Skip to main content
NCBI home page
ACS Omega logoLink to ACS Omega
. 2025 Oct 3;10(40):47535–47543. doi: 10.1021/acsomega.5c07413

Plant Maturity Differentially Affects the Phenolic Composition and Antioxidant Properties of Green Basil (Ocimum basilicum L.) Cultivars

Holly P Lawson 1, Mattigan F Aga 1, Emily D Niemeyer 1,*
PMCID: PMC12529196  PMID: 41114168

Abstract

Basil is an aromatic herb of culinary importance as well as a rich source of phenolic compounds, secondary plant metabolites with strong antioxidant properties that are associated with dietary health benefits. Although basil is cultivated worldwide, little is known about how its phytochemical content changes as the plant matures, information that is useful to identify harvest strategies that maximize the herb’s nutritional value. Therefore, the aim of this study was to determine the effects of plant development on the phenolic composition and antioxidant properties of three green basil cultivars (Genovese, Spicy, and Tuscany) that were harvested weekly throughout the vegetative stage. Average total phenolic content (TPC), individual phenolic acid concentrations, and antioxidant capacity values showed significant cultivar × plant maturity interactions, indicating that these properties change with plant growth in genotype-specific ways. TPC values and the concentrations of rosmarinic, caffeic, and caftaric acids generally increased as basil plants matured. Caffeic acid had the highest concentrations (0.71–5.00 mg/100 g dry weight, depending on the cultivar) and levels correlated strongly with basil height (r s = 0.717; p < 0.001) and leaf mass (r s = 0.676; p < 0.001), suggesting that its accumulation is closely associated with plant growth. Hierarchical cluster analysis revealed that the genotype exerts a greater influence than plant maturity on the phenolic composition of Genovese and Tuscany basils. In contrast, the chemical properties of Spicy basil varied greatly with developmental stage, implying that plant maturity plays a larger role in the phytochemical profile of this cultivar.

graphic file with name ao5c07413_0008.jpg

graphic file with name ao5c07413_0006.jpg

1. Introduction

The Ocimum genus within the Lamiaceae family includes a diverse array of aromatic plant species among which basil (Ocimum basilicum) is the most widely cultivated and studied. In addition to being a plant of global culinary importance, basil has medicinal properties that contribute to its significance in both traditional and modern herbal practices. When consumed in the diet, basil may boost memory and cognitive function, reduce inflammation, and lower the risk of cardiovascular disease and certain cancers. ,

Basil is a rich source of phenolic compounds, secondary plant metabolites with strong antioxidant properties that are also associated with numerous human health benefits. Plants such as basil synthesize secondary metabolites throughout their development to aid in fulfilling essential physiological and ecological functions such as photosynthesis, attracting pollinators, and defending against pathogens. Environmental factors, agronomic conditions, and the genetics of the plant itself also affect both the identity and concentration of secondary metabolites that a plant produces. , Basil is an herb with broad genetic diversity encompassing a wide range of cultivars that are distinguished by their unique phenolic profiles, differing in the levels of particular phenolic acids (e.g., caffeic, chicoric, rosmarinic, and caftaric acids) as well as the strength of their antioxidant properties. − Morphological traits, such as flower and leaf color, growth habit, and leaf shape, have also been associated with significant variations in essential oil composition and biological activities among basil cultivars. ,

Phenolic biosynthesis is activated at different stages of a plant’s life cycle, and pathways for secondary metabolite production are often species specific. Herbs within the Lamiaceae family such as lemon balm, oregano, and thyme exhibit changes in their phenolic content with plant development, − but the effects are varied. The total phenolic content of lemon balm correlates directly with growth: as plant height and mass increase, phenolic concentrations and rosmarinic acid content also increase. In contrast, oregano has its highest flavonoid levels near the full flowering stage, although phenolic acid concentrations steadily decrease over the same developmental period. The polyphenol content of rosemary is high throughout the vegetative stage and decreases at flowering, while flavonoid concentrations remain relatively constant as the herb matures. In basil, phenolic content differs greatly among genotypes ,− and is affected by other agronomic factors, yet little is known about the variations in secondary metabolite synthesis that may occur during basil growth and development.

To identify harvest strategies that optimize the nutritional and medicinal values of basil, it is important to establish how the phenolic content and antioxidant properties of the herb change with plant maturity. Therefore, we analyzed three green basil cultivars throughout the vegetative stage prior to flowering, when leaves are most often harvested in the commercial production of fresh and dried basil. Common cultivars were selected that represent different green basil morphotypes: a true basil with an upright growth habit (Genovese), a small-leaf basil characterized by short narrow leaves and rounded growth (Spicy), and a lettuce-leaf variety with broad blistered leaves (Tuscany). Basil is well-suited to greenhouses and controlled indoor conditions, so plants were cultivated within a growth facility to minimize the effects of environmental factors on basil chemical composition. Leaves from basil plants were harvested weekly, and their total phenolic contents, phenolic acid compositions, and antioxidant properties were quantified and compared to identify how the levels of bioactive secondary metabolites varied with plant maturity among the three cultivars.

2. Materials and Methods

2.1. Chemical Reagents

Analytical standards (trolox or 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, gallic acid, caffeic acid, chicoric acid, and rosmarinic acid) of the highest purity were purchased from MilliporeSigma (St. Louis, MO, USA) except caftaric acid, which was obtained from INDOFINE Chemical Company (Hillsborough, NJ, USA). Solvents were all high-performance liquid chromatography (HPLC) grade and were purchased from Pharmco-AAPER (Brookfield, CT, USA). Chemical reagents were purchased from MilliporeSigma, and the Folin Ciocalteu reagent was obtained from VWR International (Radnor, PA, USA).

