Introduction

Basil (Ocimum basilicum L.) is widely recognized for its medicinal, aromatic, and culinary properties, making it valuable across various sectors. The therapeutic potential of basil leaf extracts is vast, with applications in alleviating headaches, treating respiratory tract inflammation (including sore throat, laryngitis, and bronchitis), managing inflammatory bowel disease (IBD), addressing kidney dysfunction, and providing antipyretic and anticancer effects1,2,3. Furthermore, basil extracts offer beneficial pharmacological effects, including antioxidant, anti-inflammatory, analgesic, and antispasmodic activities, particularly in the application of chronic inflammatory conditions such as metabolic syndrome and inflammatory bowel diseases4,5,6. The health benefits of basil are primarily attributed to its secondary metabolites, especially phenolic and flavonoid compounds7,8.

Secondary metabolites, which are not essential for normal plant functions but play vital roles in plant defense, pollination, signaling, and coping with abiotic stress, are influenced by various environmental factors9,10. The levels of secondary metabolites in plants vary according to seasonal variations, diurnal cycles, and climate conditions11,12.

With the growing global population and rising food demand, nanotechnology applications in agriculture are gaining momentum. Nanomaterials can improve nutrient absorption efficiency in agriculture, reduce the need for frequent fertilizer applications, and minimize nutrient runoff, ultimately improving the sustainability of farming methods13. This enables more efficient utilization of plant nutrients, reduces the required amount of nutrients, and decreases application frequency14. Thus, compared with conventional fertilizers, nutrient-use efficiency can be significantly improved, and nutrient losses via leaching or volatilization are minimized15. Additionally, nanoparticles can be designed to target specific plant tissues, optimizing nutrient distribution to areas that require them the most13. The effects of nanoparticles on plants depend on factors such as the type of nanoparticle, particle size, composition, and surface area16. Plant responses to nanoparticles are shaped by various factors, including environmental conditions, plant species, age, exposure duration, and nanoparticle characteristics17, and foliar nanoparticles can be absorbed by the plant via the epidermis or stomata18. Foliar nanoparticles are regarded as an effective solution to address significant agricultural challenges. Delivering the right amounts of micro- and macronutrients through foliar sprays can reduce the damage caused by conventional soil-root application methods19.

Graphene oxide (GO) is a two-dimensional, carbon-based nanomaterial characterized by a high specific surface area, good electrical conductivity, and optical transparency. Owing to these properties, GO has been reported to modulate stress responses in plants exposed to stress factors, to affect chlorophyll and carotenoid contents, photosystem II (PSII) efficiency, and overall photosynthetic performance, and these alterations have been associated with improvements in plant growth performance and biomass20.

Conventional fertilizers contribute significantly to greenhouse gas emissions, particularly nitrous oxide (N₂O), which has a global warming potential approximately 300 times that of carbon dioxide21. On the other hand, conventional fertilizers like nitrogen-phosphorus-potassium (NPK) suffer from poor thermal stability, leading to significant nutrient loss in the soil and reducing their availability to plants22,23. Furthermore, degradation processes such as photolysis, hydrolysis, and microbial activity in the soil lead to the rapid breakdown of active ingredients in agrochemicals24,25. To overcome the limitations of existing agrochemical delivery systems, it is essential to reduce overuse, minimize nutrient loss, and improve nutrient bioavailability. Developing more efficient, innovative delivery systems is necessary to address the challenge of low effectiveness in current agricultural carriers26.

Recent years have seen a surge in nanoparticle research. Although basil has been widely studied, the impact of nanoparticles, particularly GO, on its quality characteristics has not been sufficiently explored, especially in plants grown with both organic and chemical fertilizers. This study investigates the effects of foliar-applied nGO (at varying doses) alongside farmyard manure or chemical fertilizer (NPK) on basil’s (O. basilicum L.) biological activities and essential oil composition. As one of the first to explore foliar nGO applications, it comprehensively examines the interactions between nanoparticle applications and fertilization methods, offering novel insights into sustainable agriculture.

Material and method

Experimental location

The experiment was conducted between 2022 and 2023 at the Medicinal and Aromatic Plants Research and Application Field of Afyonkarahisar Health Sciences University (38°46’ N, 30°27’ E, 1009 m). Afyonkarahisar has a harsh climate, characterized by hot, dry summers and cold, snowy winters27, with peak precipitation occurring in winter and spring. During the 2022 growing season, meteorological conditions were moderate, while 2023 experienced generally higher temperatures, increased rainfall, especially during early spring28, and greater humidity and sunshine during the 1st cutting (Table S1). Soil analysis at a 30 cm depth showed a clayey-loamy texture with low organic matter and salt levels but high lime content. Notably, organic matter increased in the second year, and soil pH shifted from standard limits to slightly alkaline. Nutrient levels (P, K, Fe, Cu, Mn) were mostly adequate, except for a Zn deficiency and excess Ca and Mg (Table S2).

Plant material, applications and design

The seeds of the Dino variety of basil, known for their rapid growth, large size, green color, and aromatic leaves, were used in the experiment. The seeds, selectively improved, were sourced from AG Seeds Industry and Trade Inc. The field experiment was designed using a randomized block split-plot layout with three replications. Plots measured 1.2 × 4 m, with 30 cm row spacing, 20 cm plant spacing (~ 166,660 plants ha⁻1), 2 m between plots, and 3 m between blocks. A total of 11 application plots were evaluated for the impact of foliar nGO doses on basil quality under organic and chemical fertilizers (Table 1).

