QTRAP LC/MS/MS of Garlic Nanoparticles and Improving Sunflower Oil Stabilization during Accelerated Shelf Life Storage

The purpose of this research was to assess and utilize the bioactive compounds of garlic nanoparticles (Ga-NPs) as a natural antioxidant in sunflower oil (SFO) stored at 65 ± 1 °C for 24 days. The garlic nanoparticles (Ga-NPs) from the Balady cultivar were prepared, characterized, and added to SFO at three concentrations: 200, 600, and 1000 ppm (w/v), and they were compared with 600 ppm garlic lyophilized powder extract (Ga-LPE), 200 ppm BHT, 200 ppm α-tocopherol, and SFO without Ga-NPs (control). The QTRAP LC/MS/MS profile of Ga-NPs revealed the presence of four organosulfur compounds. Ga-NPs exhibited the highest capacity for phenolic, flavonoid, and antioxidant compounds. In Ga-NP SFO samples, the values of peroxide, p-anisidine, totox, conjugated dienes, and conjugated trienes were significantly lower than the control. The antioxidant indices of SFO samples containing Ga-NPs were higher than the control. The Ga-NPs enhanced the sensory acceptability of SFO treatments up to day 24 of storage. The shelf life of SFO treated with Ga-NPs was substantially increased (presuming a Q10 amount). The results show that Ga-NPs are a powerful antioxidant that improves SFO stability and extends the shelf life (~384 days at 25 °C).


Introduction
Sunflower oil (SFO) is a popular vegetable oil that is commonly consumed all over the world [1]. SFO contains a high concentration of polyunsaturated fatty acids (PUFAs), particularly linoleic acid (C18:2, 68-72%) [2]. SFO is more sensitive to oxidative degradation, resulting in rancid odors, off flavors, and discoloration [3]. To improve oil stability, artificial antioxidants such as BHT (butylated hydroxytoluene) and TBHQ (tertiary butylhydroquinone) have been used as additives [4]. However, at high doses (>200 ppm), these compounds cause numerous health risks, including cancer [5]. Recently, the uses of natural

Preparation of Garlic Nanoparticles (Ga-NPs) and Garlic Extracts
Fresh garlic (Allium sativum L.), Balady cultivar, was cleaned, manually peeled, and frozen at (~−40 ± 1 • C), then dried in a lyophilizer (Labconco 74200, Kansas, KS, USA) on 0.120 mbar, at 19 ± 1 • C and a condenser at −85 ± 1 • C. The garlic was ground and transferred to nanoparticles, as stated by Khataee, et al. [26]. The garlic was crushed with a grinder (Moulinex-Grinder; MC300, France) to fine garlic lyophilized powder (Ga-LP). Then, the particles were crushed by planetary ball mill (PM 2400, Karaj, Iran) (ball:powder ratio; 10:1) under a rotation velocity of 320 rpm for 2 h at ambient temperature (25 • C) to produce garlic nanoparticles. The garlic nanoparticles (Ga-NPs) were assessed with a Zetasizer (Nano-Sight NS300, UK) that was 90 ± 7 nm. The Ga-NPs were packaged in dark bottles until utilized according to Morsy, Morsy, Elbarbary and Saad [6]. Extraction of Ga-LP and Ga-NPs to obtain Ga-LPE and Ga-NPsE was done in an ultrasonic bath (Bandelin-Super Sonorex RK-100H), as asserted by Tabaraki, et al. [27]. The 50 mL methanol (70%) was added to a 1 g sample in a conical flask and sonicated for 30 min, then cooled to 25 ± 1 • C. The garlic extracts were passed through Whatman paper No. 1 and concentrated by rotary evaporator (IKA-WERKE; Germany) at a rotation speed of 250 rpm at 40 • C under vacuum. Samples were frozen at~−40 ± 1 • C, then dried for 72 h in a lyophilizer.