2.2. Plant Growth Conditions and Sample Preparation

Seeds of three basil cultivars, Genovese (O. basilicum ‘Genovese’), Spicy (O. basilicum var. minimum), and Tuscany (O. basilicum ‘Tuscany’), were purchased from Johnny’s Selected Seeds and sown in seedling trays filled with a Miracle-Gro Moisture Control Potting mix. After 30 days, plants were moved to individual pots containing the same soil mixture and arranged in a randomized complete block design based on three basil cultivars, eight planned harvest dates, and five replicate plants per cultivar for each harvest date (n = 120 pots). Basil seedlings and plants were grown under full-spectrum hydroponic lights (SunBlaster 6400K T5HO) for 10 h/day within a growth facility (average temperature = 24.01 ± 1.32 °C). Watering occurred every other day, and an all-purpose fertilizer (Miracle-Gro) was added to the watering solution once per week. Harvesting began 22 days after germination (DAG) and continued weekly throughout the vegetative phase for 10 weeks. To harvest, all leaves were removed from the basil plants, immediately frozen using liquid nitrogen, and then stored at −80 °C. Above-ground height and total leaf mass were measured for all plants at the time of harvest, and average values were calculated for each cultivar using replicate plants.

Phenolic compounds were extracted from basil leaves using an established method ,, with minor modifications. Frozen leaves from each basil plant were ground in liquid nitrogen using a mortar and pestle, and 0.10–0.15 g of the resulting powder was placed in a microcentrifuge tube. Samples were dried for 7 h under vacuum at 35 °C, and the mass of the dried samples was recorded before 1.0 mL of 80% aqueous methanol was added to each tube. The tubes were shaken for 15 h at room temperature and then centrifuged at 13,200 rpm for 20 min. The supernatant was removed and stored at −80 °C. Sample preparation for HPLC analysis used an identical procedure with 0.5 mL of extraction solvent.

2.3. Determination of Total Phenolic Contents and Antioxidant Capacities

A version of the Folin Ciocalteu assay adapted to a microplate reader was used to determine the total phenolic content of each basil sample. Gallic acid (12.5–800.0 mg/L) was used for the calibration curve, and total phenolic content values were calculated in gallic acid equivalents (GAE) in mg/g basil dry weight (DW).

Antioxidant capacities of basil extracts were measured using both the cupric ion-reducing antioxidant capacity (CUPRAC) assay adapted to a microplate reader and a high-throughput 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. , Trolox was used as the calibration standard for both methods (49.9–3196.3 μM for CUPRAC, 5–80.0 μM for DPPH), and final values were reported as trolox equivalent antioxidant capacities (TEAC) in mmol/100 g DW.

2.4. Analysis of Individual Phenolic Compounds

Identification and quantification of the most prevalent basil phenolic acids (caffeic acid, caftaric acid, chicoric acid, and rosmarinic acid) were accomplished by modification of a previous method. , A Shimadzu HPLC system (Columbia, MD, USA) equipped with an autosampler was used with 2.5% aqueous formic acid (eluent A) and 100% acetonitrile (eluent B) at a flow rate of 1 mL/min and the following gradient conditions: 0–15 min, 15–25% B; 15–18 min, 25–90% B; 18–20 min, 90–100% B; 20–25 min, 100–15% B; and 25–30 min, 15% B. The sample injection volume was 25 μL, and separation occurred on a Waters Symmetry C-18 column (5 μm, 4.6 mm × 150 mm; Milford, MA, USA). A diode array detector was used to collect absorbance spectra of all eluting compounds from 250 to 350 nm. The four phenolic acids of interest were identified in basil samples by comparing the peak retention time (t r) to values for analytical reference standards (caftaric acid t r = 2.684 ± 0.063 min, caffeic acid t r = 4.041 ± 0.126 min, chicoric acid t r = 9.451 ± 0.390 min, and rosmarinic acid t r = 13.975 ± 0.390 min). Additionally, phenolic acid assignments were confirmed by separate HPLC analysis of samples for each basil cultivar using the same chromatographic conditions with mass spectral detection. Calibration curves were prepared for each phenolic acid over a wide concentration range (1.00–100.00 mg/L) based on chromatograms measured at 330 nm, and limits of detection (LOD) were determined for each analyte using the regression method (caftaric acid LOD = 0.72 mg/L, R 2 = 0.9986; caffeic acid LOD = 0.34 mg/L, R 2 = 0.9999; chicoric acid LOD = 0.28 mg/L, R 2 = 0.9999; and rosmarinic acid LOD = 0.59 mg/L, R 2 = 0.9998). Calibration standards were also reanalyzed at regular intervals to verify instrument stability and accuracy over time. Phenolic acid concentrations were determined by comparing integrated sample peak areas to the external calibration curves, and final concentrations within basil samples were reported in milligrams per 100 g of DW.

2.5. Statistical Analysis

All statistical analyses were conducted using SPSS Statistics, version 29. Values are reported as the mean ± standard error and were calculated by using replicate plant samples. A two-way analysis of variance (ANOVA) was used to evaluate the individual and combined effects of cultivar and plant maturity on experimental values (i.e., total phenolic content, antioxidant capacities, and individual phenolic concentrations). Before ANOVA analysis, normality was confirmed using the Shapiro-Wilk test, and homogeneity of variances was examined using Levene’s test. Most data sets met the assumption of normality; however, if a harvest date for a cultivar failed the Shapiro-Wilk test and transformation did not improve normality, then a bootstrapped two-way ANOVA was performed. Posthoc pairwise comparisons were completed, and p values were Bonferroni-adjusted. Mann–Whitney U tests were used to confirm any significant differences involving a nonnormally distributed group. Significance was set at p < 0.05 for all statistical tests.

Spearman’s rank correlation coefficients were calculated to determine relationships among total phenolic content, antioxidant capacities, concentrations of individual phenolic compounds, and growth parameters. Spearman’s rho (r s) was used for all correlations to ensure consistency since some variables were not normally distributed. The magnitude of the calculated r s value was used to determine the strength of the correlation with 0.3–0.5 considered a moderate correlation, while >0.5 indicated a strong correlation between variables. A heatmap was generated to visualize the correlation matrix with color denoting the direction of the correlation and intensity corresponding to the strength.

