The “Red Face” strawberry variety was grown in Dandong, northeast China (E 124.23′, N 40.07′, elevation 20 meters above sea level), and was subsequently cultivated in Zhaotong (E 103.72′, N 27.34′, elevation 1900 meters above sea level). For simplicity and clarity, the “Red Face” (Fragaria × ananassa) variety Dandong-grown was named as DD99, and cultivated in Zhaotong was named as ZT99, and the indigenous Zhaotong strawberry variety “Akihime” (Fragaria × ananassa Duch.) was named as ZT.

Environmental data for both locations was obtained through online link https://globalsolaratlas.info on November 30, 2023. The UV radiation differential between Zhen’an (Dandong) and Zhaoyang (Zhaotong) is attributable to distinct geographical and climatic parameters. Dandong’s higher latitude (40°N) results in reduced UV exposure due to its temperate conditions, while Zhaotong’s lower latitude (28°N) experiences enhanced UV radiation. The substantial altitude differential significantly influences UV intensity, with Zhaotong’s 1900m elevation reducing atmospheric attenuation. Furthermore, Dandong’s humid climate with high precipitation contrasts with Zhaotong’s arid conditions and predominant clear skies, particularly during autumn and winter. Consequently, Zhaoyang experiences elevated UV exposure due to the cumulative effects of elevation, latitude, aridity, and increased solar radiation compared to Zhen’an.

Experimental materials comprised ZT plants obtained from Zhaotong University research plots and DD99 seedlings sourced from Dongji Luyuan Agriculture and Animal Husbandry Co., Ltd. in Fengcheng, Dandong. In August 2023, seedlings approximately 10 cm in height were transplanted under greenhouse conditions. ZT99 and ZT were cultivated at Zhaotong University’s greenhouse facility, while DD99 was grown at Dongji Luyuan’s facility, both under natural environmental conditions following standardized cultivation protocols, optimum fertilization, sandy loam soil, and normal irrigation practices were performed (Reganold et al., 2010; Bottoms et al., 2013). Mature fruit samples were collected simultaneously from all three DD99, ZT99, and ZT samples on December 12, 2023. Each treatment consisted of five biological replicates from individual plants, which were processed via liquid nitrogen grinding, divided into technical triplicates, and preserved in cryotubes. Samples were maintained in liquid nitrogen before transfer to -80°C storage for subsequent UPLC-MS/MS analysis of anthocyanin and proanthocyanidin profiles.

Acetic acid (C₂H₄O₂), acetone (CH₃)₂CO, aluminum chloride hexabydrate (AlCl₃•6H₂O), 2-(3,4-dihydroxyphenyl)-3,4-dihydro-2H-chromene-3,5,7-triol (catechin C₁₅H₁₄O₆), 2,2-diphenyl-1-picrylhydrazyl (DPPH C₁₈H₁₂N₅O₆), ethanol C₂H₅OH, methanol (CH₃OH), hydrochloric acid (HCl), quercetin-3-O-rutinoside (rutin C₂₇H₃₀O₁₆), sodium hydroxide (NaOH), sodium nitrite (NaNO₂), and 6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (trolox C₁₄H₁₈O₄) were purchased from Sigma-Aldrich (Shanghai, China) and quercetin (C₁₅H₁₀O₇) was purchased from Xi’an ZB Biotech Co., Ltd., (Shaanxi, China). The metaphosphoric acid (HPO₃) and 2,6-dichlorophenolindophenol (C₁₂H₆Cl₂NNaO₂) were acquired from Suzhou Senfeida Chemical Co., Ltd., (Jiangsu, China) while the L-ascorbic acid (C₆H₈O₆) from Dayang Chem (Hangzhou) Co., Ltd., (Zhejiang, China).

Flavonoid analysis in strawberry fruits was conducted using UPLC-MS/MS methodology. Fruit samples were processed through freeze-drying and pulverization, after which 50 mg of the resultant powder was weighed into tubes for flavonoid analysis. The extraction procedure involved the addition of 500 μL of extraction solution (50% methanol in water containing 0.1% HCl) supplemented with Rutin as an internal standard (4000 nmol/L). The samples underwent ultrasonic extraction for 30 minutes, followed by centrifugation at 12,200×g for 5 minutes at ambient temperature. The supernatant was carefully collected and filtered through a 0.22 μm filter membrane prior to UPLC-MS/MS analysis. Analytical parameters and instrumental configuration were implemented according to established protocol (Chen et al., 2013). Flavonoids identification was achieved through comparative analysis of Q1 precursor ions, Q3 product ions, fragmentation patterns, and peak areas with reference standards under identical analytical conditions, as previously described (Chen et al., 2013). All analyses were performed in triplicate, and mass spectrometric data were processed using Analyst 1.6.3 software. Detailed specifications of the analyzed compounds are presented in Supplementary Table S1 .