Total Phenolics Content (TPC), Total Flavonoids Content (TFC), and Antioxidant Ability of Extracts
TPC was calculated using the Folin-Ciocalteu reagent, as described by Singleton et al. [29]. The results were expressed as mg GAE (gallic acid equivalent) per g −1 dw extract. Total flavonoids content (TFC) was determined in accordance with Zhishen, et al. [30] and expressed as mg RE (Rutin equivalents) per g −1 dw extract. The antioxidant ability of garlic extracts in terms of free radical scavenging potential was tested using DPPH assay. The outcomes were computed as IC 50 (µg mL −1 ) [31]. The ABTS method was also used in accordance with Re, et al. [32], which measured at a wavelength of 734 nm.

Sunflower Oil (SFO) and Ga-NPs
The SFO (without synthetic antioxidants) was divided into seven groups. Garlic nanoparticles (Ga-NPs) were added to SFO in 3 groups at different concentrations, namely, 200 ppm, 600 ppm, and 1000 ppm (w/v). The 4th group contained Ga-LPE 600 ppm (w/v). The 5th and 6th groups included BHT and α-tocopherol at 200 ppm (w/v). The final one was SFO without antioxidant (control group). The oil samples were packed into dark glass bottles and stored under accelerated oxidation conditions (65 • C/24 days). Oil samples were collected at regular intervals for analysis at 0, 4,8,12,16,20, and 24 days of storage. The peroxide value (PV) of SFO samples was performed as stated by Zhang, et al. [33]. The PV was calculated as meq kg −1 oil (milliequivalent per kilogram).
2.6.2. p-Anisidine Value (p-AnV) and Totox Value (TV) p-Anisidine value was carried out according to Chong, et al. [34] using the spectrophotometer (Model CM-5; Konica Minolta Sensing, Inc., Osaka, Japan) at 350 nm. The outcomes of p-AnV were calculated as mg kg −1 in accordance with the equation (Equation (1)): where As is the absorbance of oil sample with the p-anisidine reagent, Ab is the absorbance of blank, and W is the sample weight (g). Totox value (TV) was estimated using AOCS [35] using the Equation (2): where p-AnV is the p-anisidine value and PV is the peroxide value 2.6.3. Conjugated Dienes (CDs) and Conjugated Trienes (CTs) Determination of CDs and CTs of oil samples were performed using a spectrophotometer at a wavelength of 233 and 268 nm, respectively [36].

Induction Period (IP), Antioxidant Efficiency, and Shelf Life Prediction
The induction period (IP) of the oil samples was estimated with a Metrohm 679 Rancimat instrument (Switzerland) at 110 ± 1 • C at a flow velocity of 15 Lh −1 [37]. Protection factor (PF) and antioxidant activity (AA) values of SFO were determined according to Bandonien, et al. [38] based on the following calculations (Equations (3) and (4)): where IP S is the induction period of the sample (SFO) with an antioxidant additive, IP c is the induction period of the control sample without an antioxidant additive, and IP BHT is the induction period of the sample with BHT. The line slopes were measured by the plotting concentration, and time was used to calculate the temperature acceleration factor (Q10), which was based on the increase in oxidation level for every 10 • C increase in temperature, in accordance with [39] using the Equation (5): where e is the constant equal (2), T1 is the accelerated temperature (65 • C), and the T2 is ambient temperature (25 • C).

Sensory Evaluation
The organoleptic test of sunflower oil (SFO) was performed by an experienced and trained 12-member panel from the Biochemistry Department, according to Sadeghi, et al. [40]. The SFO treatments were placed into small dark glasses (~30 mL) with random numbers. All SFO samples were scored by the panel members using a hedonic (7-point) scale for color, aroma, and acceptability.