Table 1 Number of applications and descriptions.
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The applications were divided into two groups: four plots with Ch (NPK: 90:60:60 kg ha⁻1) + nGO and four plots with F (30 tons ha⁻1) + nGO. The remaining three plots were allocated to Ch-only, F-only, and untreated control. F properties are detailed in Table 2. Table 3 presents the characteristics of commercially procured nGO as determined by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). F was incorporated into the soil 40 days before planting, whereas NPK was also applied with sowing. The full amounts of P (P2O5), K (K2O-water-soluble), and 2/3 of N (9% urea + 9% ammonia) were administered at planting. The remaining N (46%) was supplemented after the 1st cutting. Four nGO concentrations (100, 200, 300, and 500 mg L⁻1) were tested. In studies on medicinal and aromatic species, nGO/GO doses up to 300 mg L⁻1 were reported to confer protective and growth-promoting effects29,30,31,32. A dose beyond this range (500 mg L⁻1) was used to evaluate potential upper-limit and toxicity effects33,34. Furthermore, by co-applying nGO with organic manure and NPK fertilizer, we assessed possible synergistic effects that could enhance plant tolerance to higher doses. nGO suspensions were prepared by dissolving nanoparticles in an ultrasonic water bath (Faithful FSF-080S) for 120 min and applied at 400 ± 20 mL per plot. The initial nGO foliar application was conducted at the 8–12 leaf stage. A total of two applications were administered before each cutting at 14-day intervals (Table 4). Furthermore, the control group received distilled water sprays concurrently with nGO applications.

Table 2 Physicochemical properties of farmyard manure used in the experiment.
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Table 3 Properties of nGO* used in the experiment.
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Table 4 nGO applications and cutting times over a 2-year period.
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Harvesting

Cuttings were made at the beginning of flowering stage (Table 4). After eliminating border effects, the plants were cut 6–8 cm above the soil level. Samples were dried on racks in a well-ventilated room until moisture content reached 12 ± 1%. The moisture content was measured using the weight loss method, which involved tracking weight changes from initial to final stages of the drying process.

Distillation of essential oils

The amount of EO was measured volumetrically (ml 100 g⁻1) in triplicate using dried leaves from each application. Water distillation was performed with a neo-Clevenger apparatus, where 50 g of dried leaves were mixed with distilled water at a 1:10 ratio and processed for 180 minutes35. The EO content was then expressed as a percentage.

GC–MS analyses

A GC–MS system (Shimadzu GC–MS QP2010 SE) with a flame ionization detector (FID) was used to identify EO constituents. Preparation of EO samples, fixed column specifications, and the conditions of GC–MS analysis and injection are enclosed in Table S3.

The retention indices (RI) were calculated by injecting a mixture of C7-C30 n-alkanes (Merck/Supelco) into the GC/FID-MS system under the same conditions used for the analysis of the EOs. Compound identification was based on a comparison of RI values with the National Institute of Standards and Technology (NIST), Wiley Libraries, additional published mass spectrometry data36, and our own database. The relative abundance (% area) of each component was calculated as the ratio of its peak area to the total peak area.

Extraction and analysis procedure

Five grams of milled plant sample were transferred to an Erlenmeyer flask, and 100 mL of 80% methanol–water solution (Merck/Supelco) was added for extraction. The flasks were sealed with parafilm and sonicated in an ultrasonic water bath (Faithful FSF-080S) at 35–40 °C for 180 min. The extract was filtered through black band filter paper (Whatman, 125 mm) and transferred to pre-weighed containers. These were left in an oven (CLS / CLOF-125) at 40 °C for 3–4 days until solvent evaporation. The dried extracts were weighed on a precision balance (Ohaus Pr423/E), and the yield was calculated in grams37, expressed as percentages. For antioxidant activity, phenolic, and flavonoid content analysis, 20 mg of extracts stored at + 4 °C were solubilized by vortexing in Falcon tubes with 10 mL of 80% methanol–water.

DPPH free radical scavenging activity

The antioxidant activities of 80% methanol extracts and EOs from O. basilicum were assessed using a UV–Vis spectrophotometer (Thermo Scientific™ Multiskan).

Two methods were employed. In the 1st method, the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity was measured38,39. DPPH solution (200 µL, 0.4 mM) in methanol was added to test tubes containing 50 µL of sample solutions at varying concentrations (0.5, 1, and 2 mg mL⁻1). The control contained 50 µL of methanol instead of the extract. After a 30 min incubation at room temperature in the dark, absorbance was measured at 517 nm. The DPPH radical scavenging activity was calculated using the equation: RSA (%) = [(Δ517nm control − Δ517nm sample) / Δ517nm control] × 100.

The IC50 values (mg mL⁻1) were determined at different concentrations. A calibration graph and regression equation for the trolox standard are provided in Fig. S1.

Ferric ion reducing antioxidant power (FRAP)

The FRAP assay was conducted to evaluate the antioxidant activity of O. basilicum extracts and EOs by measuring Fe3⁺/Fe2⁺ reduction potential39,40. The FRAP reagent was prepared by mixing acetate buffer (0.3 M, pH 3.6), TPTZ (10 mM), and FeCl₃ (20 mM) in 40 mM HCl at a 10:1:1 ratio. Then, 200 µL of the reagent was added to test tubes containing 10 µL of sample solutions (0.5, 1, and 2 mg mL⁻1) and incubated at room temperature for 30 min. The absorbance was measured at 593 nm. FRAP values were expressed as trolox equivalents (mg TEs g⁻1) and IC50 values (mg mL⁻1), corresponding to an absorbance of 0.500. Figure S2 presents the trolox standard calibration curve and regression equation.

