Inhibition of oxidative stress and advanced glycation end‐product formation in purified BSA/glucose glycation system by polyphenol extracts of selected nuts from Pakistan

Abstract Glycation generates advanced glycation end products (AGE) and its intermediates, thus increasing the risk of developing various ailments including diabetes mellitus. Current study was planned to explore the antioxidant and antiglycation potential of selected nuts viz, Juglans regia (Walnut), Prunus dulcis (Almond), Pistacia vera (Pistachio), and Arachis hypogaea (Peanut), locally available and readily consumed in Faisalabad, Pakistan, for their health‐promoting properties. The prepared methanolic extracts of selected nuts were tested for biological activities including the antioxidant and antiglycation potential. The effect of these extracts against oxidation and AGE formation was evaluated by in vitro method using bovine serum albumin (BSA)‐glucose system. Juglans regia, Pistacia vera, and Arachis hypogaea were found rich in phenolics and flavonoids contents with increased reducing potential and least IC50 due to the DPPH free radical scavenging inhibition. Dose‐ and time‐dependent inhibition of glucose‐induced advanced glycation end‐product (AGE) was exhibited by fruit extracts through in vitro bovine serum albumin (BSA)‐glucose system. Juglans regia and Pistacia vera were predominantly effective in the inhibition of early and intermediary glycation products at different incubation conditions. The study indicated that the extracts of selected nuts possess significant antioxidant capacity and are rich in phenolics and flavonoids, making them useful supplements as an important part of a balanced diet.


| INTRODUC TI ON
Dietary guidelines that emphasize on the dynamic characteristics of phytonutrients have valuable impact on human health against a variety of diseases (Shahidi, 2009;Zia-Ul-Haq et al., 2014). The consumption of some fruits and vegetables is inversely correlated with disease morbidity, which may be due to their antioxidant potential (Wootton-Beard & Ryan, 2011). However, it is widely agreed that some synthetic antioxidants such as butylhydroxyanisole and butylhydroxytoluene should be replaced with natural antioxidants due to their suspected toxicity and associated health hazards (Zhang & Liu, 2015).
Age-related nonenzymatic production and accumulation of AGEs (advanced glycation end products) have been linked to the increased progression of diabetes as well as the pathogenesis of a variety of other disorders including neurodegeneration and diabetic vascular complications (Lee et al., 2007). Hyperglycemia in diabetes induces oxidative stress which increases free radical production owing to glucose auto-oxidation and disturbs the electron transport chain (Ahmad et al., 2009;Mustafa et al., 2019). In humans, oxidative stress is caused by the disruption of natural antioxidant defense systems, usually consisting of reactive oxygen species (ROS) which is linked to a variety of diseases (Fatima et al., 2022;Riaz et al., 2017). Dietary phytonutrient supplementation offers a variety of defense mechanisms against different ailments, resulting in improved general health. Some developing countries like Pakistan are rich in fruits and vegetables but have not yet thoroughly tested for their health-promoting properties. The current study was planned to explore the antioxidant and anti-glycation potential of widely used and easily available nuts as dietary supplements.

| Sample collection and identification
The selected nuts, that is, Juglans regia, Prunus dulcis, Pistacia vera, and Arachis hypogaea, used for the current study were obtained from the local market of Faisalabad, Pakistan. These nuts materials were identified and authenticated from Department of Botany, University of Agriculture Faisalabad, Pakistan, and grinded to fine powder, mixed thoroughly, and stored at −20°C for further analysis.

| Extraction of crude polyphenols
The crude polyphenols from selected nuts materials were extracted following the reported method of Chandrasekara and Shahidi (2011) with slight modification. Nuts materials (25 g each nut separately) were blended in 100 mL of chilled 80% ethanol in 1:8 w/v for 10 min with the help of a warming blender and then homogenized the mixture for 5 min in a Virtis High Speed Homogenizer followed by filtration under vacuum. The mixture was then washed using n-hexane (50 mL) through centrifugation at 2500 g for 10 min. Following the removal of n-hexane fraction, the extract was washed twice and the ethanol/water mixture was evaporated to dryness under vacuum at 45°C using rotary evaporator. The extracts were stored at −40°C until further analysis.