Statistical Analysis
The statistical significance in the SFO analysis was accomplished using the ANOVA test (SPSS 19, SPSS Inc., Chicago, IL, USA) followed by Tukey's multiple comparison test, with a significance level of p < 0.05 [41] 3. Results and Discussion 3.1. Characterization of Garlic and Garlic Nanoparticles (Ga-NPs) 3.1.1. Antioxidant Capacity TPC and TFC are important constituents that can be used as an indicator of antioxidant capacities. Different cultivars of garlic such as Egyptian garlic (Balady), Italian Red, Sids-40, Chinese, and Brazilian Hozan samples were used. The TPCs in varied cultivars of garlic are presented in Table 1. The average TPCs ranged from 22.7-27.2 mg GAE g −1 extract (on dw). The TPC was highest in the Balady garlic cloves (p < 0.05) when compared to other cultivars. Petropoulos, Fernandes, Barros, Ciric, Sokovic and Ferreira [16] found that TPC values in garlic ranged from 8.59-44.85 mg GAE g −1 dw extract, while Kim, et al. [42] reported that the TPC in aged black garlic was 22.17 mg GAE g −1 dw. This variation could be attributed to cultivars or extraction methods. Total flavonoid content (TFC) in garlic cultivars ranged from 0.65-1.52 mg g −1 dw extract. These findings are consistent with those reported by Soto, et al. [43]. On the other hand, the nanoparticles of Balady garlic (Ga-NPs) had the highest TPC of 38.1 and TFC of 3 mg GAE g −1 compared to garlic (bulk shape). According to previous research, phenolics and flavonoids play an important role in antioxidant capacity [44].  Table 1 illustrates that the antioxidant activity (DPPH and ABTS) of Balady garlic extract and Ga-NPs. The data reveal that garlic extract has a high antioxidant ability of 11.8 and 10.8 µg mL −1 for DPPH and ABTS, respectively. The antioxidant ability of Balady Ga-NPsE was lower than BHT and higher than α-tocopherol. The results are consistent with data obtained by Iqbal and Bhanger [45]. The analysis of the data reveals that the antioxidant activity of garlic cultivars was correlated with TPC and TFC (R = 1). The TFC and TPC became increasingly important to antioxidant activity. [44,45] also found that antioxidant ability was positively correlated with TFC and TPC.

LC/MS/MS Fingerprint
The organosulfur compounds in garlic were determined using QTRAP LC/MS/MS analysis. The results in Figure 1 confirmed that garlic extracts have a significant number of organosulfur compounds. The LC/MS/MS recognized the major sulfur compounds (~4 peaks) in garlic extracts as alliin, allicin, S-allyl-mercapto-cysteine, and Glutamyl-(s)-Allyl-Cyste. The compounds' precursor-product ion pairs (quantification transitions) were discovered to be m/z 178.1-88, 163.1-73.2, 194.0-111.2, and 291.3-145.2, respectively. These fragments were also observed by Zhu, Kakino, Nogami, Ohnuki and Shimizu [28], who found that garlic extract have alliin, S-allyl-L-cysteine, γ-glutamyl-S-allyl-L-cysteine, and allicin. These compounds are primarily responsible for the biological activities of garlic extract, which include antioxidant, antibacterial, and anticancer properties [46]. Sulfur compounds' antioxidant activity is linked to their ability to reduce reactive oxygen species and activate antioxidative enzymes [47]. When exposed to negative conditions such as high temperatures, oxygen, and light, the organosulfur compounds found in garlic become volatile, thermally unstable, and prone to degradation and oxidation [48]. The characterization of bioactive compounds is dependent on the stability of garlic. Alliin is unstable and non-volatile due to the alliinase-catalyzed conversion into allicin when garlic is crushed. Alliinase reacts with alliin to produce reactive intermediates that combine to form allicin, an unstable alkenyl alkene thiosulfinate [49]. While allicin decomposes quickly in vitro to form a variety of organosulfur compounds, such as diallyl sulfides. This degradation was observed within hours at room temperature or during cooking [50].    Table 2 shows the addition of antioxidants and time of storage on PV had a significant impact on PV (p ≤ 0.05). In general, the PV increased for all samples with the progression of time. The PV increased in the following sequence: control > α-tocopherol > Ga-LPE 600 ppm > Ga-NPsE 200 ppm > Ga-NPsE 600 ppm > Ga-NPsE 1000 ppm > BHT meq kg −1 . Sunflower oil treatments without the antioxidant (control) achieved the highest PV of 166.1 meq kg −1 after 24 days of storage at 65 • C. The increase in PV caused by lipid hydroperoxide formation may also degrade into volatile and non-volatile compounds, causing the oil's quality to deteriorate [51]. PV was found to be significantly different between the control and garlic extract samples. The rate of peroxide formation was slowed at all concentrations where the PV recorded was 70.6, 46.4, 41.7, 38.6, 20.14, 20.2 meq kg −1 for α-tocopherol, Ga-LPE 600 ppm, Ga-NPsE 200 ppm, Ga-NPsE 600 ppm, Ga-NPsE 1000 ppm, and BHT, respectively, while the limiting value of peroxide was less than 21 meq kg −1 oil. Samples containing α-tocopherol, on the other hand, had significantly higher values than all Ga-NPsE and Ga-LPE samples combined. However, the PV in BHT-containing SFO samples was lower than in garlic-containing samples [45]. a-g : There are no significant differences between any two means in the same column that have the same lowercase superscript letter (p ≥ 0.05). A-G : There are no significant differences between any two means in the same row that have the same uppercase superscript letter (p ≥ 0.05). Ga-NPsE: garlic nanoparticles extract; Ga-LPE: garlic lyophilized powder extract.