Total phenolic content determination

The total phenolic content (TPC) of O. basilicum extracts (80% methanol) was assessed via UV–Vis spectrophotometry (Thermo Scientific™ Multiskan) using the Folin-Ciocalteu reagent (AFG Bioscience)39. The extract solution (50 µL) was mixed with diluted Folin-Ciocalteu reagent (100 µL, 1:9), then shaken vigorously. After 3 min, 75 µL of 2% Na₂CO₃ (Merck/Millipore) was added, and the mixture was incubated at room temperature for 2 h. Absorbance was measured at 760 nm, and TPC was expressed as gallic acid equivalents (GAEs). Figure S3 shows the gallic acid standard calibration curve and regression equation.

Total flavonoid content determination

The total flavonoid content (TFC) of O. basilicum extracts (80% methanol) was quantified using a UV–visible spectrophotometer (Thermo Scientific™ Multiskan) and a standard colorimetric assay39. Extracts (200 µL) were mixed with 100 µL of 2% AlCl₃ solution in methanol. A blank solution was prepared with 200 µL sample solution and 100 µL methanol, excluding AlCl₃. After a 10-min incubation at room temperature, absorbances were measured at 415 nm, with the blank absorbance subtracted from the sample39. TFC was expressed as quercetin equivalents (QEs, mg QEs g⁻1). Figure S4 shows the calibration curve and regression equation for the quercetin standard.

Determination of residual nano Graphene Oxide

To investigate the residual status of the nGO applied to the leaves, FT-IR spectra (Perkin Elmer Spectrum Two) were acquired using ATR. These spectra were recorded in the range of 650–4000 cm⁻1, with a step size of 4 cm−1, at room temperature.

Statistical analyses

Antioxidant activity [IC50 (mg mL⁻1) values], TPC, and TFC were analyzed using ANOVA followed by Duncan’s Multiple Range Test. For EOs, statistical analysis was conducted using MSTAT-C software, with mean differences compared via Duncan’s Multiple Range Test. Heatmap analysis and principal component analysis (PCA) were conducted to evaluate relationships between key examined properties and applications, using ClustVis and JMP statistical software.

Ethical compliance

This study did not involve human participants or live animals and therefore did not require ethical approval. All plant materials were handled in accordance with institutional and national guidelines.

Results and discussion

Essential oil content

The EO content values over two years are presented in Table 5. Among the applications, the control group (0.82%) exhibited the highest EO content, while the ChG4 application (0.50%) had the lowest. EO content followed the ranking: control > farmyard manure > NPK. Similar studies have reported a reduction in EO content with fertilizer application in basil cultivation41,42. Cheng et al. (2009)43 suggested that secondary metabolite production is influenced by the balance between carbohydrate sources and sinks, with an increased source capacity ratio promoting metabolite synthesis.

Table 5 Essential oil content of O. basilicum.
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Higher nGO concentrations reduced EO content, likely due to nGO enhancing yield and alleviating stress conditions. While Naderianfar et al. (2018)44 and Mehrabani et al. (2023)45 reported an increase in EO content with nano fertilizers and GO, respectively, current findings contrast with these, demonstrating a decline at higher nGO concentrations. This discrepancy may be explained by the enhanced photosynthetic pigment levels and carbonic anhydrase activity observed in plants exposed to nanoparticles, which improve light utilization and carbohydrate accumulation but may redirect metabolic pathways away from EO biosynthesis46. Overall, current findings suggest that increasing nGO concentrations reduce EO content, possibly by altering the source-sink relationship and alleviating stress conditions that are critical for secondary metabolite synthesis.

When F and NPK-treated compared to the control application, the control group exhibited the highest EO content, suggesting that plants facing nutrient limitations produced higher levels of secondary metabolites. Additionally, EO content was higher in F-treated plots compared to NPK-treated ones (Table 2). The findings are consistent with studies by Azizi et al. (2009)47, who reported that nitrogen application reduced the EO content in Origanum vulgare. Similarly, Emongor et al. (1990)48 reported that phosphorus fertilizer application reduced the EO content in Matricaria chamomilla. Similar to the researchers’ findings, our study found that chemical and organic fertilizer applications decreased the EO content. In addition, Babalar et al. (2010)49 reported that nitrogen is an essential component in the biosynthesis of secondary metabolites, while certain micronutrients contribute to their biosynthesis through distinct physiological pathways.

nGO can inhibit the EO biosynthesis via imposing dose-dependent stress in plants 50,51. In the Lamiaceae family, EOs are synthesized in foliar secretory glands; stress alters both the structure and number of these glands, thereby reducing oil production52,53,54,55. Moreover, stress decreases the activity of terpenoid-biosynthetic enzymes such as DXS, diverting metabolic flux toward defense mechanisms51. It has also been reported that GO can coat the leaf surface, limiting light transmittance and stomatal gas exchange, while inducing ROS-mediated oxidative stress that redirects energy resources toward repair and defense50. High doses of Fe₂O₃ nanoparticles increase H₂O₂ accumulation and activate antioxidant defenses, inhibiting EO production51. Conversely, simultaneous application of nGO with proline may enhance proline transport and efficacy34.

