Flavonoid Profiles in the Pulp of Different Lemon Cultivars and Their Antioxidant Activity Based on UPLC–Q–TOF–MS

Previous studies have indicated that there may be differences among the varieties of lemon flavonoids, but the details have not yet been made clear, which limits the comprehensive use of different cultivated lemon varieties. In this study, ultra-performance liquid chromatography–quadrupole–time-of-flight–mass spectrometry (UPLC–Q–TOF–MS) and ultraviolet–visible spectroscopy (UV–Vis) were used to investigate the types and contents of flavonoids in the flesh of the main cultivated variety (Eureka) and five common lemon varieties, as well as their in vitro antioxidant activity. A total of 21 compounds were identified, five of which were common compounds. Among them, Verna, Lisbon, and Bearss each have characteristic components that can serve as potential criteria for variety identification. Each of the six varieties of lemon has strong antioxidant activity. The antioxidant activity of different lemon varieties is related to flavonoids. Therefore, Eureka and the other five varieties of lemon are good natural antioxidants, and the cultivation and industrial production of lemons should consider the needs and selection of suitable varieties.


Introduction
Lemon (Citrus limon) is one of the main cultivated citrus plants in the world; it is grown in more than 60 countries and regions (FAO Statistics, 2021) and is popular among consumers for its unique flavour [1,2].Previous studies have shown that lemons likely originated in China or India from the three ancestors of the citrus genus, namely C. Reticulata, pomelo, and citron, that were hybridized [2][3][4].Then, various locally cultivated varieties (e.g., Eureka, Lisbon, Femminello, and Fino) were produced after continued human intervention.These lemon varieties are important for industrial production.In addition to being used as fruits, lemons and their extracts are also processed for the production of other commodities, such as juice/juice beverages, preserved fruits, lemon essential oils, and food supplements.Recent studies have shown that lemons are rich in polyphenolic compounds, including flavonoids, phenolic acids, limonoids, and terpenoids, which have antioxidant, anti-inflammatory, antitumour, and lipid-lowering properties.They are widely used in fields such as medicine, food, and cosmetics [5][6][7][8][9][10].However, research on different lemon varieties is limited, which leads to confusion in the cultivation and industrial production of lemon varieties.Currently, Eureka is the most commonly cultivated variety, and more research and understanding are needed to fully utilize different lemon varieties.
Flavonoids are the main phenolics in citrus plants and are the main compounds that exert biological activity [11,12].Flavonoids that have been isolated and identified from lemons include eriodictyl, hesperidin, hesperetin, naringin, apigenin, diosmin, quercetin, lipocitrin, spinacetin, and neohesperidin.These components have been shown to be associated with lemon's anti-inflammatory, antioxidant, antibacterial, antitumour, and lipid-lowering properties [5,6,8,[10][11][12].However, current research has focused on the chemical composition and pharmacological effects of individual lemon varieties, or on the composition of essential oils from the peels of multiple varieties of lemon.There have been no studies on the distribution of flavonoids in the flesh of different varieties of lemon fruit.Therefore, it is necessary to further study the composition and content of flavonoids in different varieties of lemon pulp.
Flavonoids play an important role in antioxidant activity and provide many of the health benefits of healthy food.The biological activity, composition, and content of flavonoids are closely related [13][14][15].Previous studies have shown that lemon peel extract and lemon essential oil have good antioxidant activity [6,7,13].The differences in the antioxidant activities of flavonoids in different varieties of lemon pulp are still unclear.
The aim of this study was to clarify the differences in flavonoid compounds and antioxidant activities in different varieties of lemon pulp.The composition and content of flavonoids in lemon pulp were measured using five representative lemon varieties and one main cultivated variety (Eureka).Then, the differences in antioxidant activity were compared, and the correlations between different flavonoids and antioxidant activity were analysed.