Hierarchical cluster analysis (HCA) was conducted to examine similarities in the chemical composition and antioxidant properties among basil cultivars and plant maturities. Average values for total phenolic contents, antioxidant capacities (CUPRAC and DPPH), and concentrations of individual phenolic compounds (rosmarinic, caffeic, caftaric, and chicoric acids) were standardized using Z-scores and analyzed using Ward’s method with squared Euclidean distance. Final HCA results were visualized by using a dendrogram plot.

3. Results and Discussion

3.1. Total Phenolic Content

Phenolic compounds are the most common secondary metabolites found within plants, and these nonvolatile bioactive species confer antioxidant and pharmacological properties to basil leaves. The total phenolic content (TPC) of green basil cultivars (Figure a) ranged from 1.73 GAE mg/g for Spicy basil at 36 DAG to 6.75 GAE mg/g for Genovese basil at 57 DAG. The TPC values of basil in this study were similar in magnitude to those reported in the literature, ranging from 3.1 to 7.4 GAE mg/g DW. Genotype may influence phenolic compound production within plants, and our results confirmed that cultivar affected basil total phenolic contents (p < 0.001) with Genovese basil having a significantly higher TPC (average = 4.95 ± 0.22 GAE mg/g) than both Spicy (average = 3.21 ± 0.18 GAE mg/g) and Tuscany (average = 3.02 ± 0.12 GAE mg/g) basils. Examining individual harvest dates, Genovese basil also exhibited statistically greater TPC values than Tuscany at all plant maturities except 36 DAG and significantly higher phenolic contents than Spicy basil at all harvest dates except 57 and 64 DAG (statistical differences among cultivars at different plant maturities are compiled in the Supporting Information, Figure S1). TPC varied with plant maturity (p < 0.001), and a significant interaction between harvest date and cultivar (p = 0.008) confirmed that total phenolic content was differentially affected by plant development among the three basil morphotypes. For example, average TPC values were not statistically different for Tuscany basil plants with different maturities. However, both Spicy and Genovese basils exhibited total phenolic contents that increased as the herb matured, reaching a maximum at 57 DAG, then declining; average TPC values for both cultivars at 57 DAG were significantly higher than the phenolic contents of younger basil plants at 29 and 36 DAG.

1.

1

Open in a new tab

Average values (±standard error) for total phenolic content ((a) in gallic acid equivalents, GAE), plant height (b), and total leaf mass (c) of basil cultivars as a function of plant maturity.

The growth-differentiation balance (GDB) hypothesis proposes that plants experience trade-offs between growth and the production of secondary metabolites throughout their development. When plants direct energy and resources to biomass production, the GDB predicts that the synthesis of secondary metabolites, such as phenolic compounds, will slow. In this study, basil total phenolic content exhibited a moderate (r s = 0.511) significant (p < 0.001) correlation with average plant height (Figure b) and a lower correlation (r s = 0.204; p = 0.028) with total leaf mass (Figure c). This result indicates that as basil plants matured, grew taller, and to a lesser extent accumulated leaf mass, TPC values also generally increased. Spicy basil, for example, exhibited its largest total phenolic content at 57 DAG, a developmental period in which plants were still growing substantially, in terms of both height (from 5.96 ± 0.95 cm at 50 DAG to 7.58 ± 0.94 cm at 57 DAG, a 27% increase) and total leaf mass (2.245 ± 0.420 6 at 50 DAG to 3.923 ± 0.822 at 57 DAG, a 74% increase). The indoor agronomic conditions used in our experiment likely provided basil plants with sufficient resources to simultaneously support both growth and phenolic synthesis, resulting in a deviation from the predictions of the GDB hypothesis. Alternatively, the production of phenolic compounds within these cultivars may be more strongly regulated by their developmental stage than resource allocation, increasing as basil plants age within the vegetative state before flowering regardless of their growth rate.

3.2. Individual Phenolic Acid Concentrations

Phenolic acids are the most abundant nonvolatile secondary metabolites within basil leaves and are key contributors to the herb’s antioxidant properties. In addition to being potent free radical scavengers, phenolic acids have high bioavailability and can act as strong inhibitors of oxidation through several biochemical mechanisms. , We quantified the four most prevalent phenolic acids (caffeic, caftaric, chicoric, and rosmarinic acids) within the three basil cultivars as a function of plant maturity, and the results are presented in Table . Phenolic acid concentrations were affected by cultivar and plant maturity at the time of harvest (for both variables and all phenolic acids, p < 0.001). Additionally, a significant interaction between the two variables (cultivar × plant maturity, p < 0.001 for all phenolic acids) indicated that the phenolic acid levels of the three basil genotypes varied in response to plant development in different ways. For example, caffeic acid was the phenolic acid found in highest concentrations within all three basil cultivars (Genovese, 5.00 ± 1.03 mg/100 g DW at 57 DAG; Spicy, 3.35 ± 0.21 mg/100 g DW at 57 DAG; and Tuscany, 1.62 ± 0.19 mg/100 g DW at 64 DAG), yet the predominant phenolic acid changed as basil plants matured with distinctions between genotypes. Chicoric acid was the most abundant phenolic acid within Spicy basil at 29 and 36 DAG, but as plants aged, the chicoric acid content decreased. Conversely, the levels of other phenolics, such as caffeic, caftaric, and rosmarinic acids, notably increased. For Tuscany basil, rosmarinic acid was the predominant phenolic acid throughout early stages of development, and although rosmarinic acid concentrations continued to gradually rise after 50 DAG, caffeic acid levels increased to a greater extent, causing it to become the most abundant phenolic acid in more mature Tuscany plants. Genovese basil maintained a relatively consistent phenolic acid profile, with caffeic acid found in the highest concentration at nearly all stages of maturity except 36 DAG, when rosmarinic acid was detected in higher abundance. Maturation-related changes in the levels of phenolic compounds within other herbaceous plants are known to occur based on phenological (i.e., vegetative versus flowering) and developmental stages. Our results indicate that the genotype may also be a critical factor influencing the phytochemical profiles of aromatic medicinal plants as they mature.