In this study, nGO appears to trigger ROS-mediated oxidative stress and thereby prioritizing the defense/repair pathways, whereas NPK fertilizer and F reinforced the growth-oriented metabolism. The combined effects of potential stomatal blockage and constrained photosynthesis may have prevented the allocation of carbon skeletons and energy toward EO biosynthesis. Furthermore, nGO may have functioned as a carrier to enhance fertilizer efficacy, thereby further suppressing EO production.

nGO modulates stress-responsive gene expression, hormonal balance, reactive oxygen species (ROS) and antioxidant homeostasis, photosynthetic performance (PSII and chlorophyll content), and carbon–nitrogen metabolism; it also channels additional substrate into the MEP and MVA pathways, thereby altering the plant’s primary and secondary metabolite production45,56,57,58,59,60.

Essential oil components

Total 35 EO components were identified in O. basilicum L. var. dino (Tables 6, 7, 8, 9). Linalool (48.8–57.92%) was the predominant compound, reaching its highest concentration (57.92%) in the 1st cutting of the 1st year under the FG4 application, while the lowest (48.8%) was recorded under ChG2. Linalool levels were consistently elevated in the 1st cuttings of F-treated plots and in FG applications across both years. Moreover, in all ChG applications except ChG1, linalool content was higher in the 2nd cuttings. Eugenol (16.62–26.24%), the second major component, peaked under ChG4 in the 1st cutting of the 1st year but showed no significant response to nGO. In the control and Ch applications, eugenol levels were generally higher in the 1st cutting, whereas F applications exhibited elevated levels in the 2nd cutting. Additionally, other prominent constituents included 1,8-cineole (5.11–10.66%), cis-α-bergamotene (3.37–8.05%), and γ-cadinene (1.38–2.67%), with 1,8-cineole being highest in the control group and γ-cadinene reaching its maximum in FG3 (1st cutting, 1st year). Analysis of Tables 6, 7, 8, 9 revealed that certain EO components were absent in some repetitions of the four cuttings and eleven applications over the two experimental years. However, 11 components, myrcene, 1,8-cineole, linalool, bornyl acetate, cis-α-bergamotene, β-elemene, α-terpineol, germacrene D, γ-cadinene, epicubenol, and eugenol, were consistently detected across all cuttings and applications.

Table 6 Essential oil components of O. basilicum (1st year, 1st cutting).
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Table 7 Essential oil components of O. basilicum (1st year, 2nd cutting).
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Table 8 Essential oil components of O. basilicum (2nd year, 1st cutting).
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Table 9 Essential oil components of O. basilicum (2nd year, 2nd cutting).
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The identified EO constituents were classified into six categories: oxygenated monoterpenes (76.77–88.43%), sesquiterpene hydrocarbons (7.44–18.59%), monoterpene hydrocarbons (0.21–3.59%), oxygenated sesquiterpenes (0.57–2.5%), esters (0.77–1.55%), and alcohols (detected only in the 2nd cutting as a single compound; Table 7). Oxygenated monoterpenes were the most abundant, with the highest levels recorded in the ChG1 application and the lowest in FG3 during the 1st cutting of the 1st year (Table 6). On the other hand, sesquiterpene hydrocarbons encompassed the largest number of compounds. Overall, applications did not significantly alter the component composition, though cutting time and fertilizer type influenced specific concentrations (e.g., F enhanced linalool in 1st cuttings, while ChG applications increased eugenol). These findings align with prior reports of linalool- and eugenol-rich basil chemotypes and emphasize agronomic-driven variations, such as FG applications boosting linalool and ChG applications promoting eugenol, underscoring the role of cultivation strategies in modulating oil profiles61,62,63,64,65,66. In addition, this observation aligns with the findings of Sarmoum et al. (2019)61 and Raffo et al. (2020)62, highlighting that the biosynthesis of terpenoids happens through different pathways.

The EO of O. basilicum is not listed in globally recognized pharmacopoeias or monographs. Its composition is influenced by factors such as variety, geographical origin, and harvest season, which significantly impact its aroma and flavor profile.

Likewise, seasonal changes that occur depending on the changing harvest dates also affect the rate and composition of the EO63. Climatic changes, occurring during the approximately 1-month time difference between the 1st and 2nd cuttings, have influenced the composition of the EO.

Comparison with existing literature revealed that the EO composition in this study largely aligned with Lee et al. (2005)64 and partially corresponded with the findings of Brada et al. (2011)65 and Ilić et al. (2019)66. EOs in aromatic plants are synthesized through complex biochemical pathways, primarily composed of terpenes, while bioactive compounds such as phenylpropanoids shape their aroma and flavor. Phenolic compounds, fatty acid derivatives, and isoprenoids also contribute to EO biosynthesis67,68. The synthesis of bioactive compounds in medicinal plants, is governed by genetic factors and dynamically shaped by environmental variables (climate, soil, cultivation practices) and developmental stage. Epigenetic modifications regulate metabolite biosynthesis pathways, whereas post-harvest processing techniques critically determine their final chemical composition and stability69. Although no prior studies have investigated the effects of nGO on basil EOs, conflicting evidence exists regarding the influence of nanoparticles on EO production. Some studies report increased oil content and major components following nanoparticle applications70,71,72,73 whereas others found no significant impact74. In this study, EO yield and composition varied across fertilization applications, with distinct patterns observed in linalool and eugenol concentrations. External factors may further modify EO profiles through biochemical transformations such as oxidation, isomerization, and dehydrogenation75.