p-Anisidine Value (p-AnV)
The p-anisidine value (p-AnV) is a measurement of the secondary fat oxidation output such as aliphatic aldehydes, ketones, alcohols, acids, and hydrocarbons. In this case, the edible oils develop off flavors and off odors. Table 2 shows the results, which reveal increases in p-AnV for all the SFO samples. The p-AnV concentration in the control group significantly increased from 4.20 to 25.26 mg kg −1 until day 24 of storage. Ga-NPsE (1000 ppm) and BHT-incorporated oil showed the lowest amount of p-AnV compared to α-tocopherol, Ga-LPE 600 ppm, Ga-NPsE 200 ppm, and Ga-NPsE 600 ppm. The threshold of p-AnV in oil is less than 10 mg kg −1 . These findings are consistent with the findings reported by Nyam, et al. [52], who found that the natural extracts have a notable reduction impact against the oxidation of SFO under accelerated storage. Although Ga-NP extract was significantly lower in the p-AnV, the efficiency of BHT synthetic antioxidants was slightly higher than Ga-NPsE.

Totox Value (TV)
Totox value (TV) indicates the presence of primary and secondary oxidation compounds, indicating the early and later stages of oxidative rancidity. As a result, it provides a more accurate estimate of oil deterioration and quality. There was a noticeable difference in the totox value of the treated SFO and the control ( Table 3). After 24 days of storage, the maximum values were 357.5 and 160.7 meq kg −1 for the control samples and α-tocopherolenriched sample, respectively, while those of oil samples enriched with BHT and Ga-NPs 1000 ppm Ga-NPsE had the lowest TV. a-g : There are no significant differences between any two means in the same column that have the same lowercase superscript letter (p ≥ 0.05). A-G : There are no significant differences between any two means in the same row that have the same uppercase superscript letter (p ≥ 0.05). Ga-NPsE: garlic nanoparticles extract; Ga-LPE: garlic lyophilized powder extract. These results could be attributed to bioactive compounds of garlic such as allicin, 1,2-vinyldithiin, allicin, and S-allyl-cysteine [46]. Table 4 shows the formation of CDs and CTs in the control and stabilized SFO treatments during storage. Generally, the CD values of all groups increased. The highest contents were observed for the control sample, while the addition of garlic reduced the number of CDs. BHT recorded the lowest value (6.15). Ga-NPsE (1000 ppm) had a low value compared to the samples containing non-synthetic antioxidants. Furthermore, the values of CTs were increased significantly with storage time in all treatments (Table 4). CT values were lowest in oil samples supplemented with BHT and Ga-NPs 1000. The CDs and CTs of the SFO samples supplemented with BHT were significantly lower than those given garlic extract-treated samples [52]. a-d : There are no significant differences between any two means in the same column that have the same lowercase superscript letter (p ≥ 0.05). A-G : There are no significant differences between any two means in the same row that have the same uppercase superscript letter (p ≥ 0.05). Ga-NPsE: garlic nanoparticles extract; Ga-LPE: garlic lyophilized powder extract. Table 5 demonstrates that all SFO samples were significantly different (p ≤ 0.05) in IP values than that of the control. IP values in samples containing Ga-NPs were 15.4, 18.7, and 22.2 h, for Ga-NPs concentrations of 200 ppm, 600 ppm, and 1000 ppm, respectively. It was noted that there are significant differences in the samples' IP scores, including different concentrations of Ga-NPs. The SFO sample containing BHT exhibited the highest IP (25.3 h). The findings agree with those reported by Carelli, et al. [53], who found that natural antioxidants increased the IP of SFO. Table 5 shows that the values of the protection factor (PF) of SFO samples including garlic extracts ranged from 1.21 to 3.33. The high concentrations of Ga-NPsE showed a significant increase in PF value. Moreover, the antioxidant activity (AA) of samples containing BHT and Ga-NPsE 1000 ppm did not differ significantly (p ≥ 0.05). In addition, we observed that AA increased up to 1 in SFO containing BHT. Generally, adding Ga-NPsE to SFO samples significantly increased the antioxidant efficiency (AE).    Figure 3 depicts the sensory parameters, such as color, aroma, and acceptability, of sunflower oil (SFO) treatments during storage. The sensory assessment of SFO samples was done for different storage periods caused by the ending of shelf life with the intelligible development of an off flavor. Panelists generally approved of all sensory properties of SFO treatments at time zero; as the storage progressed, the sensory acceptability decreased. The control sample scored the lowest color value during the storage periods, while the oil sample incorporating Ga-NPs at different levels were accepted up to day 24. The aroma value differed significantly (p ≤ 0.05) between the oil treatments and the control group. SFO samples incorporating BHT, α-tocopherol or Ga-NPS 1000 ppm were accepted until day 24 of storage; however, the control was rejected on day 12 due to an off flavor. SFO samples' acceptability values decreased significantly over time, whereas BHT and Ga-NPS 1000 ppm samples did not differ after 24 days. The sensory data demonstrate that the control sample was rejected after day 12 of storage; however, the oil samples incorporating BHT, α-tocopherol, and Ga-NPS 1000 ppm were accepted up to day 24. These changes match well with chemical indicators, i.e., PV, p-AnV, and TV. These findings are consistent with those noted by Iqbal and Bhanger [45], who found that the chemical and sensory parameters are important to evaluate the efficacy of garlic antioxidants in sunflower oil.

