Pistachio green hull and pomegranate peel extracts as two natural antiglycation agents

Abstract Advanced glycation end products (AGEs) are formed in the final step of the nonenzymatic Maillard reaction, which can contribute to various health problems such as diabetes mellitus, renal failure, and chronic inflammation. Bioactive compounds with antiglycation properties have the potential to inhibit AGE‐related diseases. This study investigated the antiglycation potential of pistachio green hull (PGH) and pomegranate peel (PP) extracts, which are polyphenol‐rich agro‐residues, against fluorescent AGE formation and compared the results with pyridoxine (vitamin B6), metformin, and EDTA (as usual chemical antiglycation agents). The results showed that PGH and PP effectively inhibited the formation of AGEs in bovine serum albumin–glucose (BSA‐Glu) and BSA–fructose (BSA‐Fru) with antiglycation activities ranging from 92% to 97%. PP extract (with an IC50 of 94 mg ml−1) had a greater antiglycation ability than PGH extract (with an IC50 of 142 mg ml−1). Also, results indicated that the antiglycation activities of the extracts were comparable to that of pyridoxine, and higher than metformin and EDTA. These findings suggest that the two studied extracts can be used for sustainable production of high‐added‐value food products with a positive effect on consumers' health.

metformin, and pyridoxine, can prevent AGEs-induced diseases, but they may also have some side effects on the body (Anwar et al., 2021;Kazeem et al., 2019).
Currently, many studies are being conducted on the application of natural antiglycation agents obtained from plants.The inhibitory activity of red grape skin extract (RGSE) was tested in the bovine serum albumin-fructose (BSA-Fru) model system.Results indicated that RGSE has a good antiglycation activity which correlated with the radical scavenging activity of the extract (Jariyapamornkoon et al., 2013).In another study, Calendula officinalis L. and Juglans regia L. extracts were added into the bovine serum albumin-glucose (BSA-Glu) reaction mixture, and a good antiglycation activity was observed.
These extracts had the same activity as aminoguanidine (Ahmad et al., 2012).Antiglycation activities of 14 plant extracts in the BSA-Glu and bovine serum albumin-methylglyoxal (BSA-MGO) model systems were studied in an in vitro assay, and the highest activities were found in star anise, cinnamon, allspice, and cloves, respectively (Starowicz & Zieliński, 2019).Additionally, polyphenolic compounds in hazelnut skin extract were found to inhibit the formation of fluorescent AGEs in the BSA-MGO model system.The antiglycation activity of hazelnut skin extract was higher than that of aminoguanidine and gallic acid (Spagnuolo et al., 2021).Other natural sources, such as leaf extracts from Chilean bean landraces (Ávila et al., 2022), sorghum bicolor leaf sheath extract (Adetayo et al., 2021), crude and purified extracts of tomato varieties (Błaszczak et al., 2020), barnyard millet phenolics (Anis & Sreerama, 2020), and peanut skin extract (Zhao, Zhu, et al., 2021a), have also been studied for their antiglycation activity by other scientists.
Based on our literature review, there are no published reports on the antiglycation activities of PGH and PP extracts.Also, considering that pistachio and pomegranate peels are widely produced around the world, with a significant portion of them increasingly becoming solid waste, utilizing these peels as natural antiglycation agents in the food industry can be a sustainable and cost-effective solution.
Therefore, we designed this research to investigate the potential of PGH and PP extracts as natural antiglycation agents for preventing the formation of harmful glycation products in two model systems (BSA-Glu and BSA-Fru).
Pyridoxine hydrochloride (vitamin B 6 ) and metformin were provided by Alborz Darou Company (Qazvin, Iran).All of the other chemicals and solvents used in this study were of analytical grade.