The extraction of total flavonoids was initiated by homogenizing 50 mg of strawberry fruit tissue with 2.5 mL of 80% aqueous methanol solution. Following homogenization, a 0.5 mL aliquot of the supernatant was collected and gently mixed with 2.25 mL of deionized water. The mixture was subsequently treated with 0.15 mL of 5% sodium nitrite solution and incubated at room temperature for 6 minutes. After incubation, 0.3 mL of 10% aluminum chloride hexahydrate solution was added and allowed to react at ambient temperature. The reaction was completed by adding 1 mL of 1M sodium hydroxide, followed by a 2-minute incubation period. Spectrophotometric analysis was performed at 510 nm using a UV-1800 (Shimadzu Corporation, Japan) spectrophotometer (Dewanto et al., 2002). Total flavonoid content was quantified through comparison with a standard rutin calibration curve, and results were expressed as mg/g of rutin.

Total soluble solids (TSS) were measured using a digital refractometer (Magwaza and Opara, 2015). The instrument was calibrated to zero using distilled water prior to analysis. Strawberry juice was obtained through gentle compression of the fruit, and a single drop was placed on the refractometer’s prism. Measurements were performed in triplicate at ambient temperature (21 ± 2°C) and results are presented as Brix.

Ascorbic acid content was determined via titration (Rekha et al., 2012). Sample preparation consisted of homogenizing 10 g of strawberry tissue with 90 mL of 3% metaphosphoric acid solution, followed by filtration. A 10 mL filtrate aliquot was titrated against 2,6-dichlorophenolindophenol until a stable pink coloration persisted for 15 seconds. Vitamin C concentrations were calculated using an L-ascorbic acid standard curve and expressed as mg/g fresh weight (Cordenunsi et al., 2003).

Titratable acidity (TA) was assessed by combining 10 g of strawberry puree with 90 mL of distilled water (Cordenunsi et al., 2003). The mixture was titrated with 0.1 N sodium hydroxide to the appropriate pH endpoint. TA percentage was calculated according to the following Equation 1:

Where 0.064 represents the citric acid milliequivalent factor.

Tissue mechanical resistance was evaluated using a GY-4 fruit firmness analyzer (GY-4 model, TOP Cloud-agri, Guangzhou, China). Following removal of a small epidermal section, measurements were conducted at two opposing points along the fruit’s equatorial region (Cordenunsi et al., 2003). The maximum force (N) required for a 2 mm compression was recorded using a 4 mm diameter stainless steel cylindrical probe.

The assessment of antioxidant properties was performed utilizing the DPPH radical scavenging assay (Dudonne et al., 2009). A 0.1 g sample was subjected to extraction using a solvent system comprising ethanol (70%), water (29%), and acetic acid (1%). All reagents required for DPPH analysis were obtained from Sigma-Aldrich. The extraction procedure involved combining 1 mL of the solvent solution with 100 mg of strawberry sample. Following thorough homogenization, the mixture was centrifuged at 12,208×g for 8 minutes. The reaction mixture was prepared by combining 2.97 mL of 0.1 mM DPPH solution with 0.03 mL of the supernatant from the extracted sample. The reaction was allowed to proceed under dark conditions for 30 minutes, after which absorbance measurements were recorded at 517 nm using a UV-1800 spectrophotometer. A control solution was prepared following the same protocol, substituting the sample extract with deionized water (0.03 mL). Results were expressed as millimolar trolox equivalents per 100 mg sample. The radical scavenging activity was calculated according to the following Equation 2:

