Reactions of polyphenols in pomegranate peel with nitrite under simulated stomach conditions

Abstract Punicalagin and ellagic acid are the major polyphenols present in pomegranate peels. The contents of α‐punicalagin, β‐punicalagin, and ellagic acid in the pomegranate peels were approximately 75, 72, and 20 µM, respectively. The reactions of polyphenols in pomegranate peels with sodium nitrite under simulated stomach conditions were studied. The reactions decreased the polyphenolic contents of the pomegranate peels and accompanied the formation of nitroso compounds. The oxidation rates followed the order ellagic acid ＜α‐punicalagin ≈ β‐punicalagin. The results suggested that the reactions can occur in the stomach after a meal, while the pH changes from 2 to 4.5.

& Veljovic-Jovanovic, 2016; Peri et al., 2005;Takahama, Yamauchi, & Hirota, 2013;Takahama, Yamauchi, & Hirota, 2016) has been reported under the conditions simulating the stomach. The reduction of nitrous acid to NO by the above components has been investigated in acidic buffer solutions and acidified saliva.
This report explores and analyzes the reactions of polyphenols in pomegranate peel with nitrite under simulated stomach conditions to discuss the functions of pomegranate peels.

| Reagents and material
Pomegranate fruits were purchased from the local market of Chengdu City, Sichuan Province, China. The peels were manually separated by hand from the seeds, and then rinsed with distilled water. After being dried in an oven with air circulation at 40°C, the peels were grounded in a laboratory grinder. Particles with a particle size of 0.42 mm were screened according to China Pharmacopoeia.
Ellagic acid and punicalagins were purchased from Sigma-Aldrich.
The other chemicals were purchased from Kelong Chemical Co, Ltd.
All the chemical reagents were analytical grade except for the acetonitrile, which was chromatographic grade. All solutions were prepared in deionized water.

| Nitrite-induced oxidation of polyphenols
Pomegranate peel powder (1.0 g) was sonicated in distilled water (250 ml) for 30 min at 40°C. Then, the pomegranate peel extract was centrifuged at 7,000 g for 10 min, and the pH was adjusted to 2.0 by adding 2 M HCl. Then, the solution was divided into 1 ml test tubes, different concentrations of sodium nitrite were added to the solutions, and the solutions were placed into a thermostat water bath for incubation. After the incubation, 5 ml of ethyl acetate was added to each incubated sample. The ethyl acetate extract was evaporated under nitrogen in a water bath, and the residue was dissolved in 1 ml mobiles phases. The solutions were centrifuged at 10,000 g for 2 min and then used for HPLC analysis.

| Statistical analysis
All experiments were repeated in triplicate. The data are presented as the mean ± SD.  (Çam & Hışıl, 2010;Živković, Šavikin, Janković, Ćujić, & Menković, 2018). Under the HPLC conditions, α-punicalagin, β-punicalagin, and ellagic acid were separated as a major component. The concentrations of the above polyphenols were calculated using calibration curves. The results are presented as micromoles per gram of dry weight (µM/g dw) ( Table 1). The mean concentrations of ellagic acid were much lower than those of α-punicalagin and β-punicalagin. The presence of these polyphenols in pomegranate peels has been reported in many previous studies (Akhtar et al., 2015;Mushtaq, Sultana, Anwar, Adnan, & Rizvi, 2015).

| Reactions of pomegranate peel polyphenols with nitrite
It has been reported that the concentration of salivary nitrite is approximately 0.05 to 1.0 mM during the stay in the stomach (Pannala et al., 2003). The pomegranate peel extract was incubated with sodium nitrite (0.1-1.0 mM) for 30 min. The color turned yellow, and as the concentration of sodium nitrite increased, the color gradually deepened to brown, which was the same as the reactions of polyphenols in masticated apple fruit with nitrite (Hirota & Takahama, 2015).
Pomegranate peel extracts were incubated with 0.5 mM sodium nitrite for 30 min. The typical HPLC chromatogram of the reaction products is shown in Figure 2b (378 nm). The concentrations of α-punicalagin and β-punicalagin decreased remarkably, but the decrease in the concentration of ellagic acid was slight. The decreases in polyphenol concentrations were accompanied by the production of I, II, and III, whose retention times were 12.1 min, 12.7 min, and 13.1 min, respectively. The data indicate that product II is the main product. Figure 2c shows the UV/visible absorption spectra of the three productions. The spectrum of product Ⅰ was essentially the same as that of products Ⅱ and Ⅲ, which had an absorption peak at approximately 260 nm and a shoulder at approximately 378nm. This indicates that the three components were supposed to be isomers. The ESI-MS data displayed the molecular ion at m/z 1,117 and the fragment ions at m/z 1,055, m/z 781 (punicalin moiety) and m/z 601 (gallagic acid moiety). Figure 3a shows the changes in α-punicalagin, β-punicalagin, and ellagic acid with increasing nitrite concentrations. The results indicated that with increasing nitrite concentration, the concentrations of α-punicalagin, β-punicalagin, and ellagic acid decreased gradually.