1. Average Basil Phenolic Acid Concentrations (± Standard Error) in mg/100 g Dry Weight, DW,

3.2.

Open in a new tab
a

BDL indicates that the phenolic acid was below the method limit of detection, LOD.

b

The phenolic acid found in the highest concentration at each plant maturity is noted in the bold colored type (green for Genovese, blue for Spicy, and purple for Tuscany).

Rosmarinic acid, a caffeic acid ester with strong antioxidant, anti-inflammatory, and pharmacological properties, is often noted as the most prevalent phenolic compound within basil. , However, we found that for the basil cultivars in this study, rosmarinic acid was the most abundant phenolic acid only at earlier stages of plant growth, 50 DAG or less. Across all plant maturities, Spicy basil had a significantly lower (p < 0.001) rosmarinic acid concentration (average = 0.415 ± 0.083 mg/100 g DW) than both Tuscany (average = 0.875 ± 0.056 mg/100 g DW) and Genovese (average = 0.914 ± 0.062 mg/100 g DW) basil. Overall increases in rosmarinic acid levels occurred for all basil cultivars as plants matured, with basil from later harvests (57, 64, and 71 DAG) exhibiting significantly greater rosmarinic acid contents than earlier ones (29 and 36 DAG; Supporting Information, Figure S2). Birenboim et al. also found that rosmarinic acid levels increased with plant development in six green and purple Israeli basil cultivars; however, most of their genotypes reached a maximum rosmarinic content at the third or fourth weekly harvest, and their basil rosmarinic acid concentrations were substantially higher, likely due to their hydroponic growing conditions or their analysis of only upper leaves.

Like rosmarinic acid, caffeic acid concentrations were statistically lower at earlier harvests (29, 36, and 43 DAG) compared to those at later harvests (57, 64, and 71) in Tuscany and Genovese basils. In contrast, Spicy basil showed fewer significant differences in caffeic acid levels across maturities (Supporting Information, Figure S3). Genovese basil had significantly greater (p < 0.001) caffeic acid concentrations overall (average = 2.217 ± 0.260 mg/100 g DW) than both Tuscany (average = 1.755 ± 0.189 mg/100 g) and Spicy (average = 0.880 ± 0.083 mg/100 g) basils, and caffeic acid contents were similar to those previously reported for basil within the literature, 1.62–5.57 mg/100 g DW. Caftaric acid also generally increased as basil plants matured, but with differences found among cultivars. At 57 DAG, Genovese and Spicy basil had their highest caftaric acid concentrations, and they were significantly greater than the caftaric acid content for all earlier harvests (Supporting Information, Figure S4) while caftaric acid concentrations for Tuscany basil continually increased with plant growth to their highest levels at the final harvest, 71 DAG. Caftaric acid content is known to depend on plant maturity within purple basil. However, the highest caftaric acid levels occur slightly earlier in purple cultivars, at 35 and 49 DAG, compared to the green basil genotypes in our study. Tuscany basil had the lowest caftaric acid levels across all plant maturities (average = 0.597 ± 0.048 mg/100 g DW), and its caftaric acid content was significantly less (p < 0.001) than both Genovese (average = 0.802 ± 0.065 mg/100 g DW) and Spicy basil (average = 0.913 ± 0.090 mg/100 g DW). Overall, caftaric acid levels for these green basil cultivars were similar to those reported for dried basil samples of an unknown genotype, ranging from 0.47 to 1.75 mg/100 g DW.

In contrast to the general increases in rosmarinic, caftaric, and caffeic acid concentrations that occurred as basil plants matured, chicoric acid displayed greater variation with basil growth, particularly among cultivars. Spicy basil exhibited significantly higher concentrations of chicoric acid at earlier harvests (29 and 36 DAG) compared with more mature plants (50, 57, and 71 DAG; Supporting Information, Figure S5). For Tuscany and Genovese basil, however, chicoric acid levels varied but with no discernible trend among different plant maturities. Spicy basil also had much greater chicoric acid content overall (average = 1.235 ± 0.176 mg/100 g DW) with significantly higher levels (p < 0.001) than Genovese (average = 0.516 ± 0.042 mg/100 g DW) and Tuscany (average = 0.466 ± 0.031 mg/100 g) basils.

Among the four phenolic acids analyzed, caffeic acid showed the strongest positive correlation with both plant height (r s = 0.717; p < 0.001) and total leaf mass (r s = 0.676; p < 0.001), suggesting that this phenolic acid is more closely associated with plant development (Figure ). As an intermediate in the phenylpropanoid biosynthetic pathway that produces secondary metabolites, caffeic acid may serve a functional role in growth-related processes within basil.

2.

2

Open in a new tab

Heatmap correlation matrix among basil total phenolic contents, antioxidant capacities, individual phenolic acid concentrations, and growth parameters. Dark green indicates a strong positive correlation, dark purple indicates a strong negative correlation, and white indicates that there is no significant correlation. Spearman’s rho values (r s) are included in each cell, and statistical significance is denoted at the p < 0.05 (*) and p < 0.001 (**) levels.

Cinnamic acid derivatives such as caffeic acid are widely distributed in plants and contribute to the biosynthesis of structural components, function as defense compounds, and serve as signaling molecules. These diverse biological roles may explain the prevalence of caffeic acid within these basil cultivars and its strong relationship with plant development. Rosmarinic and caftaric acids showed weaker correlations, implying their accumulation within basil leaves relates to plant growth, but other factors may play a larger role in regulating their concentrations. Chicoric acid had a weak but statistically significant negative correlation with both plant height (r s = −0.287; p = 0.008) and total leaf mass (r s = −0.277; p = 0.011), suggesting that its concentration may decline with plant maturation, a trend that was particularly noticeable for the Spicy basil cultivar. The biosynthetic pathway of chicoric acid is not well documented, and our results indicate that basil leaves may regulate chicoric acid production and accumulation differently than other common phenolic acids.