Extract yield

The extract yields obtained from various cuttings are presented in Table 10. As shown, the highest extract yield (20.88%) was recorded in the 2nd cutting of the 1st year, while the lowest yield (15.25%) was observed in the 2nd cutting of the 2nd year. Statistical analysis indicated no significant effect of the applications on extract yield values. A comparison of the experimental data with literature values revealed that current results were consistent with those reported by Yaldiz et al. (2018)76 (9–33.86%) and Yaldiz and Camlica (2022)77 (13.69–29.47%).

Table 10 Extract yield values (%) of O. basilicum for different cuttings.
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In our study, the extract yield varied significantly across cuttings and years. However, differences among fertilizers, nGO, and combined applications were not found to be statistically significant, suggesting that cuttings and inter-annual variability exert a dominant influence on extract yield. Since the basil cuttings were completed in early August and in September in the 1st application year and in late August and in September in the 2nd application year, the extract yield may have been affected by inter-annual climatic differences (Table 4). Furthermore, the changes in soil mineral content between the two experimental years may have affected the extract yield (Table S2).

DPPH free radical scavenging activity

The DPPH radical scavenging activity (%) of extracts and EOs is summarized in Table 11. The highest extract activity (88.35%) was observed in the 1st cutting of the 1st year, while the lowest (82.27–83.88%) occurred in both cuttings of the 2nd year. For EOs, the highest activity (67.53–68.22%) was recorded in the 2nd cutting of the 1st year and the 1st cutting of the 2nd year, with the lowest (63.64%) in the 1st cutting of the 1st year. The ChG2 and control groups exhibited the highest activity (68.50–68.60%), whereas the Ch application showed the lowest (62.89%).

Table 11 DPPH radical scavenging activity of O. basilicum extract and essential oil according to different cuttings
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As can be seen from Table 11, the DPPH radical scavenging activity of the EOs were found to be statistically significant at the level of 0.05% (p < 0.05) between fertilizer and nGO applications and the cuttings. Especially, the highest activity was recorded in the 1st cutting of the F application and the 2nd cutting of the FG4 application (73.9–73.8%), while the lowest was observed in the 1st cutting of the FG3 application (49.3%). So, the the DPPH radical scavenging activity values showed differences among the applications depending on the cuttings. The DPPH radical scavenging activity values at 2nd cutting showed a regular decrease among all applications and the highest values were determined in the 1st cutting in two experimental years. In addition, when all applications were compared over the two years, FG4 and the control showed higher values than the other applications. Moreover, FG4 application had the maximum DPPH radical scavenging activity for the two experimental years. The results of Javed et al. (2017)78 and Dey et al. (2013)79 also confirmed that antioxidant activity was increased by using GO.

Extract activity surpassed values reported by Naidu et al. (2016)80 (73.75%) and Yaldiz and Camlica (2022)77 (8.14–54.69%). EO activity aligned with Farouk et al. (2016)81 (22.2–86.9%) but diverged from those reported by Khelifa et al. (2012)82, Mahendran and Vimolmangkang (2023)83, and Kanmaz et al. (2023)84. These discrepancies likely reflect compositional differences, particularly in eugenol and linalool content, as demonstrated in prior studies85,86.

Antioxidant activity in plants is linked to stress-induced secondary metabolites87 and is associated with volatile phenolic compounds such as eugenol, as well as flavanols, flavonoids, and anthocyanins88, and is affected by solvent polarity during extraction89,90. Politeo et al. (2007)85 reported lower DPPH activity (1.378 mg mL⁻1) compared to Stanojević et al. (2017)91 (2.38 mg mL⁻1), with the latter’s enhanced activity attributed to higher eugenol concentrations despite comparable linalool and methyl chavicol levels. Furthermore, increasing solvent polarity by adding water, which causes the plant material to swell, improves the solvent’s penetration of the solid matrix and enhances the extraction of phenolic compounds92.

Moreover, nanoparticle applications can affect antioxidant properties; for instance, Baali et al. (2019)93 reported high DPPH scavenging activity for pure GO, while Guo et al. (2021)94 and Jiao et al. (2016)95 observed that graphene enhanced the antiradical activity of iron oxide nanoparticles. GO may enhance activity by improving compound translocation and stress resilience96,97, as evidenced by increased limonene and cis-ocimene levels in Artemisia dracunculus98. Given that the antioxidant activity of EOs is linked to their constituent compounds, and considering that GO is known to influence these components, it is suggested that the observed differences in DPPH free radical scavenging activity in this study may result from the nGO application. Nonetheless, the variations in DPPH values likely stem from a combination of nGO application, genetic factors, differences in extraction methods, environmental conditions, and agronomic practices. Additionally, Table 11 indicates that extracts exhibit higher activity than EOs, which is consistent with Ahmed et al. (2019)99.