The antioxidant capacity of strawberry fruits was assessed utilizing the Ferric Reducing Antioxidant Power (FRAP) assay (Benzie and Strain, 1996). The FRAP reagent was prepared by combining three components in a 1:10:1 ratio: ferric chloride hexahydrate (FeCl₃•6H₂O, 20 mmol in 40 mmol/L HCl), acetate buffer (300 mmol, pH 3.6), and 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ) solution (100 mmol/L in 40 mmol/L HCl). For the analysis, strawberry fruit tissue samples (100 mg) were homogenized, and an aliquot of 25 μL supernatant was combined with 175 μL FRAP reagent. The mixture was incubated at room temperature in darkness for 30 minutes. The absorbance of the resultant blue Fe²⁺-TPTZ complex was measured spectrophotometrically at 593 nm using a UV-1800 spectrophotometer. A blank measurement was conducted using acetate buffer as a reference. The final FRAP value was calculated by subtracting the blank absorbance from the sample absorbance. This methodology quantified the antioxidant potential through the assessment of ferric ion reduction capacity, with results expressed as mmol Fe²⁺ per gram of strawberry fruits tissue (Benzie and Strain, 1996).

The antioxidant activity was evaluated utilizing an adapted protocol based on the ABTS radical scavenging method (Dudonne et al., 2009). The ABTS•+ stock solution was prepared by combining equimolar volumes of ABTS (7 mmol/L) and potassium persulfate (2.45 mmol/L) solutions. The reaction mixture was incubated in darkness at an ambient temperature for 12 hours to ensure complete radical generation. Subsequently, the stock solution was diluted with methanol to achieve an absorbance of 0.70 ± 0.02 at 734 nm, measured using a UV-1800 spectrophotometer. The experimental protocol consisted of combining 2 mL of ABTS•+ solution with 1 mL of sample extracts at concentrations ranging from 0.5-5.0 mg/mL. The mixture was incubated under dark conditions at room temperature for 10 minutes. A control was prepared by combining 2 mL ABTS•+ solution with 1 mL double-distilled water, while butylated hydroxytoluene (BHT) was used as the reference standard. Absorbance measurements were conducted in triplicate at 734 nm against a blank using the UV-1800 spectrophotometer. The radical scavenging capacity was calculated using the following Equation 3:

Metabolomic data underwent Z-score normalization procedures prior to hierarchical cluster analysis (HCA). The statistical framework encompassed HCA and principal component analysis (PCA), following standardized protocols outlined in reference (Mu et al., 2024). Graphical visualizations and standard error computations were executed using Microsoft Excel software. The least significant difference test was utilized to assess variations in antioxidant concentrations and total flavonoid levels across different strawberry varieties. All statistical analyses of antioxidant activities and flavonoids content were performed using Statistix 8.1 software, with triplicate biological samples.

Hierarchical cluster analysis (HCA) was achieved to assess the variation and abundance of flavonols across different strawberries grown in diverse environments ( Figure 1A ). The analysis revealed that the DD99 variety, grown in a lower-altitude environment, formed a distinct cluster compared to ZT99 and ZT variety grown at higher altitudes ( Figure 1A ). This finding indicates that altitude-ecological conditions significantly influence flavonol biosynthesis in strawberry fruits. Approximately 20 flavonols exhibited higher abundance in the DD99 variety compared to ZT and ZT99 ( Figure 1A ). This observation underscores the significant impact of diverse ecological conditions on the synthesis of beneficial bioactive compounds within the plants. The ZT variety demonstrated a higher abundance of 18 flavonol compounds, while ZT99 showed a higher abundance of 33 flavonols compared to DD99, and 24 flavonols were more abundant in ZT99 fruits than in the ZT variety ( Figure 1A ). Notably, cultivation of the “Red Face” strawberry, introduced from Dandong to Zhaotong resulted in a marked increase in the concentration of several health-promoting flavonols in ZT99 fruits compared to the native Zhaotong ZT variety.

The variety-wise principal component analysis (PCA) revealed distinct separations of ZT, ZT99, and DD99 varieties ( Figure 1B ). The ZT variety clustered in the upper-left quadrant of the PCA plot, while DD99 was positioned on the right side on the x-axis. The ZT99 was located in the lower-left quadrant ( Figure 1B ). This finding highlights the substantial influence of diverse environmental conditions on the production of these beneficial compounds within the strawberry fruits. PC1 accounted for 54.3% of the variation along the x-axis, while PC2 explained 22.8% of the variation. These results indicate significant variations in the flavonol profiles among the three strawberries.