Total Phenolic Content
The detection results of TPC in different varieties of lemon pulp are shown in Table 1.The results showed that the total phenolic content in the lemon pulp ranged from 27.60 to 46.19 mg GAE/g DW, and the total phenolic content of the main cultivated variety (Eureka) was 35.59 mg GAE/g DW.The total phenolic content in the Fino lemon was the highest, followed by that in Lisbon, Femminello, and Verna lemons, all of which were significantly greater than the total phenolic content in Eureka and Bearss lemons (p < 0.05).This may be related to the extraction method and testing variety of phenolic compounds [14,16,17].Compared with the distribution of phenolic compounds in different parts of lemon fruit [18], our results support that lemon pulp, especially that of Fino and Lisbon lemons, is a good source of phenolic compounds.

Total Flavonoid Content
The detection results of TFC in different varieties of lemon pulp are shown in Table 1.The results showed that the total flavonoid content in the lemon pulp ranged from 2.52 to 5.27 mg RE/g DW, and the total flavonoid content in the main cultivated variety (Eureka) was 3.74 mg RE/g DW.The TFC in the Fino lemon was the highest, followed by that in Femminello, Lisbon, and Eureka lemons, all of which were significantly greater than the TFC in Verna and Bearss lemons (p < 0.05).This may be related to the distribution of flavonoids in citrus fruits and the tested varieties [5,8,17,[19][20][21]. Flavonoids are most abundant in the peel of citrus fruits and have a lower content in the flesh.Our results indicate that the composition of flavonoids strongly depends on the lemon variety, and further analysis is needed to determine the differences in the types and contents of flavonoids.
Principal component analysis (PCA) of samples helps us understand the differences between group samples and the degree of variation within group samples, as shown in Figure 1.In the 3D PCA graph, samples from different groups are clustered separately, indicating that the metabolic profiles of different lemon varieties are different.The clustering of samples within the same group indicates a uniform distribution of metabolites, which confirms the repeatability and reliability of this experiment.According to the 2D PCA plot, Verna was significantly different from the five other lemons, indicating the uniqueness of its flavonoid compounds.To visualize the distribution of flavonoids in different varieties of lemon, we conducted a cluster heatmap analysis, as shown in Figure 2. Verna can be clustered separately from the other five types of lemons, consistent with the PCA results, and some flavonoid compounds have relatively high contents.The clustering of samples within the same group indicates that the composition and content of flavonoids in lemon pulp may be influenced by genotype.

Antioxidant Activity
The antioxidant activity of the six lemon varieties was evaluated using DPPH, ABTS, and FRAP, as shown in Figure 3.The results showed that the IC50 values of the DPPH and

Antioxidant Activity
The antioxidant activity of the six lemon varieties was evaluated using DPPH, ABTS, and FRAP, as shown in Figure 3.The results showed that the IC50 values of the DPPH and