Ferric ion reducing antioxidant power (FRAP)

As can be seen from Table 12, the FRAP reducing power values of extracts and EOs were found to be statistically significant at the level of 0.05% in terms of applications and cutting time (p < 0.05). For extracts, the highest FRAP value was observed in the 1st cutting of the 1st year (250.30 µg TEs mg⁻1) and the lowest in the 2nd cutting of the 2nd year (199.61 µg TEs mg⁻1). In addition, the 1st cuttings showed significantly higher activity than 2nd cutting in both years. In addition, the highest FRAP reducing power values of extracts was obtained in FG4 (247.3 µg TEs mg-1) application, followed by the FG2 (242 µg TEs mg-1) application in the both years while the lowest FRAP reducing power values of extracts was obtained in Ch (208.7 µg TEs mg-1) application (Table 12). In EOs, the highest FRAP reducing power values was obtained in Ch2 (255.3 µg TEs ml-1) application, followed by the control (254.9 µg TEs ml-1) application in the both years while the lowest FRAP reducing power values was obtained in Ch (232.8 µg TEs ml-1) application (Table 12). So, FRAP reducing power values amount increased with the F and nGO applications likely due to the high concentrations of Cu, and Zn present in F content (Table 2).

Table 12 Ferric ion reducing antioxidant power of O. basilicum extract and essential oil according to different cuttings.
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The extract values obtained this study differed from Nadeem et al. (2022)100 (237 µmol Fe g⁻1) and Kanmaz et al. (2023)84 (146.36 µmol Fe g⁻1). On the other hand, antioxidant activity is influenced by various factors, including storage period, geoclimatic conditions, environmental factors, extraction techniques, solvents, analytical methods, calculation standards, and other technological considerations101. Additionally, drying techniques have been shown to affect the bioactive compounds in plants and preserve those compounds exhibiting antioxidant activity by preventing potential oxidation102. Furthermore, it has been emphasized that factors such as solvent polarity, pH, temperature, and concentration play crucial roles in determining the solubility of phenolic compounds in the solvent103. Antioxidant assays may also differ depending on the solubility of phenolic compounds. While applications did not alter total phenolics/flavonoids, cuttings significantly influenced these metrics.

Current findings suggest that the observed FRAP variations are attributable to nGO application along with genetic, methodological, and environmental differences. For EOs, the highest FRAP value was observed in the 2nd cutting of the 1st year (253.5 µg TEs ml⁻1) and the lowest in the 1st cutting of the 1st year (236.2 µg TEs ml⁻1). Among applications, ChG2 (255.3 µg TEs ml⁻1) and Ch (232.8 µg TEs ml⁻1) displayed the highest and lowest values, respectively. The FRAP reducing power values in EOs were significantly affected by the interaction between fertilizer and nGO applications and cuttings. Specifically, the highest value was recorded in the 1st cutting of the F application of the 1st year (276.0 µg TEs ml⁻1), while the lowest was observed in the 1st cutting of the FG3 application of the 1st year (181.0 µg TEs ml⁻1). A comparison with previous studies revealed that the values obtained from the EOs in this research differed from those reported by Mohammed et al. (2020)104 (86.30 mg TEs g⁻1) and Kanmaz et al. (2023)84 (47.88 mmol FE), respectively. Antioxidant activity in basil is influenced by multiple cuttings per season105, with cutting time affecting phenolic/flavonoid content106. These discrepancies likely stem from genotypic variation, cutting timing, environmental/climatic conditions, and methodological differences (drying, extraction solvents, nGO doses). Stress responses, such as terpenoid accumulation under adverse conditions107, and agronomic practices (soil, climate, post-harvest handling) further modulate metabolite profiles108. Additionally, Table 12 indicates that extracts consistently exhibit higher FRAP activity than EOs, which is in agreement with previous reports.

Total phenolic content determination

Table 13 and Fig. 1 present the total phenolic content (TPC) of the extracts from different cuttings. As can be seen from Table 13, the TPC values were not found to be statistically significant. The highest TPC values (81.25–96.09 µg GAEs mg⁻1) were observed in the 1st and 2nd cuttings of the 1st year and the 1st cutting of the 2nd year, whereas the 2nd cutting of the 2nd year yielded the lowest (81.25 µg GAEs mg⁻1). The TPC of O. basilicum extracts varied significantly across cuttings, supporting the hypothesis that cutting time influences phenolic accumulation.

Table 13 Total phenolic content and flavonoid levels according to different cuttings in O. basilicum extracts.
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Fig. 1
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Total phenolic content (µg GAEs mg-1 sample) according to cuttings in O. basilicum extracts. GAEs: gallic acid equivalents. Superscript lowercase letters (a, b, c) indicate statistically significant differences among values (p < 0.05), as determined by one-way ANOVA followed by Duncan’s multiple range test.

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Based on data obtained from this study, especially FG4 application showed high TPC in two experimental years that may enhance agronomic traits, photosynthetic pigments, chlorophyll fluorescence parameters, membrane stability index (MSI), proline, phenols, antioxidant enzymes activities and dominant constituents of essential oils and decreasing MDA and H2O33. In addition, positive effect of GO nanoparticles may be referred to enhance the effect of GO in the synthesized nanostructure33. In our study, the total phenolic content increased with the foliar use of nGO and F applications. This increase could be due to the high concentrations of Fe, Ca, Mg and Zn present in F content and GO can enhance plant improvement and nutrient uptake by minimizing nutrient losses or enhancing the plant defense system45. However, Hassanpouraghdam et al. (2023)98 reported elevated TPC under GO application (77.8 vs. 64.8 mg g⁻1 FW) in the control.

Consistent with previous findings, Gavrić et al. (2018)106 reported that 2nd cuttings in basil exhibited higher TPC. The seasonal decrease in phenolic content may be attributed to their conversion into insoluble cell wall components or their transformation into oligomeric and polymeric compounds such as tannins or lignans109.