| Preparation of PGH and PP extracts
Pomegranate peels (Malase torshe saveh variety) and pistachio green hulls (Ahmad aghaei variety) were obtained from the Saveh and Yazd Agricultural Research Centers in Iran, respectively.The peels were manually removed, sun-dried, and maximum drying temperature was 38°C, and ground in a grinder to a 40-mesh size.Phenolic compounds were extracted from the hulls using distilled water as the solvent, with a liquid-to-solid ratio of 15:1, for 8 h at 25°C.The extracts were filtered through Whatman filter paper to remove fine particles.After extraction, the extracts were freeze-dried and stored at −20°C until further use (Aliyari et al., 2020;Rajaei et al., 2010).Extraction yields of PGH and PP were 14.0% and 12%, respectively.

| Determination of total phenolic content
The total phenolic content (TPC) was determined using Folin-Ciocalteu colorimetric method with minor modification (Rajaei et al., 2010).Twenty microliters of each extract was added to 1.4 mL of distilled water and then, 100 μL of Folin-Ciocalteu reagent was added to the mixture.After that, 0.3 mL of Na 2 CO 3 (7.5%)was added and well mixed.This mixture was incubated for 30 min at 40°C.A ultraviolet-visible spectrophotometer (Agilent Cary 60, USA) was used for measuring the absorbance at 765 nm.Gallic acid was used as a standard for drawing calibration curve.The calibration curve's equation of gallic acid was Y = 0.2725x-0.2554;r = 0.9991.Results were expressed as milligrams of gallic acid equivalents per gram dry weight (mg GAE/gdw) (Ghandahari Yazdi et al., 2019).

| Antioxidant activity assay
The antioxidant activities of extracts were determined by the DPPH • method (Ghandahari Yazdi et al., 2019) with some modifications.
Then, the absorbance was read at 517 nm against a blank.The scavenging activity was calculated using the Equation 1: where A c and A s represent the absorbance of the control and sample.
The concentration of the extract which is required to scavenge 50% of the DPPH free radicals was considered as IC 50 .

| Glycation reaction of BSA with fructose and glucose
Glycation reactions of BSA with fructose and glucose were done as previously described by Błaszczak et al. (2020), with minor modifications.Briefly, 0.5 mL of BSA (5% w/v) was incubated with 0.5 mL of fructose or glucose (0.8 M) in PBS buffer (0.2 M, pH = 7.4) containing sodium azide (0.002% w/w) in darkness at 37°C for 6 days in the absence or presence of two freeze-dried extracts (0.1%, 0.5%, 1.0%, 1.5%, 2.0%, and 6.0% w/v) and some reference compounds such as metformin, pyridoxine, and EDTA (100 μL, 1.0 Mm).Test samples were dissolved in PBS (0.2 M, pH = 7.4).The control sample contained all the reaction components without any inhibitor.

| Statistical analysis
All analyses were performed in triplicate, and the results were reported as mean ± standard deviation.Mean comparisons were performed using the one-way ANOVA method, and a p-value lower than 0.05 was considered statistically significant.Statistical analysis was performed using SAS software (version 13).

| Determination of antioxidant activity
Results of DPPH • free radical assay showed that PGH and PP extracts have significant radical scavenging activity with IC 50 of 142.01 ± 1.0 μg mL −1 and 94.0 ± 1.6 μg mL −1 , respectively.By comparing the results, it can be found that the antioxidant activity of PP extract is higher than PGH, but both extracts are a rich source of bioactive compounds compared to other plant sources (Table 1).