Antioxidant Activity
The antioxidant activity of the six lemon varieties was evaluated using DPPH, ABTS, and FRAP, as shown in Figure 3.The results showed that the IC 50 values of the DPPH and ABTS clearance rates of these lemons were 2.10-3.79mg/mL and 3.62-6.49mg/mL, respectively, with a total antioxidant capacity of 97.80-114.35mg FeSO 4 /g DW, indicating differences in antioxidant activity among the different lemon varieties.The DPPH clearance rate of the Fino lemon was the highest, followed by those of the Lisbon, Verna, Femminello, and Eureka lemons, all of which were significantly greater than that of the Bearss lemon (p < 0.05).The ABTS clearance rate of the Verna lemon was the highest, followed by those of the Fino, Femminello, Lisbon, and Eureka lemons, all of which were significantly greater than that of the Bearss lemon (p < 0.05).The total antioxidant capacities of the Lisbon and Verna lemons were comparable, as were those of the Fino and Femminello lemons.The total antioxidant capacity of the Bearss lemon was significantly lower than that of the other five lemon varieties.
ences in antioxidant activity among the different lemon varieties.The DPPH clearance rate of the Fino lemon was the highest, followed by those of the Lisbon, Verna, Femminello, and Eureka lemons, all of which were significantly greater than that of the Bearss lemon (p < 0.05).The ABTS clearance rate of the Verna lemon was the highest, followed by those of the Fino, Femminello, Lisbon, and Eureka lemons, all of which were significantly greater than that of the Bearss lemon (p < 0.05).The total antioxidant capacities of the Lisbon and Verna lemons were comparable, as were those of the Fino and Femminello lemons.The total antioxidant capacity of the Bearss lemon was significantly lower than that of the other five lemon varieties.
Our results indicate that the antioxidant activity reflected by different methods is not entirely consistent, which may be related to phenolic substances.There are differences in the types and contents of phenolic substances in different varieties of lemon pulp, resulting in different antioxidant activity detection results for different indicators [14,22].Overall, the differences in antioxidant activity among the different varieties of lemon pulp were consistent with the trends in the total phenolic and flavonoid contents.The antioxidant activity of the different varieties of lemons is lower than that of oranges but greater than that of fruits such as pomelo, grapefruit, pear, watermelon, and mango [14,15,23].

Correlations between TPC, TFC, Flavonoids, and Antioxidant Activity
To investigate the correlation between flavonoids and antioxidant differences in different varieties of lemon, we conducted a Pearson correlation analysis, as shown in  Our results indicate that the antioxidant activity reflected by different methods is not entirely consistent, which may be related to phenolic substances.There are differences in the types and contents of phenolic substances in different varieties of lemon pulp, resulting in different antioxidant activity detection results for different indicators [14,22].Overall, the differences in antioxidant activity among the different varieties of lemon pulp were consistent with the trends in the total phenolic and flavonoid contents.The antioxidant activity of the different varieties of lemons is lower than that of oranges but greater than that of fruits such as pomelo, grapefruit, pear, watermelon, and mango [14,15,23].

Correlations between TPC, TFC, Flavonoids, and Antioxidant Activity
To investigate the correlation between flavonoids and antioxidant differences in different varieties of lemon, we conducted a Pearson correlation analysis, as shown in Figure 4

Plant Material
All lemon samples were collected from the planting base of the Lemon Industry Bureau in Anyue County, Sichuan Province, China, in November 2023.Four adjacent lemon trees of high yield and stable quality were randomly selected, and 6 fresh fruits of similar size that were free from pests and diseases were randomly selected from each tree.The varieties (Eureka, Lisbon, Femminello, Fino, Bearss, and Verna) were numbered and transported to the laboratory using dry ice.The samples were washed, the peel and pulp were manually separated, and the pulp was freeze-dried using the following method: The lemon pulp was evenly spread in a single layer and placed in a vacuum freeze dryer, cooled to −30℃, and pre-frozen for 5 h before the heating plate temperature of the freeze dryer was set to 50 °C and the condenser temperature to −50 °C.After sealing, the pump was set to a pressure of 10 Pa and the sample dried for 72 h.The pulp was crushed with a grinder after drying, and all the samples were stored in a dry place at room temperature.

Plant Material
All lemon samples were collected from the planting base of the Lemon Industry Bureau in Anyue County, Sichuan Province, China, in November 2023.Four adjacent lemon trees of high yield and stable quality were randomly selected, and 6 fresh fruits of similar size that were free from pests and diseases were randomly selected from each tree.The varieties (Eureka, Lisbon, Femminello, Fino, Bearss, and Verna) were numbered and transported to the laboratory using dry ice.The samples were washed, the peel and pulp were manually separated, and the pulp was freeze-dried using the following method: The lemon pulp was evenly spread in a single layer and placed in a vacuum freeze dryer, cooled to −30 • C, and pre-frozen for 5 h before the heating plate temperature of the freeze dryer was set to 50 • C and the condenser temperature to −50 • C.After sealing, the pump was set to a pressure of 10 Pa and the sample dried for 72 h.The pulp was crushed with a grinder after drying, and all the samples were stored in a dry place at room temperature.