| Total phenolic contents (TPC)
Standard Folin-Ciocalteu method was used for the determination of TPC using gallic acid as the standard. Briefly, equal volume of extract (500 μL; 5 mg of extract dissolved in 5 mL of 1:1 v/v methanol/water) was mixed with Folin-Ciocalteu reagent (500 μL) and kept for 5 min at room temperature. Following the addition of 20% Na 2 CO 3 (1.0 mL), the mixture was incubated at room temperature for 10 min and centrifuged at 150 g for 8 min. Then, 200 μL supernatant was transferred to micro-well plate followed by measuring the absorbance of each well at 730 nm. The total phenolic content was expressed as gallic acid equivalents (GAE) in mg/g of sample (Noor et al., 2014).

| Total flavonoids contents (TFC)
For the determination of TFC, 1 mL extract of each selected nut (10 mg/mL) was placed in a 10-mL volumetric flask followed by the addition of 5 mL distilled water and 0.3 mL of 5% NaNo 2 . After 5 min, 0.6 mL of 10% AlCl 3 was added to the mixture followed by the addition of 2 mL of 1 M NaoH. Absorbance of the mixture was measured at 510 nm photometrically and the amount of TFC was expressed as mgCE/g dry weight of the sample (Barros et al., 2011).
Following 30 min incubation at room temperature, absorbance of phenolics and flavonoids, making them useful supplements as an important part of a balanced diet.

K E Y W O R D S
antiglycation, antioxidants, free radicals, hyperglycemia, polyphenols the mixture was measured photometrically at 517 nm against reagent blank.
Where %I: percent inhibition, A blank: absorbance of blank, A sample: absorbance of sample.

| Determination of reducing power
The reducing power of the extracts was determined as described by Barros et al. (2011). Briefly, 5 mL of sample was mixed with 5 mL of 0.2 M phosphate buffer (pH: 6.6) followed by the addition of 5 mL of 1% potassium ferricyanide. After 20 min of incubation at 50°C, 5 mL of 10% trichloroacetic acid was added to the mixture and then centrifuged at 12,000 g for 10 min. The upper layer of the solution (2.5 mL) was mixed with distilled water (5.0 mL) and 0.1% ferric chloride solution (0.5 mL). The absorbance of the mixture was measured at 700 nm.

| Nitro-blue tetrazolium (NBT) reductive assay
For NBT reductive assay, 0.5 mL of glycated material and 2 mL of 0.3 mM NBT reagent in 100 mM Na 2 CO 3 were incubated at room temperature for 15 min. Absorbance of the mixture was measured at 530 nm against a blank (Zhang et al., 2011).

| AGEs analysis
The glycated material (0.5 mL) was diluted with distilled water making the volume up to 10 mL. The fluorescence of the sample was measured at 370 and 440 nm for excitation and emission, respectively, using an F-5301 spectrofluorometer (Shimadzu; Zhang et al., 2011).

| Statistical analysis
Mean ± SD of replicate measurements were computed using Microsoft Excel 7.0. One-way analysis of variance (ANOVA) followed by multiple comparison test was applied to determine the statistically significant differences of tested biological activities for the studied nuts extract using Minitab 13.1 statistical software. p < .05 was considered statistically significant.

| Antioxidant potential of polyphenolic extracts of nuts
Antioxidants are the agents or chemicals that have the free radical scavenging potential and prevent the production of ROS. Medicinal plants, herbs, and vegetables are the natural sources of variety of substances called bioactives, which have many biological activities including antioxidant potential (Kauser et al., 2018). Recently, the use of natural antioxidants in food industry and health care has gained much importance. A large number of phytobioactive compounds such as phenolics and flavonoids are found in nonedible and edible plants including nuts and are associated with various biopharmacological activities . Because of the significant role of natural antioxidants in improving the general health, we in the current research evaluated the antioxidant potential of selected nuts extract.

| Total phenolics contents (TPC) and total flavonoids contents (TFC)
Nuts are high in several therapeutically active constituents, particularly polyphenols (Carvalho et al., 2010). The results of TPC and TFC of studied nuts are given in Table 1. All studied nuts extracts possess significant amounts of total phenolics ranging from 1.68 to 8.44 mg GAE/g, and total flavonoids ranged from 0.73 to 3.78 mg CE/g. The highest phenolics (8.44 mg GAE/g) and flavonoids (3.78 mg CE/g) contents were found in Juglans regia, while the least phenolic contents (1.68 ± 0.01 mg GAE/g) and flavonoids contents (0.73 ± 0.02 mg CE/g) were found in Prunus dulcis. The tannin content of walnuts and almonds was previously reported in published study of Amarowicz and Pegg (2008). Juglans regia (Walnut) methanolic extracts have the highest total flavonoids contents, which agrees with various other research studies that demonstrated J. regia seeds to be a rich dietary source of phenolics (Chen & Blumberg, 2008;Yang et al., 2009). However, seasonal, agronomic practice variables, genomics, moisture levels, extraction method, and the standard used, among some other factors, could explain the discrepancies in polyphenolic content of fruit (Kalpna & Mital, 2011).