Compound-wise PCA analysis demonstrated that the majority of compounds clustered at the intersection of the x- and y-axis, with the exception of 10 flavonol compounds ( Figure 1C ). These 10 flavonols were distinctly separated from the main cluster and accounted for the maximum variation in both PC1 and PC2. PC1 explained 78.6% of the variation along the x-axis, while PC2 accounted for 20.9% of the variation along the y-axis. These findings suggest that strawberry plants undergo biochemical adaptations in response to varying growth environments, potentially enhancing their nutritional value and pharmaceutical applications.

A comparative analysis of flavonoid profiles (including 37 flavanones, 33 flavones, and 8 flavanonols) in strawberries cultivated under varying environmental conditions was conducted using HCA ( Figure 2A ). The results reveal distinct patterns in flavonoids abundance and distribution across different strawberry varieties. Similar to flavonol HCA analysis, the flavonoids profiles of DD99 exhibited a unique clustering pattern when compared to the ZT99 and ZT varieties ( Figure 2A ). The HCA analysis identified approximately 28 flavonoids that were more prevalent in the DD99 variety compared to ZT and ZT99 varieties ( Figure 2A ). Furthermore, the ZT variety showed elevated levels of 13 flavonoid compounds than DD99 and ZT99, while the ZT99 strawberry demonstrated higher concentrations of 43 flavonoids relative to DD99. Additionally, 22 flavonoids were found to be more abundant in ZT99 fruits compared to the ZT ( Figure 2A ). The Red Face variety cultivated in Zhaotong (ZT99), resulted in a significant increase in the levels of various health-promoting flavonoids, than those found in the native ZT strawberry variety. Also, these results suggest that the altitude-associated ecological factors play a crucial role in modulating flavonoids biosynthesis in strawberry fruits.

The PCA of flavonoid varieties revealed that the PC1 accounted for 54.3% of the total variance along the horizontal axis, while the PC2 explained 24% of the variance on vertically axis ( Figure 2B ). In the PCA plot, the ZT variety clustered in the upper-left quadrant, DD99 was positioned on the right side near to the horizontal axis, and ZT99 was located in the lower-left region ( Figure 2B ). A separate PCA focusing on individual compounds demonstrated that the majority of flavonoids clustered near the origin, with the exception of six distinct compounds ( Figure 2C ). Among these, four were situated adjacent to the main cluster, while two were notably distant and contributed significant variation in the PCA plot ( Figure 2C ). In this analysis, PC1 represented 74% of the variance along the x-axis, and PC2 accounted for 25.6% of the variance on the y-axis ( Figure 2C ). The observed variations in flavonoid profiles among the strawberries demonstrate the significant impact of diverse altitude-associated ecological conditions on the biosynthesis and accumulation of these bioactive compounds within strawberry fruits.

Among the 163 identified flavonoids, 115 flavonoids exhibited more than a two-fold change, either up-regulated or down-regulated, across distinct strawberry varieties ( Table 1 ). The results indicate that 55 flavonoids were more than two-fold higher in ZT99 samples compared to DD99, while 30 flavonoids were more than two-fold down-regulated in ZT99 relative to DD99 samples ( Table 1 ). Furthermore, 34 flavonoids were found to be elevated (more than 2-folds) in ZT99 samples compared to ZT, whereas 14 flavonoids were more than two-fold down-regulated in ZT99 samples compared to ZT ( Table 1 ). Additionally, 43 flavonoids exhibited more than a two-fold increase in ZT compared to DD99, while 37 flavonoids were more than two-fold down-regulated in ZT compared to DD99 ( Table 1 ). These results showed that the high-altitude-associated conditions of Zhaotong facilitate ZT99 fruits to accumulate more flavonoid compounds than the DD99 fruits grown at lower-altitude-associated conditions of Dandong.

The 115 flavonoid compounds that were 2-fold or more than 2-fold altered among strawberry varieties ( Table 1 ). The DD99 strawberry variety which is cultivated at lower-altitude showed that the chrysoeriol-7-O-glucoside 53.9, 6-hydroxykaempferol-6,7-O-diglucoside 36.3, eucalyptin 9.7, quercetin-3-O-robinobioside 8.8, and hamiltone A 8.3, were fold higher than ZT99 strawberry, which is cultivated at higher-altitude environment of Zhaotong ( Table 1 ). The ZT99 strawberry showed that the neohesperidin 20.4, tamarixetin-3-O-glucoside-7-O-rhamnoside 17.7, isovitexin 9.1, hesperidin 8.5, tamarixetin-3-O-rutinoside 5.4, quercetin-3-O-sambubioside-7-O-rhamnoside 6.9, quercetin 3-O-glucoside 7-O-xyloside 6.1, narcissin 5.8, and patuletin-3-O-glucoside 5.2, were fold higher than DD99 strawberry variety ( Table 1 ). This differential concentrations of flavonoids in response to varying environmental conditions, providing valuable insights into the metabolic adaptations of these strawberries.