Sample Preparation and Extraction
The sample processing followed the method of Ledesma Escobar et al. [31].Lemon pulp powder (0.15 g) was accurately weighed, 5 mL of 80% methanol solution was added, and the mixture was mixed evenly.The sample was extracted using ultrasonication for 60 min (vortexing every 15 min).Then, the mixture was centrifuged for 15 min (4000 rpm/min), the supernatant was collected, and 0.22 µM filter membrane filtration was used.The filtrate was stored in an injection bottle for ultra-performance liquid chromatography-quadrupole-time-of-flight-mass spectrometry (UPLC-Q-TOF-MS) analysis.

Measurement of Total Phenolic Content
The Folin-Ciocalteu colorimetric method [32] was used to determine the total phenolic content (TPC), and the results are presented as mg gallic acid equivalents (GAE)/g dry weight (DW) of the pulp sample.Gallic acid solutions with concentrations of 0, 0.004, 0.005, 0.006, 0.008, 0.009, and 0.01 mg/mL were prepared.Then, 0.25 mL of phenol reagent, 4.75 mL of 20% sodium carbonate solution, 0.5 mL of gallic acid solution, or different concentrations of lemon extract were added, the solution was mixed well, 80% methanol was added to bring the volume to 15 mL, and the mixture was placed in a water bath at 75 • C for 30 min.The solution was cooled to room temperature, and the absorbance was measured at 760 nm.The standard curve was y = 10.697x− 0.0324 (R 2 = 0.9994).

Measurement of Total Flavonoid Content
The total flavonoid content (TFC) was determined using the sodium nitrite-aluminium nitrate-sodium hydroxide colorimetric method [33].A 0.2 mg/mL rutin solution consisting of 0 mL, 0.5 mL, 1.0 mL, 2 mL, 2.5 mL, 4.0 mL, 5.0 mL, or 3.0 mL of lemon extract was prepared.Then, the following solutions were added: 0.75 mL of 5% sodium nitrite, 0.75 mL of 10% aluminium nitrate, 6 mL of 4% sodium hydroxide, and, finally, 80% methanol to adjust the volume to 15 mL.After each addition, the mixture was mixed well and allowed to stand for 6 min, and for 15 min after methanol addition.The absorbance was measured at 510 nm.The standard curve was y = 12.55x − 0.0095 (R 2 = 0.9998).The results are presented as mg rutin equivalent (RE)/g DW of the pulp sample.
The collected raw data were imported into Compound Discoverer 3.0 software, the peak areas and peak alignments were extracted, and the measured spectra of secondary fragments were matched with the mzCloud network database and the local traditional Chinese medicine ingredient database (OTCML).The set filtering parameters for the matching results were as follows: peak area threshold value: 80,000, primary and secondary quality deviation: 5 ppm, and matching degree score: above 80.Compounds were analysed and identified by comparing the filtered ions with the compound information in the database.