Current TPC values were lower than those reported by Yaldiz and Camlica (2022)77 (4.47–42.94 mg GAE g⁻1), Kanmaz et al. (2023)84 (55.64 mg GAE g⁻1), and other studies. These discrepancies are likely due to factors influencing phenolic content, including the plant’s ontogenetic stage, biotic/abiotic stress (e.g., pest exposure, drought)110, biodiversity, seasonal dynamics111, UV radiation112,113, and rainfall114.

These findings underscore the role of agronomic practices, environmental conditions, and analytical methods in shaping phenolic profiles, aligning with literature trends while highlighting context-specific variability.

Total flavonoid content determination

Table 13 and Fig. 2 present the total flavonoid content (TFC) of the extracts from various cuttings. The highest TFC was observed in the 1st cutting of the 2nd year (8.78 µg QEs/mg), while the 2nd cutting of the same year had the lowest (3.78 µg QEs/mg). Different applications were not found to be statistically significant at the level of 0.05% (p < 0.05).

Fig. 2
figure 2

Total flavonoid content (µg QEs mg-1 sample) according to cuttings in O. basilicum extracts. QEs: quercetin equivalents. Superscript lowercase letters (a, b, c) indicate statistically significant differences among values (p < 0.05), as determined by one-way ANOVA followed by Duncan’s multiple range test.

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Among the all applications, the highest TFC was obtained from ChG1 (6.86 µg QEs mg-1) application and followed by FG4 (6.27 µg QEs mg-1) and FG1 (6.07 µg QEs mg-1) applications. The lowest contents of TFC were recorded at FG2 (5.63 µg QEs mg-1) application in two experimental years. Therefore, compared with the Ch applications, the F applications increased the TFC. Likewise TPC, TFC increased with the foliar use of nGO and F applications due to the high concentrations of Fe, Ca, Mg, and Zn present in F content and GO can enhance plant improvement and nutrient uptake by minimizing nutrient losses or enhancing the plant defense system45.

Compared to the literature, current TFC values were lower than those reported by Yaldiz and Camlica (2022)77 (4.42–14.33 mg QE g-1 dry weight), Balanescu et al. (2020)115 (619.34–689.05 mg Qeq g-1), and Kanmaz et al. (2023)84 (19.90 mg GAE g-1). Phenol and flavonoid concentrations are influenced by environmental conditions (water, air, soil, altitude, light, ultraviolet radiation, temperature, salinity, drought), species variation, and extraction methods116,117. For instance, Hassanpouraghdam et al. (2023)98 found that GO application reduced flavonoid content in tarragon to 4.14 mg g-1, while Gavrić et al. (2018)106 reported higher TFC in the 2nd cuttings of O. basilicum, contrasting current results. These variations underscore the impact of species-specific traits, seasonal dynamics, and abiotic stressors on flavonoid synthesis.

Status of nano graphene oxide residues

FT-IR spectra (Photograph S1-4) revealed no peaks corresponding to nGO residues. Specifically, no changes were observed in the C–C stretching region (~ 1009 cm⁻1), CH₂/CH₃ peaks (2850–2950 cm⁻1), or CN/CO vibrational bands. Minor differences in the OH peak were attributed to moisture retention variations rather than nGO presence.

Literature indicates that nanoparticles, depending on their size, can enter plant cells via apoplastic uptake and endocytosis118, spread through symplastic transport and vascular tissues119. During foliar application, nanoparticles primarily penetrate stomata, migrate to mesophyll cells, and translocate via xylem and phloem120. They may also reach subcellular organelles121,122 and induce toxic effects via reactive oxygen species (ROS) generation123,124.

However, FT-IR analysis detected no nGO residues, suggesting that the application method (e.g., combined use with manure/NPK), plant developmental stage, or applied concentration may have influenced the outcome.

Heatmap analysis

To determine the relationship between the applications and the examined properties, a heatmap analysis was conducted (Fig. 3). The results of the heatmap cluster analysis showed two types of dendrograms: one for applications (horizontally positioned) and one for examined properties (vertically positioned). The analysis demonstrated that the primary chemical properties of basil were significantly influenced by the applications. Significant differences were observed among the applications, with the heatmap segmented into four groups based on the examined properties. Notably, FRAP-extract and TPC-extract were found in the same cluster, and this cluster was separated by the applications of FG2 and FG4. The remaining applications clustered together, though FRAP-EO formed a separate cluster associated with FG2, FG3, and FG4. Furthermore, DPPH-EO, DPPH-extract, and FRAP-EO were noted in the same cluster with three major EO components: linalool, 1,8-cineole, and eugenol. In general, extract-related values clustered together, with the exception of DPPH-extract. Incorporating the applications, the heatmap analysis was divided into four different groups. FG2 and FG4 formed separate groups, while ChG2, Ctrl, and ChG4 clustered together. The remaining applications clustered together in the fourth group (Fig. 3).