The relative abundance of major flavonoid compounds was calculated as a percentage of total flavonoids ( Figure 3 ). Tricetin 3’-glucuronide exhibited the highest percentage, accounting for 24.49%, 24.14%, and 15.31% in ZT99, ZT, and DD99 samples, respectively. This was closely followed by quercetin-4’-O-glucuronide, which constituted 24.15%, 23.88%, and 15.59% in ZT99, ZT, and DD99 samples, respectively ( Figure 3 ). These two compounds emerged as the predominant flavonoids in the strawberry fruits analyzed. Notably, the relative abundance of these compounds was significantly higher in fruits cultivated at higher altitudes (ZT99 and ZT) compared to those grown at lower elevations (DD99) ( Figure 3 ). This observation suggests a potential correlation between altitude-associated ecological conditions and flavonoid accumulation in strawberry fruits. Similarly, the percentage of quercetin-5-O-glucuronide was significantly higher in ZT99 (11.26%) and ZT (10.36%) compared to DD99 (6.02%) ( Figure 3 ). This trend further corroborates the hypothesis that altitude-associated ecological conditions may play a crucial role in modulating flavonoid biosynthesis and accumulation in strawberry fruits.

The concentrations of specific flavonoids such as apigenin-5-O-glucoside, aromadendrin-7-O-glucoside, and luteolin-7-O-(6’’-malonyl)glucoside were notably higher in DD99 (11.12%, 3.62%, and 2.34%, respectively) compared to ZT99 (2.24%, 1.5%, and 1.28%, respectively) and ZT (7.63%, 1.66%, and 0.71%, respectively) ( Figure 3 ). These findings indicate that the Red Face variety, when cultivated at lower altitudes (DD99), exhibited elevated levels of these compounds. Conversely, their concentrations decreased significantly in samples of the same variety grown under higher-altitude (ZT99) ecological conditions in Zhaotong. Interestingly, poncirin levels did not show significant altitude-dependent variation between DD99 (2.35%) and ZT99 (2.27%); however, the ZT variety demonstrated a significantly lower poncirin concentration (0.96%) ( Figure 3 ). These observed variations in flavonoid profiles suggest potential adaptive mechanisms in response to altitude-associated environmental factors. Such insights could prove valuable for breeding programs aimed at enhancing the nutritional and therapeutic value of strawberries.

A correlation heat map analysis was conducted to examine the relationship between flavonoid compounds and altitude ( Figure 4 ). The analysis revealed distinct distribution patterns of flavonoid metabolites, with 36 compounds showing positive correlations with elevation (indicated in red) ( Figure 4 ). Among these positively correlated compounds, four flavonoids - 3’-methoxyquercetin-3-O-L-rhamnosyl(1→2)-glucopyranoside, robinetin, gossypetin-8-O-β-D-glucuronide, and narcissin - demonstrated particularly strong and statistically significant correlations. These compounds exhibited consistent altitude-dependent accumulation patterns, with higher concentrations observed in strawberry samples harvested from the high-altitude ecological conditions of Zhaotong compared to the lower-altitude conditions of Dandong.

Conversely, 29 flavonoid compounds exhibited negative correlations with altitude, indicating an inverse relationship between their abundance and elevation. Among these, quercetin-3-O-robinobioside, kaempferol-3-O-(2’’-O-acetyl)glucoside, and luteolin-7-O-rutinoside demonstrated strong and significantly negative correlations (highlighted in blue color) ( Figure 4 ). The concentrations of these metabolites showed a significant decrease in strawberry fruits harvested from the high-altitude ecological conditions of Zhaotong compared to the lower-altitude conditions of Dandong, suggesting adaptive responses to elevation-associated environmental conditions. These bidirectional correlation patterns provide valuable insights into how altitude-dependent ecological conditions influence the regulation of endogenous flavonoid metabolism.