Sensory Evaluation of SFO Incorporating Ga-NPsE
The shelf life of SFO at 65 °C and predictable shelf life during storage at 25 °C are shown in (Figure 4). The data reveal that the shelf life of SFO at 65 °C was 12 days for the control sample, 16 days for Ga-NPsE 200 ppm, 20 days for Ga-NPsE 600 ppm, 24 days for  Figure 3 depicts the sensory parameters, such as color, aroma, and acceptability, of sunflower oil (SFO) treatments during storage. The sensory assessment of SFO samples was done for different storage periods caused by the ending of shelf life with the intelligible development of an off flavor. Panelists generally approved of all sensory properties of SFO treatments at time zero; as the storage progressed, the sensory acceptability decreased. The control sample scored the lowest color value during the storage periods, while the oil sample incorporating Ga-NPs at different levels were accepted up to day 24. The aroma value differed significantly (p ≤ 0.05) between the oil treatments and the control group. SFO samples incorporating BHT, α-tocopherol or Ga-NPS 1000 ppm were accepted until day 24 of storage; however, the control was rejected on day 12 due to an off flavor. SFO samples' acceptability values decreased significantly over time, whereas BHT and Ga-NPS 1000 ppm samples did not differ after 24 days. The sensory data demonstrate that the control sample was rejected after day 12 of storage; however, the oil samples incorporating BHT, α-tocopherol, and Ga-NPS 1000 ppm were accepted up to day 24. These changes match well with chemical indicators, i.e., PV, p-AnV, and TV. These findings are consistent with those noted by Iqbal and Bhanger [45], who found that the chemical and sensory parameters are important to evaluate the efficacy of garlic antioxidants in sunflower oil.  conjugated dienes, and conjugated trienes were significantly lower than in the control. The antioxidant indices of SFO samples containing Ga-NPs were higher than the control. The Ga-NPsE enhanced the sensory acceptability of SFO treatments up to 24 days of storage. The garlic nanoparticles are more active due to their small size, widespread distribution, and interaction with various antioxidant modes of action. The results show that Ga-NPsE is an effective antioxidant for enhancing the stability of SFO and extending its shelf life (~384 days at 25 • C). However, more experiments are required to test the efficacy of Ga-NPsE antioxidants such as biological-based antioxidant mechanisms (electron transfer or inhibition of lipid peroxidation).