| Antiglycation activity
As shown in Figure 1, the PP extract inhibited the glycation reaction in a concentration-dependent manner.The percentage of inhibition in the BSA-Glu system was slightly less than 25.5% initially, but it increased significantly and reached to 95.7% at the highest concentration (6.0% w/v, p < 0.05).
In the BSA-Fru system, the PP extract's antiglycation activity was about 58.66% at 0.1% w/v, and it reached ~92.7% and then remained constant up to 2.0% w/v.The highest activity (97%) was observed at 6.0% w/v of PP, which was significantly higher than the other concentrations (p < 0.05).
As can be seen in Figure 2, there is a direct correlation between the concentration of the PGH extract and its antiglycation activity.
The results of the BSA-Glu system revealed that the initial antiglycation activity was 31.7% (at 0.1% w/v of extract) and increased to 95% at 6.0% w/v.In addition, the PGH extract showed a similar behavior in the BSA-Fru model system, with an initial antiglycation activity of 35.66% (at 0.1 w/v %) that reached to 92% at 6.0% w/v.
In this research, PP and PGH extracts exhibited different antiglycation activities.In this research, PPE and PGHE exhibited different antiglycation activities.The PPE exhibited greater activity in comparison with the PGHE.This difference in activity could be attributed to the distinct profile and quantity of phenolic compounds found in each extract.Pomegranate peel is rich in ellagitannins (Saroj et al., 2020), while pistachio hull mainly contains proanthocyanidins (Mandalari et al., 2021).Also, pomegranate peel has higher levels of flavonoids, as well as anthocyanins compared to pistachio hull (Zhao, Shen, et al., 2021b).To confirm the potential inhibitory effect of PGH and PP extracts (at 6.0% w/v), their activities were evaluated and compared with two common inhibitors (e.g., metformin and pyridoxine) and a chelator (EDTA) (at 1.0 mM).As shown in Figure 3, pyridoxine was able to completely inhibit the glycation reaction.Schalkwijk and Miyata (2012) reported that pyridoxine (1) Free radical scavenging activity ( % ) = A s − A c A c × 100 (2) reacts with the produced active carbonyl compounds and oxygen free radicals during the glycation process.In contrast, metformin did not significantly affect the glycation process in the BSA-Glu system but had a slightly inhibitory effect on the BSA-Fru system.This result is in agreement with the results reported by Sadowska-Bartosz et al. ( 2014), who found that metformin slightly decreases the rate of glycoxidation in systems containing glucose, but has a moderate inhibitory effect on the glycoxidation in the presence of fructose.
EDTA did not show a significant effect on the two studied systems.Interestingly, the antiglycation activity of the pomegranate peel and pistachio green hull were comparable to vitamin B 6 and significantly higher than EDTA and metformin in both systems.
It seems that the antiglycation activities of the two studied extracts are attributed to their high contents of phenolic compounds.
Phenolic compounds are believed to inhibit glycation and its propagation through various mechanisms, including reducing oxidative pathways by scavenging free radicals, blocking reactive carbonyl groups that can react with different biomolecules, leading to oxidative stress, and chelating metal ions (Khan et al., 2020)  carbonyl species and prevent their reaction with other molecules (Chen et al., 2014;Thilavech et al., 2015).Li et al. (2014) have reported that quercetin can suppress α-dicarbonyl compounds that induce protein glycation and trap methylglyoxal.Another study reported that epicatechin, p-coumaric acid, and gallic acid are able to decrease protein carbonyl, thiol oxidation, and fluorescence AGE formation (Khan et al., 2020).Moreover, gallic acid has been found to reduce the levels of oxidative stress markers (Gao et al., 2019).
Another study found that phloroglucinol inhibits the formation of AGEs due to its antioxidant activity (Drygalski et al., 2021).Many studies have reported that ellagitannins, gallotannins, and gallagyl esters in the PP extract (Singh et al., 2018) and gallic acid, quercetin, phloroglucinol, theogallin, galloyl derivatives, catechin, and pyrogallol in the PGH extract (Arjeh et al., 2020) are responsible for their antioxidant activity.Atta et al., 2023 reported that pistachio extract (at a dose of 1 mg/mL) showed the highest inhibitory activity on the formation of dicarbonyl chemicals in BSA glycation processes compared to other studied nuts.Therefore, the present data are consistent with the literature regarding the antiglycation mechanism of phenolic compounds.
Table 2 presents a selection of natural inhibitors whose inhibitory effects can be compared to those of the studied extracts.
The use of these extracts is significant, as they not only reduce waste and promote resource efficiency but also offer significant potential as functional ingredients for controlling AGEs in food processing.