The ZT and ZT99 strawberries exhibited significantly higher total flavonoid contents (19.28 mg/g and 19.26 mg/g of rutin, respectively) compared to DD99 (14.05 mg/g of rutin) ( Figure 5 ). The ZT99 strawberries demonstrated a significantly higher percentage of flavonols (54.61%), followed by ZT (49.65%), while DD99 exhibited a significantly lower percentage of flavonol content (43.37%) ( Figure 5 ). These findings indicate that the “Red Face” strawberries harvested from higher-altitudes (ZT99) conditions of Zhaotong significantly enhances total flavonols accumulation compared to samples harvested from lower-altitudes conditions (DD99) of Dandong. Regarding flavones, ZT displayed a higher percentage (40.75%), followed by DD99 (38.88%), whereas ZT99 strawberry fruits showed lower percentage of flavones (34.45%); however, the flavone variations among strawberry samples were insignificant ( Figure 5 ).

Antioxidant profiling was conducted to evaluate the antioxidant potential and free radical scavenging capacities of strawberry fruits using multiple assays ( Figure 6 ). The “Red Face” strawberry samples obtained from the high-altitude region of Zhaotong (ZT99) demonstrated superior antioxidant properties across all antioxidant assays. Specifically, ZT99 samples exhibited enhanced DPPH activity of 65.34% and antioxidant capacity of 91.39 mm TE/100 mg, followed by ZT samples. In contrast, “Red Face” strawberry samples from Dandong (DD99) exhibited significantly lower DPPH activity (35.47%) and antioxidant capacity (49.46 mm TE/100 mg). Furthermore, FRAP analysis revealed elevated reducing capacity of 27.76 mmol Fe²⁺/g and ABTS•+ radical cation scavenging activity of 79.92 PS% in ZT99 samples. DD99 samples, however, demonstrated significantly reduced antioxidant capabilities, with FRAP values of 11.45 mmol Fe²⁺/g and ABTS•+ scavenging activity of 48.28 PS% ( Figure 6 ). These findings suggest that the cultivation of the Red Face variety under high-altitude environmental conditions in Zhaotong resulted in significant increment of antioxidant scavenging activities, potentially attributed to environmental responses and adaptive mechanisms induced by high-altitude ecological conditions.

Strawberry fruit quality attributes assessment was conducted to evaluate key physicochemical parameters, including soluble solids content (SSC), ascorbic acid concentration, titratable acidity, and fruit firmness ( Table 2 ). The ZT99 fruit samples demonstrated significantly superior quality characteristics, particularly regarding SSC (15.30°Brix) and ascorbic acid content (15.73 mg/g), compared to DD99 fruit samples, which exhibited markedly lower values of 10.96°Brix and 8.53 mg/g, respectively ( Table 2 ). Notably, the ZT strawberry samples showed reduced levels of ascorbic acid (7.82 mg/g), titratable acidity (0.31%), and fruit firmness (8.46 N) ( Table 2 ). No significant variations in titratable acidity and firmness were observed between DD99 and ZT99 samples. Comparative assessment of the “Red Face” strawberry variety under contrasting ecological conditions demonstrated that high-altitude cultivation significantly enhanced both soluble solid content and ascorbic acid synthesis. Specifically, fruits harvested from the ZT99 high-altitude site exhibited higher levels of these compounds compared to those from the DD99 low-altitude environments in Dandong ( Table 2 ). These findings exhibit the substantial influence of ecological conditions on the development of key quality attributes in strawberry fruits.

The phytochemical correlation analysis revealed complex interconnections among diverse bioactive compounds and quality parameters. Total concentrations of flavonoids, flavonols, and flavones demonstrated statistically significant positive correlations with all variables, except the ascorbic acid, titratable acid, and firmness ( Table 3 ). This observation suggests coordinated biosynthetic pathways among these flavonoid compounds. All antioxidant scavenging assays exhibited statistically significant positive correlations with one another, as well as with flavonoids, flavonols, and sugars ( Table 3 ), indicating these metabolites’ substantial contribution to the samples’ overall antioxidant capacity. Ascorbic acid exhibited non-significant positive correlations with all variables except flavones, where a slight negative correlation was observed ( Table 3 ). Titratable acid and firmness demonstrated negative correlations with all variables except ascorbic acid. Notably, titratable acid and ascorbic acid displayed a significant positive correlation ( Table 3 ), suggesting potential shared regulatory mechanisms or complementary roles in fruit acid metabolism. These findings provide insights into the intricate interactions between strawberry fruits quality parameters and their bioactive compounds, which may have significant implications for future breeding programs and postharvest management strategies.