composition related to that of pomegranate, a peel and seed extract from jaboticaba, rich in anthocyanins and hydrolyzable tannins, limited TBARS formation during the storage of fresh sausages while slightly reducing aerobic psychrotrophics (Baldin et al., 2016). Extracts of olive pomace combined with either grape seed or chestnut (1 % w/w) similarly limited the formation of TBARS and volatile compounds as 30 ppm of nitrite in Cinta Senese dry-fermented sausages. The overall acceptability was not affected except for color-related traits while major foodborne pathogens were below the limit (Aquilani et al., 2018). Besides, an olive leaf extract rich in oleuropein (0.025 % w/w) compared well with ellagic acid (0.03 % w/w) in TBARS limitation for raw and cooked pork sausages (Hayes, Stepanyan, Allen, O'Grady and Kerry, 2011). Olive phenols as additives in the meat sector appear to have great potentiality due to color retention, microbial growth retardation, fat preservation, and ultimately extended shelf-life (Galanakis, 2018). On the other hand, proanthocyanin-rich extracts (1 % w/w) from grape seed and pine bark were found to limit lipid oxidation (TBARS, hexanal) in cooked beef. They also efficiently reduced the growth of several food pathogens when rosemary did not (Ahn, Grun and Mustapha, 2007). Moreover, a rosemary extract (1 % w/w), rich in carnosic acid and carnosol, and a green tea extract (0.25 % w/w) were found to largely inhibit TBARS formation and the smell of rancid in Bologna-type fresh sausages (Jongberg, Tørngren, Gunvig, Skibsted and Lund, 2013). Although differing in weight contents added, these extracts presented close contents in total phenolic compounds, 500 ppm and 400 ppm, respectively, pointing to the key importance of extract standardization before the assessment of any physicochemical, nutritional or sensory property.

### 5. Conclusion

A new test was developed to probe the antinitrosating capacity of pure phenolic compounds and plant extracts. It is based on the *N*-nitrosation of *N*-acetyltryptophan as secondary amine target in an o/w emulsion containing heme and non-heme irons. Extracts from green tea, a fruit mix, and an olive-grape mix, evaluated at the level of 1 mM GAE, were highly antinitrosating and their superior capacity was mainly attributed to flavanols. In the absence of nitrite, extracts from green tea, pomegranate skin or grape seed markedly inhibited the metmyoglobin-initiated lipid oxidation. This comparison further outlines that the phenolic composition rather than the antioxidant activity is the key driver for the antinitrosating capacity.

Given the re-evaluation of nitrites as food additives by EFSA (2017), the EU Commission recently revised the maximum levels of nitrites in cured and cooked meat products, with a reduction from 150 ppm to 120 ppm applicable in October 2025 (Commission decision (EU) 2024/1225). At the same time, the French Code of Practice will evolve from 120 ppm to 90 ppm. Besides, maximum residual levels from all sources were established to better monitor the overall exposure to nitrite vis-à-vis the Acceptable Daily Intake (ADI). Altogether, our findings clearly support a beneficial role for polyphenol-rich plant extracts in these strategies of reduction or suppression of nitrate/nitrite in cured meat products. Efforts should now be devoted to practical applications in cooked and dry cured meat products with the further investigation of color, flavor, and microbiological safety after the addition of plants or
their corresponding extracts.

## Abbreviation

<table><tr><td>NDMA</td><td>N-nitrosodimethylamine</td></tr><tr><td>NDEA</td><td>N-nitrosodiethylamine</td></tr><tr><td>NPYR</td><td>N-nitrosopyrrolidine</td></tr><tr><td>NMOR</td><td>N-nitrosomorpholine</td></tr></table>

## CRediT authorship contribution statement

**Charlène Sirvins:** Writing – review & editing, Writing – original draft, Visualization, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. **Pascale Goupy:** Writing – review & editing, Visualization, Validation, Supervision, Project administration, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. **Aurélie Promeyrat:** Writing – review & editing, Resources, Project administration, Funding acquisition, Conceptualization. **Christian Ginies:** Writing – review & editing, Methodology. **Claire Dufour:** Writing – review & editing, Writing – original draft, Visualization, Validation, Supervision, Resources, Project administration, Funding acquisition, Formal analysis, Data curation, Conceptualization.

## Funding source

This research was funded by INRAE and the consortium ADDUITS. Charlène Sirvins received a PhD grant (CIFRE n° 2020/0473).

## Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.foodres.2025.117387.

## Data availability

Data will be made available on request.

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