| Effect of nitrite concentration and reaction time on the consumption of punicalagin and ellagic acid
In addition, α-punicalagin and β-punicalagin reacted more rapidly F I G U R E 2 HPLC chromatogram and UV-visible absorption spectrogram of polyphenols in pomegranate peel. Pomegranate peel extracts were incubated at 37°C for 30 min (a) without sodium nitrite; (b) with 0.5 mM sodium nitrite. (c) UV-visible absorption spectrogram of the production Ⅰ, Ⅱ, and Ⅲ TA and easily with nitrite than ellagic acid. The data in Table 1 also confirmed this result.

| Effect of pH on the reactions of pomegranate peel polyphenols with nitrite
Gastric pH increases from approximately 2～4.5 while eating a meal.
Approximately 0.5～1 hr is needed to decrease the pH to below 4, and 1～1.5 hr to approximately 2 after eating (Robinson, 2002). As shown in Figure 4, pomegranate peel polyphenols reacted easily with the nitrites at pH 2. As the pH value increased, the reaction rate decreased, and almost no reactions were observed at pH 4.5. Nitrites decreased the concentrations of α-punicalagin and β-punicalagin remarkably compared to ellagic acid. The results suggest that the reactions can occur in the stomach after a meal, while the pH changed from 2 to 4.5. Nitrous acid (pK a = 3.3) may be one of the reasons for the reactions between nitrite and pomegranate peel polyphenols.

| D ISCUSS I ON
Punicalagin, the main ingredient of polyphenol from pomegranate peels. The content of punicalagin may reach 88.70 to 118.60 mg/g dry weight extract from different regions (Khalil, Khan, Shabbir, & Rahman, 2017), while Carbone (Carbone et al., 2016) reported the content of punicalagin in the pomegranate peels ranged from 10.50 to 98.00 mg/g of the total polyphenols.
Nitrates in the body come mainly from green leafy vegetables in the diet and their own synthesis (Hsu, Arcot, & Alice Lee, 2009).
Nitrates can be reduced to nitrite in the mouth and stomach by the nitrate-reducing bacteria (Duncan et al., 1997). Nitrite is found widely in nature and is usually added to food as a preservative (Lu, F I G U R E 3 Effects of nitrite concentrations and time on the reactions of pomegranate peel polyphenols with nitrite. (a) and (b) Consumption of pomegranate peel polyphenols and formation of production Ⅰ, Ⅱ, and Ⅲ . The pomegranate peel extracts were incubated at 37°C and pH 2.0 for 30 min with 0-1.0 mmol NaNO 2 ; (c) and (d) Time courses of the polyphenols decreasing and the productions forming. The pomegranate peel extracts were incubated with 0.5 mmol NaNO 2 at 37°C and pH 2.0 Dong, Li, & He, 2016). Considering that the pH of the gastric lumen decreases to below 4 after eating 0.5-1 hr (Gardner et al., 2002), the results of the study suggested that the reactions of nitrite with polyphenols in pomegranate peel may be possible in the stomach ( Figure 4). The rate constants in Table 1 show that α-punicalagin and β-punicalagin may react rapidly with nitrites. This reaction is because of its good water solubility. The inhibition of the formation of N-nitrosoamines might be beneficial for humans.
In recent years, the free radical reaction pathways of plant polyphenols and nitrites in acidic environments have been accepted and recognized by international academic circles. Previous studies by Peri et al. (2005) proved that •NO and •NO 2 are formed in solutions containing acidic NO − 2 ( Figure 5). Nitration and nitrosation of polyphenols results from scavenging •NO and •NO 2 free radicals.
Semiquinone radicals were supposed to be the initial reaction products. As the fragment ions of (m/z 1055), punicalin moiety (m/z 781), and gallagic acid moiety (m/z 601) were found by ESI-MS, the reaction is most likely to occur at position 1 and (or) 2 ( Figure 1). Three adjacent phenolic hydroxyl groups on the benzene ring are unable to form quinones and cannot form a semiquinone radical.

| CON CLUS IONS
This study showed that the polyphenols in pomegranate peel can react with nitrite under simulated stomach conditions. α-punicalagin and β-punicalagin reacted more rapidly and easily with nitrite than ellagic acid. The reaction may be accompanied by the formation of three compounds, which were supposed to be isomers. The reactions of pomegranate peel polyphenols with nitrite at pH 2 react easily. As the pH value increased, the reaction decreased, and almost no reactions were observed at pH 4.5. This finding suggests that the reactions can occur in the stomach after a meal.

ACK N OWLED G M ENTS
The authors thank Dr. Chen Yang from Sichuan Industrial Institute of Antibiotics for the use of HPLC-MS. The research was financially supported by the Education Department of Sichuan Province (Grant No. 15ZB0381).
F I G U R E 4 Effects of pH on the reactions of pomegranate peel polyphenols with nitrite F I G U R E 5 Chemical cycle of nitrite involved in phenolic compounds in acid solution

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.

E TH I C A L A PPROVA L
This study does not involve any human or animal testing.

I N FO R M E D CO N S E NT
Written informed consent was obtained from all study participants.