3.3. Antioxidant Properties

Herbs such as basil contain a range of phenolic antioxidants, and because these species vary in their chemical structures, they may inhibit oxidative processes differently. When assessing antioxidant properties using in vivo assays, it is therefore important to select methods with complementary mechanisms. As shown in Figure , we measured the antioxidant capacities of basil samples in this study using two methods: the CUPRAC assay, a robust ligand-based method utilizing a single electron transfer (SET) mechanism that assesses reducing capacity, and the DPPH method, which is based on a metastable free radical reaction and occurs through a mixed-mode (SET and hydrogen-atom transfer, HAT) mechanism.

3.

3

Open in a new tab

Average CUPRAC (cupric ion-reducing antioxidant capacity) and DPPH (2,2-diphenyl-1-picrylhydrazyl) values (±standard error) for Genovese (a), Spicy (b), and Tuscany (c) basils. Values are reported as trolox equivalent antioxidant capacities, TEAC.

Cultivar and plant maturity significantly affected antioxidant capacities measured using both the CUPRAC and DPPH methods (p < 0.001). As with total phenolic content and individual phenolic acid concentrations, a significant cultivar × harvest date interaction was observed for both assays (p < 0.001), indicating that the effects of the genotype on antioxidant capacity varied with plant maturity. For Spicy basil, CUPRAC and DPPH antioxidant capacities generally increased as plants developed, reaching a maximum at 57 DAG that was significantly greater than all previous and subsequent harvest dates (Supporting Information, Figures S6 and S7). Tuscany basil, however, maintained relatively constant CUPRAC and DPPH values throughout plant growth until the final harvest date, 71 DAG, when antioxidant capacities reached a maximum that was significantly greater than the values for young plants (harvests <50 DAG for CUPRAC and <43 DAG for DPPH). In contrast to Spicy and Tuscany basils, the Genovese cultivar exhibited no discernible trends in CUPRAC or DPPH antioxidant capacities over the growth period in this study, yet antioxidant capacities for Genovese basil were consistently higher than those of the Tuscany and Spicy cultivars. For example, Genovese had CUPRAC antioxidant capacities that were significantly greater than the values for Spicy and Tuscany at every plant maturity except 50 DAG. Genovese basil also had overall CUPRAC (average = 4.32 ± 0.04 TEAC mmol/100 g DW) and DPPH values (average = 1.26 ± 0.04 TEAC mmol/100 g DW) that were both significantly higher (p > 0.001) than the antioxidant capacities for Spicy (CUPRAC average = 2.58 ± 0.16 TEAC mmol/100 g DW, DPPH average = 1.03 ± 0.07 TEAC mmol/100 g DW) and Tuscany (CUPRAC average = 2.26 ± 0.13 TEAC mmol/100 g DW, DPPH average = 0.82 ± 0.06 TEAC mmol/100 g DW) basils.

Little data exist within the literature regarding the effects of plant development on the antioxidant properties of basil. McCance et al. reported that the reducing capacity of purple basil cultivars measured by the FRAP (ferric reducing antioxidant power) assay generally increased as plants matured and found that antioxidant properties correlated with total phenolic content. In our study, DPPH (r s = 0.676, p < 0.001) and CUPRAC (r s = 0.757, p < 0.001) values both exhibited strong significant correlations with total phenolic content (Figure ), although CUPRAC antioxidant capacities correlated more strongly. This result is not unexpected because the Folin Ciocalteu assay (used to measure basil total phenolic contents in this study) operates via an electron transfer mechanism, and TPC values often correlate with antioxidant reducing capacities measured by other SET-based assays such as CUPRAC. Interestingly, although the DPPH assay utilizes a mixed-mode mechanism, DPPH antioxidant capacities not only correlated with TPC values but also exhibited a strong significant (r s = 0.800, p < 0.001) correlation with CUPRAC values. The robust correlation between these two assays suggests that the phenolics accumulating within basil leaves as the plants mature are similarly effective as antioxidants by SET and HAT mechanisms, a result consistent with the presence of nonflavonoid compounds such as rosmarinic acid, which has phenolic hydroxyls in positions that promote both hydrogen abstraction (HAT) and stabilization of formed free radicals (SET).

3.4. Cluster Analysis

Hierarchical cluster analysis was used to identify patterns in phenolic composition and antioxidant capacities across basil cultivars and plant maturities, resulting in the five-cluster solution shown in Figure . Cluster 1 included mostly Genovese basil harvested at both the earliest (22 DAG) and latest plant maturities (all harvests >43 DAG except 50 DAG) as well as Spicy basil at 57 DAG. This cluster exhibited the greatest total phenolic content, the largest DPPH and CUPRAC antioxidant capacities, the highest caffeic acid concentrations (average = 3.105 ± 0.429 mg/100 g), and the second highest caftaric acid levels (average = 1.093 ± 0.165 mg/100 g) among clusters. Cluster 2 was the largest among HCA groupings and contained all three basil cultivars at intermediate to late plant maturities: the remaining Genovese harvests not in cluster 1, Spicy basil at 43 and 50 DAG, and late-harvest Tuscany basil. Cluster 2 had the second highest DPPH antioxidant capacity (average = 1.22 ± 0.04 TEAC mmol/100 g) and intermediate TPC and phenolic acid levels relative to other clusters. Cluster 3 contained mostly Tuscany basil, at both earlier (all harvests <43 DAG) and later (64 DAG) plant maturities, and the youngest Spicy basil (22 DAG). Although cluster 3 exhibited the lowest antioxidant capacities of any grouping, it also showed intermediate rosmarinic acid content (average = 0.745 ± 0.106 mg/100 g) and TPC values. Clusters 4 and 5 each included only two plant maturities of the Spicy cultivar: early harvest Spicy basil (29 and 36 DAG; cluster 4) had the lowest TPC among clusters, relatively low antioxidant capacity values, and the highest chicoric acid content (average = 2.558 ± 0.354 mg/100 g); Spicy basil harvested at the latest maturity (64 and 71 DAG; cluster 5), however, had the highest caftaric (average = 1.414 ± 0.112 mg/100 g) and rosmarinic (average = 1.190 ± 0.020 mg/100 g) acid contents and intermediate TPC and antioxidant capacities.