Antioxidant Activity
DPPH.The DPPH assay was conducted following the protocol outlined by Wang et al. [15] with adjustments.The lemon extracts of different concentrations (100 µL) were combined with DPPH solution (0.008 mg/mL) (100 µL) in a 96-well plate.The mixtures underwent a 30-min reaction at room temperature in darkness, and the measurement of absorbance value at 517 nm was conducted using a microplate reader.In the control group, substituting the DPPH solution with 80% methanol, the blank group was similarly treated with 80% methanol.The IC 50 value represents the DPPH clearance rate.
ABTS.Refer to Wang et al.'s method for ABTS determination, which was followed with slight modifications [15].The K 2 S 2 O 8 solution (2.6 mmol/L) was added to the ABTS solution (7.00 mmol/L) and allowed to react thoroughly in a cool place for 12-16 h.The solution obtained after dilution of the mixture seven times with 80% methanol (with an absorbance of 7.00 ± 0.02 at 734 nm) was used to prepare the ABTS working solution.Following that, 25 µL aliquots of lemon extract of different concentrations were introduced into the 96-well plate, the working solution of ABTS, a volume of 175 µL, was thoroughly mixed and allowed to react in darkness for a duration of 40 min, followed by measuring the absorbance value at 734 nm using a microplate reader; within the control group, substituting ABTS solution with 80% methanol, the blank group was also treated with 80% methanol.The IC 50 value represents the ABTS clearance rate.
FRAP.The FRAP clearance rate was determined using a total antioxidant assay kit (Nanjing Jiancheng Bioengineering Research Institute, China).Overall, the standard determination curves of FeSO 4 solutions with concentrations of 0.15, 0.3, 0.6, 0.9, 1.2, and 1.5 mmol/L were prepared.Then, 180 µL of FRAP working fluid was introduced into the wells of the 96-well plate, and 5 µL each of different concentrations of FeSO 4 solutions and lemon extracts were added.This was followed by the addition of 5 µL of each solution in the blank group; the solution extraction was substituted with distilled water.Incubation of the mixture was carried out at 37 • C for 3-5 min, and the absorbance value was measured at 593 nm using a microplate reader.The standard curve is: y = 0.007x − 0.0067, R 2 = 0.9974.The results are presented as mg FeSO 4 /g DW of the pulp sample.The standard curve is: y = 0.007x − 0.0067, R 2 = 0.9974.

Statistical Analysis
The statistical function prcomp in R version 3.3.1 for principal component analysis, and the ComplexHeatmap package in R version 3.3.1 for hierarchical cluster analysis (HCA) and production of a heatmap were used.GraphPad Prism 8.4.3 (GraphPad Software company, La Jolla, CA, USA) was used for statistical analysis, plotting bar charts, and ANOVA analysis (p < 0.05 indicated statistical significance).All results are the average of three experiments.

Conclusions
This study evaluated the differences in TPC, TFC, and antioxidant activity among different lemon varieties.Fino had the highest total phenolic content, followed by Lisbon, Femminello, and Eureka, all of which were significantly greater than those of Verna and Bearss.Fino lemon had the highest total flavonoid content, followed by Femminello, Lisbon, and Eureka, all of which were significantly greater than those of Verna and Bearss.Fino had the highest DPPH clearance rate, followed by Lisbon, Verna, Femminello, and Eureka, all of which were significantly greater than that of Bearss.The ABTS clearance rate of Verna was the highest, followed by those of Fino, Femminello, Lisbon, and Eureka, all of which were significantly greater than that of Bearss.The total antioxidant capacities of Lisbon and Verna were comparable, as were those of Fino and Femminello.The total antioxidant capacity of Bearss was significantly lower than that of the other five lemon varieties.

Figure 3 .
Figure 3.The antioxidant activity of the different lemon varieties.(A) ABTS measurement; (B) DPPH measurement; (C) The FRAP method was used to determine the total antioxidant activity of the different lemon varieties.The different lowercase le ers indicate significant differences (p < 0.05)

Figure 3 .
Figure 3.The antioxidant activity of the different lemon varieties.(A) ABTS measurement; (B) DPPH measurement; (C) The FRAP method was used to determine the total antioxidant activity of the different lemon varieties.The different lowercase letters indicate significant differences (p < 0.05).

Figure 4 .
Figure 4. Pearson correlation analysis heatmap of flavonoids in different lemon varieties.

Figure 4 .
Figure 4. Pearson correlation analysis heatmap of flavonoids in different lemon varieties.

Table 1 .
The TPC and TFC in different varieties of lemon.

Table 2 .
Volatile organic compounds identified in dry peel of Zangju at different ripening stages, sampled by GC-IMS.