| Reducing power assay of selected nuts
The reducing potential of the studied dry fruits extracts is increased with increasing concentration. Present study was planned to compare the reducing potential of the methanolic extract of studied nuts with BHA and the results are given in Table 1. Dose-response investigations revealed a positive linear relationship between the concentration of nut extracts and the reducing power prior to reaching the threshold level. All the studied nuts extracts give absorbance values above 0.65 except for the J. regia extract which gives absorbance value of 2.21. Significant similarities were observed between almond and peanut extracts with the lowest reducing potential of almond.
According to the published reports, the reducing potential is associated with the development of reductones, acting as antioxidants to disrupt the free radical chain by donating a hydrogen atom (Nisha et al., 2009;Ribeiro et al., 2008). These findings of our study are in agreement with the findings of published study which reported the highest reducing potential of P. dulcis and J. regia (Mishra et al., 2010).

| DPPH radical scavenging activity of nuts
The antioxidant activity results of dry fruits extract are given in Table 1. Methanolic extract of almond showed lowest radical scavenging potential, while the extract of walnut and pistachio were found to be the most potent DPPH radical scavengers, with IC50 values of 0.98 ± 0.48 mg of extract/g of dry weight and 1.55 ± 0.04, respectively. This can be attributed to their higher total phenolic content when compared to certain other fruits. The selected fruits were ranked according to their scavenging capacity, based on absolute numbers rather than statistical differences as >pistachio >walnut >peanut >almond. Deve et al. (2014) reported that phenolics and flavonoids in nuts and peanuts account for a substantial portion of total antioxidant activity, implying that a combined effect of phytochemical and synergistic mechanisms in the nut matrix may be able to take responsibility for their potent antioxidant activities.

| Antiglycation potential of polyphenolic extracts of selected nuts
The Maillard process is a complex set of reactions that involves in reducing the sugars and proteins and eventually giving rise to AGEs.
The events that contribute to the breakdown of proteins produced by glucose can be classified into three types: (i) those that overlap and produce early Amadori products, (ii) those that are intermediate (cross linking of protein and creation of carbonyl groups), and (iii) those that are final and make post-Amadori adducts (Miroliaei et al., 2011). These AGEs have the potential to aggravate diabetic issues as well as other neurodegenerative conditions such as Alzheimer's disease. As a result, AGE inhibitors may have therapeutic properties against various disorders. AGE inhibitors naturally derived from foods have gained popularity, and the basic concept appears to form the basis for the reduction of glycol-oxidation, reactive 1,2-dicarbonyl compound oxidative stress, reactive nitrogen, and oxygen species (Ho et al., 2010). Indeed, numerous dietary plants and their constituents have better anti-glycation properties to aminoguanidine (Peng et al., 2008;Spagnuolo et al., 2021).

| Effect of selected nuts extract on the reduction of NBT
The NBT reduction test was used to determine the inhibitory potential of studied nuts extract on the production of an early-stage glycation product (Amadori product). The results showed that all the edible components exhibited higher inhibitory effects in a dosedependent manner, with the peak inhibitory activity occurring on the eighteenth day of incubation at a dose of 5 mg/mL. Walnut (46%), pistachio (43%), and peanut (43%), as compared to aminoguanidine (38%), were found to have considerable inhibitory activity on the reduction of NBT at 5 mg/mL. Almond, on the other hand, was 35% as efficient as aminoguanidine in reducing the inhibition. The ability of tested samples to inhibit NBT reduction was in the following sequence: walnut (19%-46%) > pistachio (24%-43%) > peanut (19%-43%) > aminoguanidine (10%-38%) > almond (11%-35%) ( Table 2).
Because albumin and other proteins have a half-life of 2 weeks, and measuring early-stage glycation product (Miroliaei et al., 2011) seems to be the method utilized to quantify protein AGE adducts (Jeppsson et al., 2002;Lapolla et al., 2005). The capacity of ketoamine family to reduce the dye nitro blue tetrazolium (NBT) and synthesize a molecule that absorbs at 525 nm is used by scientists TA B L E 1 Antioxidant potential of polyphenolic extracts of selected nuts.
to detect fructosamine (Lapolla et al., 2005). The lower absorbance of the treated samples of various plant extracts suggests their efficiency in reducing glycation (Zhang et al., 2011). A considerable reduction in glycation reaction (percent inhibition) demonstrates the extract's potential to behave as a natural inhibitor attenuating Millard response at the early stage (Matsuura et al., 2002).