4.

4

Open in a new tab

Dendrogram plot displaying clustering of Genovese, Spicy, and Tuscany basils at different plant maturities (where DAG = days after germination) based on total phenolic content (TPC), antioxidant capacities, and individual phenolic acid concentrations.

Hierarchical cluster analysis revealed the strong differential effects of basil genotype on phytochemical composition and antioxidant capacity. Genovese and Tuscany basils were primarily clustered with plants of the same cultivar across multiple maturity levels: both genotypes formed two main clusters that were distinguished only by their relative phytochemical levels (higher or lower), and all harvest dates for each cultivar were split between these two groups. Although the earliest (22 DAG) and latest (>50 DAG) harvest dates for Genovese and Tuscany basils generally exhibited higher phenolic contents and stronger antioxidant properties compared to intermediate maturities, clustering analysis confirmed a relatively consistent phenolic composition for these cultivars as plants developed. This result suggests that phenolic accumulation and antioxidant capacities within these cultivars remained relatively constant with plant maturity, and the plant genotype was the more influential factor. In contrast, Spicy basil harvest dates were distributed across all five clusters, demonstrating that phenolic levels and antioxidant capacities varied much more extensively with developmental stage, and plant maturity played a greater role. Cultivar − and plant maturity , are known to independently influence the accumulation of basil secondary metabolites. Our findings highlight the importance of considering both factors, as their relative effects on the level of bioactive compounds within basil may vary substantially among genotypes.

4. Conclusions

Results from this study showed that although plant maturity influences the phenolic composition and antioxidant properties of green basil, the effects are differential and can vary greatly among genotypes. For Genovese and Tuscany basil, TPC and antioxidant capacity values were highest at the earliest and latest stages of plant maturity, yet phenolic composition remained relatively constant, indicating that the genotype has a stronger influence on phytochemical composition within these cultivars. In contrast, chemical properties varied extensively in Spicy basil with the developmental stage, implying that plant maturity plays a more important role. For all cultivars, TPC and the concentrations of rosmarinic, caffeic, and caftaric acids generally increased as basil plants matured. Our findings suggest that growers must evaluate how plant maturity affects the phytochemical content of their specific basil cultivar to select the best harvest time for the maximum nutritional value.

Supplementary Material

ao5c07413_si_001.pdf (1.2MB, pdf)

Acknowledgments

This work was supported by The Welch Foundation under grant number AF-0005 and Southwestern University’s Herbert and Kate Dishman endowment. We also wish to thank Dr. Ian Riddington of the Mass Spectrometry Facility at the University of Texas at Austin for his contribution in confirming phenolic acid identities in basil samples with mass spectrometry. The authors acknowledge ChatGPT (OpenAI) for support with reviewing statistical analysis plans and reference formatting. The graphical abstract for this article was created in BioRender (https://BioRender.com/rc0gc2x).

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c07413.

  • Statistical differences in average total phenolic contents, individual phenolic acid concentrations (rosmarinic, caffeic, caftaric, and chicoric acids), and antioxidant capacities (CUPRAC and DPPH) among basil cultivars at different plant maturities (PDF)

The authors declare no competing financial interest.

Published as part of ACS Omega special issue “Undergraduate Research as the Stimulus for Scientific Progress in the USA”.