| Antiglycation activity of the mixed extracts
According to the results presented in Figure 4, the mixture of extracts increased the antiglycation activity up to 97%.In the BSA-Fru system, Similar results were obtained in the BSA-Glu system, where increasing ratios of PPE to PGHE resulted in higher antiglycation activity.
However, the lowest antiglycation activity was observed at the 20:80 ratio of PPE:PGHE.These findings are consistent with previous studies that have reported higher TPC and antioxidant activity in PP extract compared to PGH extract (Aliyari et al., 2020).
It has been recently reported that mixing different phenolic compounds can exhibit synergistic effects in biochemical processes (Spagnuolo et al., 2021).For instance, combining gallic acid with caffeic acid, as well as quercetin, gallic acid, caffeic acid, quercetin, gallic acid, and rutin with each other demonstrated high synergistic effects (antioxidant activity).Therefore, the antioxidant and antiglycation properties of plant extracts could be explained by the synergistic effect of phenolic compounds present in the extracts (Ramkissoon et al., 2013).Additionally, from an economic standpoint, mixing compounds may prove cost-effective since less material will be consumed.

| CON CLUS ION
In this study, the antiglycation activities of two natural agricultural wastes, PGH and PP extracts, were investigated in two model systems.The results indicated that the studied extracts, either individually or in combination, could significantly inhibit the formation of fluorescent AGEs, with activities comparable to that of vitamin B 6 .Furthermore, the PP extract exhibited higher antiglycation activity than the PGH extract, likely due to its higher total phenolic content and antioxidant activity.Therefore, these extracts may have the potential for use as a rich source of bioactive molecules in food Peanut skin extract At the concentration of 0.2 mg/mL, the inhibition activity was about 75%.Zhao, Zhu, et al. (2021a) BSA (10 mg/mL), Fructose (500 mM)

Barnyard millet (Echinochloa frumentacea)
A 73% reduction in AGE formation was observed at a concentration of 100 μg/mL.
After incubation, 0.3 mL of the reaction mixture was transferred into a 96-well ELISA plate to measure the amount of produced fluorescent AGEs using a Cytation three multimode plate reader (BioTek Instruments, Winooski, VT, USA) by measuring fluorescence intensity at excitation and emission wavelengths of 340 and 435 nm, respectively.The inhibitory activity was evaluated by Equation 2: where F s and F c are the fluorescence intensities of the test sample and control, respectively.

F
Antiglycation activity of pistachio green hull (PGH) extract (6.0% w/v) in the two model systems.Results are presented as mean ± s.d.(n = 3).For abbreviations and other conditions see Figure 1 legend.Antiglycation activities of PGH and PP extracts (6.0% w/v) compared to metformin and pyridoxine, EDTA in two model systems.Results are presented as mean ± s.d.(n = 3).For abbreviations and other conditions, see Figure 1 legend.Concentration of metformin, pyridoxine, and EDTA was 1was observed for the 0:100 ratio (PPE:PGHE), and no significant difference was found among the other ratios (p < 0.05).
Total phenolic content and antioxidant activities of some plant extracts.
. Muthenna et al. (2012) indicated that ellagic acid can inhibit the formation of TA B L E 1 Rose 2.75 g GAEs/L of infusion 422 mmole TEs/L of infusion (ORAC) Sweet Osmanthus 2.13 g GAEs/L of infusion 634 mmole TEs/L of infusion (ORAC) Pistachio green hull and pomegranate peel extracts Comparison of antiglycation activities of our studied extracts with some recent reports.Leaf extracts from Chilean bean landraces IC 50 values of antiglycation activity of Coscorrón, Frutilla, Magnum, Peumo, Sapito, and Tórtola bean leaf extracts were 336, 681.9, 585.8, 462.7, 326.3, and 373.3 μg/mL, respectively.
TA B L E 2