| Effect of selected nuts on the formation of α-dicarbonyls
Published studies reported that dicarbonyl chemicals (glyoxal, methyl glyoxal, and deoxyglucosones) accelerate the protein cross-linking and formation of stable AGEs. According to a study, dicarbonyl chemicals account for 45%-50% of AGEs (Ruggiero-Lopez et al., 1999). We, in the current study, found that the quantity of dicarbonyl compounds is increased during first 18 days of incubating the dried fruit extracts with the glucose/ BSA model. All the studied nuts extract and aminoguanidine at a concentration of 5 mg/mL inhibited the synthesis of dicarbonyl compounds on the eighteenth day of incubation.

| Effect of selected dry fruits extract on the formation of AGEs
In vitro inhibitory potential of methanolic extract of dry fruits on the formation of AGEs was assessed by determining the fluorescence emission using aminoguanidine as standard AGEs inhibitor. All the studied extracts inhibited the formation of AGEs in BSA glycation systems ( Table 4). Fluorescence intensity of albumin in various glycation systems of selected dry fruits at different concentrations is plotted in Figure 1 showing (1, 2, 3, 4, 5 mg/mL), and (E) BSA incubated with glucose (1 M) and Aminoguanidine (1, 2, 3, 4, 5 mg/mL). All the graphs are plotted from the mean values of replicate measurements. The fluorescence of the BSA/glucose system rises by several orders of magnitude after 24 days of incubation at 37°C (Figure 1). However, the fluorescence produced by AGE was inhibited clearly when sample extracts and aminoguanidine were introduced to the incubating system. Walnut (49.46%) and pistachio (47.89%), in particular, have higher antiglycative effects than aminoguanidine (45.83%). In the glycated BSA model, all the studied nuts except pistachio showed their maximum inhibitory antiglycative activities at eighteenth day of incubation (not indicated in the Table 4).
At 1 mg/mL, walnut and peanuts had the highest inhibitory effects, followed by almond at 4 mg/mL and pistachio at 5 mg/mL (Figure 1).
The ability of the studied nuts extract to function as natural inhibitors attenuating Millard reaction is demonstrated by a significant decrease in glycation reaction at low doses, notably in the case of walnut and peanuts (Matsuura et al., 2002).
Thus, according to Lunceford and Gugliucci (2005), the decrease in AGE production in natural substances was mostly inhibiting the activity of the second stage of glycation mechanisms, such as the free-radical induced conversion of Amadori products to AGE. Despite the reality that no oxidation process is believed to be involved in the formation of Amadori recombination products, it is hypothesized that oxidation is linked to the generation of fluorescence and molecular crosslinking, both of which are AGE characteristics (Fu et al., 1994). Several investigations have indicated that plants extracts including ginseng (Bae & Lee, 2004), Luobuma tea (Yokozawa & Nakagawa, 2004), Finger millet (Eleusine coracana), and Kodo millet (Paspalum scrobiculatum) (Hegde et al., 2002) inhibit the formation of glycated proteins. Therefore, the inhibiting effect of selected

| CON CLUS ION
The current study concluded that all the studied nuts particularly

FU N D I N G I N FO R M ATI O N
Higher Education Commission (HEC), Government of Pakistan provided the financial support under the indigenous PhD program.

CO N FLI C T O F I NTER E S T S TATEM ENT
Authors declare that they have no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data are available from the authors upon reasonable request.

CO N S E NT FO R PU B LI C ATI O N
Not applicable.

E TH I C A L A PPROVA L
The research work was approved by the research scrutiny committee of the University of Agriculture, Faisalabad, Pakistan.