References

  1. Putievsky, E. ; Galambosi, B. . Production systems of sweet basil. In Basil: The Genus Ocimum; Harwood Academic Publishers: 1999; Vol. 10, pp 39–65. [Google Scholar]
  2. Gross M.. Flavonoids and cardiovascular disease. Pharm. Biol. 2004;42(sup1):21–35. doi: 10.3109/13880200490893483. [DOI] [Google Scholar]
  3. Singletary K. W.. Basil: A brief summary of potential health benefits. Nutr. Today. 2018;53:92–97. doi: 10.1097/NT.0000000000000267. [DOI] [Google Scholar]
  4. Capecka E., Mareczek A., Leja M.. Antioxidant activity of fresh and dry herbs of some Lamiaceae species. Food Chem. 2005;93:223–226. doi: 10.1016/j.foodchem.2004.09.020. [DOI] [Google Scholar]
  5. Crozier, A. ; Jaganath, I. B. ; Clifford, M. N. . Phenols, polyphenols and tannins: An overview. In Plant Secondary Metabolites; Crozier, A. , Clifford, M. N. , Ashihara, H. , Eds.; Wiley: 2006; pp 1–24. 10.1002/9780470988558.ch1. [DOI] [Google Scholar]
  6. Carović-Stanko K., Šalinović A., Grdiša M., Liber Z., Kolak I., Satovic Z.. Efficiency of morphological trait descriptors in discrimination of Ocimum basilicum L. accessions. Plant Biosyst. 2011;145(2):298–305. doi: 10.1080/11263504.2011.558677. [DOI] [Google Scholar]
  7. Qin L., Li C., Li D., Wang J., Yang L., Qu A., Wu Q.. Physiological, metabolic and transcriptional responses of basil (Ocimum basilicum Linn. var. Pilosum (Willd.) Benth.) to heat stress. Agronomy. 2022;12(6):1434. doi: 10.3390/agronomy12061434. [DOI] [Google Scholar]
  8. Kwee E. M., Niemeyer E. D.. Variations in phenolic composition and antioxidant properties among 15 basil (Ocimum basilicum L.) cultivars. Food Chem. 2011;128:1044–1050. doi: 10.1016/j.foodchem.2011.04.011. [DOI] [Google Scholar]
  9. Bajomo E. M., Aing M. S., Ford L. S., Niemeyer E. D.. Chemotyping of commercially available basil (Ocimum basilicum L.) varieties: Cultivar and morphotype influence phenolic acid composition and antioxidant properties. NFS J. 2022;26:1–9. doi: 10.1016/j.nfs.2022.01.001. [DOI] [Google Scholar]
  10. Ćavar Zeljković S., Komzáková K., Šišková J., Karalija E., Smékalová K., Tarkowski P.. Phytochemical variability of selected basil genotypes. Ind. Crops Prod. 2020;157:112910. doi: 10.1016/j.indcrop.2020.112910. [DOI] [Google Scholar]
  11. Labra M., Miele M., Ledda B., Grassi F., Mazzei M., Sala F.. Morphological characterization, essential oil composition and DNA genotyping of Ocimum basilicum L. cultivars. Plant Sci. 2004;167:725–731. doi: 10.1016/j.plantsci.2004.04.026. [DOI] [Google Scholar]
  12. Carović-Stanko K., Orlić S., Politeo O., Strikić F., Kolak I., Milos M., Satovic Z.. Composition and antibacterial activities of essential oils of seven Ocimum taxa. Food Chem. 2010;119:196–201. doi: 10.1016/j.foodchem.2009.06.010. [DOI] [Google Scholar]
  13. Rutkowska M., Balcerczak E., Świechowski R., Dubicka M., Olszewska M. A.. Seasonal variation in phenylpropanoid biosynthesis and in vitro antioxidant activity of Sorbus domestica leaves: Harvesting time optimization for medicinal application. Ind. Crops Prod. 2020;156:112858. doi: 10.1016/j.indcrop.2020.112858. [DOI] [Google Scholar]
  14. Ryu D.-H., Cho J.-Y., Yang S.-H., Kim H.-Y.. Effects of harvest timing on phytochemical composition in Lamiaceae plants under an environment-controlled system. Antioxidants. 2023;12:1909. doi: 10.3390/antiox12111909. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Grevsen K., Fretté X. C., Christensen L. P.. Content and composition of volatile terpenes, flavonoids and phenolic acids in Greek oregano (Origanum vulgare L. ssp. hirtum) at different development stages during cultivation in cool temperate climate. Eur. J. Hortic. Sci. 2009;74:193–203. doi: 10.1079/ejhs.2009/1182840. [DOI] [Google Scholar]
  16. Yosr Z., Hnia C., Rim T., Mohamed B.. Changes in essential oil composition and phenolic fraction in Rosmarinus officinalis L. var. typicus Batt. organs during growth and incidence on the antioxidant activity. Ind. Crops Prod. 2013;43:412–419. doi: 10.1016/j.indcrop.2012.07.044. [DOI] [Google Scholar]
  17. Flanigan P. M., Niemeyer E. D.. Effect of cultivar on phenolic levels, anthocyanin composition, and antioxidant properties in purple basil (Ocimum basilicum L.) Food Chem. 2014;164:518–526. doi: 10.1016/j.foodchem.2014.05.061. [DOI] [PubMed] [Google Scholar]
  18. Srivastava S., Cahill D. M., Conlan X. A., Adholeya A.. A novel in vitro whole plant system for analysis of polyphenolics and their antioxidant potential in cultivars of Ocimum basilicum . J. Agric. Food Chem. 2014;62:10064–10075. doi: 10.1021/jf502709e. [DOI] [PubMed] [Google Scholar]
  19. McCance K. R., Flanigan P. M., Quick M. M., Niemeyer E. D.. Influence of plant maturity on anthocyanin concentrations, phenolic composition, and antioxidant properties of 3 purple basil (Ocimum basilicum L.) cultivars. J. Food Compos. Anal. 2016;53:30–39. doi: 10.1016/j.jfca.2016.08.009. [DOI] [Google Scholar]
  20. McDowell, D. ; Niemeyer, E. . Factors influencing the production of phenolic compounds within basil (Ocimum basilicum L.). In Ocimum: An Overview; Nova Science Publishers, Incorporated, 2020. [Google Scholar]
  21. Simon, J. E. Sweet basil: A production guide; Purdue University, Cooperative Extension Service: Jan 1985. Accessed July 23, 2025. [Google Scholar]
  22. Sipos L., Balázs L., Székely G., Jung A., Sárosi S., Radácsi P., Csambalik L.. Optimization of basil (Ocimum basilicum L.) production in LED light environments – a review. Sci. Hortic. 2021;289:110486. doi: 10.1016/j.scienta.2021.110486. [DOI] [Google Scholar]
  23. Nguyen P. M., Niemeyer E. D.. Effects of nitrogen fertilization on the phenolic composition and antioxidant properties of basil (Ocimum basilicum L.) J. Agric. Food Chem. 2008;56:8685–8691. doi: 10.1021/jf801485u. [DOI] [PubMed] [Google Scholar]
  24. Singleton V. L., Rossi J. A. Jr.. Colorimetry of total phenolics with phosphomolybdic–phosphotungstic acid reagents. Am. J. Enol. Vitic. 1965;16(3):144–158. doi: 10.5344/ajev.1965.16.3.144. [DOI] [Google Scholar]
  25. Apak R., Güçlü K., Özyürek M., Karademir S. E.. Novel total antioxidant capacity index for dietary polyphenols and vitamins C and E, using their cupric ion reducing capability in the presence of neocuproine: CUPRAC method. J. Agric. Food Chem. 2004;52:7970–7981. doi: 10.1021/jf048741x. [DOI] [PubMed] [Google Scholar]
  26. Cheng Z., Moore J., Yu L.. High-throughput relative DPPH radical scavenging capacity assay. J. Agric. Food Chem. 2006;54:7429–7436. doi: 10.1021/jf0611668. [DOI] [PubMed] [Google Scholar]
  27. Ahn J., Alford A. R., Niemeyer E. D.. Variation in phenolic profiles and antioxidant properties among medicinal and culinary herbs of the Lamiaceae family. J. Food Meas. Charact. 2020;14:1720–1732. doi: 10.1007/s11694-020-00420-5. [DOI] [Google Scholar]
  28. Lv Q.-Z., Long J.-T., Gong Z.-F., Nong K.-Y., Liang X.-M., Qin T., Huang W., Yang L.. Current state of knowledge on the antioxidant effects and mechanisms of action of polyphenolic compounds. Nat. Prod. Commun. 2021;16(7):1–13. doi: 10.1177/1934578X211027745. [DOI] [Google Scholar]
  29. Sestili P., Ismail T., Calcabrini C., Guescini M., Catanzaro E., Turrini E., Layla A., Akhtar S., Fimognari C.. The potential effects of Ocimum basilicum on health: a review of pharmacological and toxicological studies. Expert Opin. Drug Metab. Toxicol. 2018;14(7):679–692. doi: 10.1080/17425255.2018.1484450. [DOI] [PubMed] [Google Scholar]
  30. Proestos C., Chorianopoulos N., Nychas G.-J. E., Komaitis M.. RP-HPLC analysis of the phenolic compounds of plant extracts. Investigation of their antioxidant capacity and antimicrobial activity. J. Agric. Food Chem. 2005;53:1190–1195. doi: 10.1021/jf040083t. [DOI] [PubMed] [Google Scholar]
  31. Treutter D.. Managing phenol contents in crop plants by phytochemical farming and breedingvisions and constraints. Int. J. Mol. Sci. 2010;11:807–857. doi: 10.3390/ijms11030807. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Herms D. A., Mattson W. J.. The dilemma of plants: To grow or defend. Q. Rev. Biol. 1992;67(3):283–335. doi: 10.1086/417659. [DOI] [Google Scholar]
  33. Zeljković S. C., Komzáková K., Šišková J., Karalijac E., Smékalová K., Tarkowski P.. Phytochemical variability of selected basil genotypes. Ind. Crops Prod. 2020;157:112910. doi: 10.1016/j.indcrop.2020.112910. [DOI] [Google Scholar]
  34. Chen J., Yang J., Ma L., Li J., Shahzad N., Kim C. K.. Structure–antioxidant activity relationship of methoxy, phenolic hydroxyl, and carboxylic acid groups of phenolic acids. Sci. Rep. 2020;10:2611. doi: 10.1038/s41598-020-59451-z. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Kiokias S., Proestos C., Oreopoulou V.. Phenolic acids of plant originA review on their antioxidant activity in vitro (O/W emulsion systems) along with their in vivo health biochemical properties. Foods. 2020;9:534. doi: 10.3390/foods9040534. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Hazrati S., Mousavi Z., Nicola S.. Harvest time optimization for medicinal and aromatic plant secondary metabolites. Plant Physiol. Biochem. 2024;212:108735. doi: 10.1016/j.plaphy.2024.108735. [DOI] [PubMed] [Google Scholar]
  37. Guan H., Luo W., Bao B., Cao Y., Cheng F., Yu S., Fan Q., Zhang L., Wu Q., Shan M.. A comprehensive review of rosmarinic acid: From phytochemistry to pharmacology and its new insight. Molecules. 2022;27:3292. doi: 10.3390/molecules27103292. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Birenboim M., Chalupowicz D., Maurer D., Barel S., Chen Y., Falik E., Kengisbuch D., Shimshoni J. A.. Optimization of sweet basil harvest time and cultivar characterization using near-infrared spectroscopy, liquid and gas chromatography, and chemometric statistical methods. J. Sci. Food Agric. 2022;102:3325–3335. doi: 10.1002/jsfa.11679. [DOI] [PubMed] [Google Scholar]
  39. Ciriello M., Formisano L., El-Nakhel C., Corrado G., Pannico A., De Pascale S., Rouphael Y.. Morpho-physiological responses and secondary metabolites modulation by preharvest factors of three hydroponically grown Genovese basil cultivars. Front. Plant Sci. 2021;12:671026. doi: 10.3389/fpls.2021.671026. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Lee J.. Caffeic acid derivatives in dried Lamiaceae and Echinacea purpurea products. J. Funct. Foods. 2010;2(3):158–162. doi: 10.1016/j.jff.2010.02.003. [DOI] [Google Scholar]
  41. Crozier, A. ; Yokota, T. ; Jaganath, I. B. ; Marks, S. C. ; Saltmarsh, M. ; Clifford, M. N. . Secondary metabolites in fruits, vegetables, beverages and other plant-based dietary components. In Plant Secondary Metabolites: Occurrence, Structure and Role in the Human Diet; Crozier, A. , Clifford, M. N. , Ashihara, H. , Eds.; Wiley Blackwell: Hoboken, 2006; pp 208–302. [Google Scholar]
  42. Marchiosi R., dos Santos W. D., Constantin R. P., de Lima R. B., Soares A. R., Finger-Teixeira A., Mota T. R., de Oliveira D. M., Foletto-Felipe M. P., Abrahão J., Ferrarese-Filho O.. Biosynthesis and metabolic actions of simple phenolic acids in plants. Phytochem. Rev. 2020;19:865–906. doi: 10.1007/s11101-020-09689-2. [DOI] [Google Scholar]
  43. Lee J., Scagel C. F.. Chicoric acid: Chemistry, distribution, and production. Front. Chem. 2013;1:40. doi: 10.3389/fchem.2013.00040. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Munteanu I. G., Apetrei C.. Analytical methods used in determining antioxidant activity: A review. Int. J. Mol. Sci. 2021;22:3380. doi: 10.3390/ijms22073380. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Huang D., Ou B., Prior R. L.. The chemistry behind antioxidant capacity assays. J. Agric. Food Chem. 2005;53:1841–1856. doi: 10.1021/jf030723c. [DOI] [PubMed] [Google Scholar]
  46. Cao H., Cheng W.-X., Li C., Pan X.-L., Xie X.-G., Li T.-H.. DFT study on the antioxidant activity of rosmarinic acid. J. Mol. Struct. 2005;719:177–183. doi: 10.1016/j.theochem.2005.01.029. [DOI] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

ao5c07413_si_001.pdf (1.2MB, pdf)

Articles from ACS Omega are provided here courtesy of American Chemical Society

RESOURCES