Fig. 3
figure 3

Heatmap analysis result of the Ocimum basilicum depending on the examined properties. From orange to red (between 1 and 2) colors indicate higher data values, and from light shades of yellow (between 0 and 1) to blue (between -0.5 and 0) colors represent lower data values. DPPH: 1,1-diphenyl-2-picrylhydrazyl, FRAP: Ferric reducing antioxidant power, TPC: Total phenolic content, TFC: Total flavonoid content, EO: Essential oil, Ctrl: Control, F: Farmyard manure, FG1: Farmyard manure + nGO100, FG2: Farmyard manure + nGO200, FG3: Farmyard manure + nGO300, FG4: Farmyard manure + nGO500, Ch: NPK, ChG1: NPK + nGO100, ChG2: NPK + nGO200, ChG3: NPK + nGO300, ChG4: NPK + nGO500.

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Principal component analysis (PCA)

To classify the different application types based on the examined properties, a principal component analysis (PCA) was performed using data from basil treated with nGO foliar application and chemical-organic fertilizers. The PCA results explained 60.5% of the total variance, with PC1 accounting for 35.9% and PC2 contributing 24.6% (Fig. 4). PC1 was strongly associated with TFC-extract, FRAP-extract, and EY-average, whereas PC2 correlated with the averages of EOC, 1,8-cineole, and eugenol (Fig. 4). The applications were separated into distinct groups based on the examined properties. DPPH-extract exhibited correlations with both DPPH-EO and FRAP-EO activities. The cis-α-bergamotene was noted in a different area of the PCA. In the application-group analysis, FG1, FG2, and FG4 exhibited strong positive correlations with FRAP-extract, TFC-extract, and linalool content. ChG4 and F applications correlated with eugenol average, 1,8-cineole average, and EOC average. Ch and FG3 applications displayed marked associations with cis-α-bergamotene average (Fig. 4).

Fig. 4
figure 4

Principal component analysis result of the examined properties in Ocimum basilicum. DPPH: 1,1-diphenyl-2-picrylhydrazyl, FRAP: Ferric reducing antioxidant power, TFC: Total flavonoid content, EO: Essential oil, EOC: Essential oil components, Ctrl: Control, F: Farmyard manure, FG1: Farmyard manure + nGO100, FG2: Farmyard manure + nGO200, FG3: Farmyard manure + nGO300, FG4: Farmyard manure + nGO500, Ch: NPK, ChG1: NPK + nGO100, ChG2: NPK + nGO200, ChG4: NPK + nGO500.

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Strengths and limitations of the study

This split-plot randomized complete block design (RCBD) with three replications evaluated interactions among nGO doses (100, 200, 300, 500 mg L⁻1), fertilizer types (chemical/organic), harvest years, and cutting cycles across 11 applications. The two-year field trial (2022–2023, two cuttings per year) provides realistic data for agricultural application, with comparative assessment of NPK and farmyard manure enhancing practical relevance. Phytochemical profiling included analyses of EO composition (GC/FID-MS), extract yield, antioxidant activities (DPPH, FRAP), and total phenolic/flavonoid contents. Multivariate analyses (PCA, heatmap) explained 60.5% of the variance in antioxidant and essential oil profiles, supporting application grouping. No nGO residues were detected in dried leaves via FT-IR, though confirmation with more sensitive quantitative methods is recommended due to FT-IR’s detection limits. Overall, the study’s multi-factor field design, comprehensive chemical-biochemical evaluation, and integration of advanced statistical analyses constitute notable strengths that enhance the robustness and applicability of its findings.

Several limitations should be acknowledged in this study. The tested nGO dose range (100, 200, 300, 500 mg L⁻1) did not encompass lower biostimulant levels (< 100 mg L⁻1) applied via foliar spraying. Physiological and biochemical mechanisms underlying the nGO dose-dependent reduction in essential oil content (e.g., gene expression, stress signaling pathways) remain uninvestigated. Although no detectable levels of nGO were found in leaves by FT-IR, trace residues cannot be ruled out due to the detection limits of the method; thus, more sensitive quantitative techniques are recommended. Finally, potential long-term environmental impacts, effects on nutritional composition, and human health implications (in vitro/in vivo toxicity) of nGO applications were not assessed, warranting further toxicological investigation.

Conclusions

This study identified linalool as the predominant component of basil’s EO, with eugenol, 1,8-cineole, and cis-α-bergamotene as secondary constituents. The cutting process exerted a significant impact on extract yield, antioxidant capacities (DPPH, FRAP), and phytochemical composition (TPC, TFC). In contrast, fertilizer and nGO applications primarily influenced the antioxidant characteristics of EOs. Notably, certain cuttings yielded superior extract performance and antioxidant potential, while synergistic effects of fertilizer and nGO (e.g., F and FG4) enhanced EO bioactivity.

The results emphasize the differential regulatory roles of agricultural practices, particularly the contribution of nGO, in shaping the bioactive profiles of EOs and plant extracts. Furthermore, FT-IR spectroscopy confirmed the absence of detectable nGO residues in dried leaves. However, the potential risks associated with nanoparticle toxicity necessitate further genetic and toxicological investigations to ensure the safe implementation of nanomaterials in agriculture. Additionally, it is suggested that future research should include testing of foliar applications at lower doses (below 100 mg L⁻1).

PCA and heatmap analyses grouped the applications into four clusters, explaining 60.5% of the total variance, with FG2 and FG4 linked to antioxidants (FRAP, TFC), and ChG4 and F to EOs (eugenol, 1,8-cineole). Antioxidant activity and EO composition varied notably among applications, especially in FG groups (FG1, FG2, FG4) and Ch-related applications (Ch, ChG4), indicating the distinct biochemical responses to different applications. The distinct clustering of DPPH-extract and cis-α-bergamotene further underscores the application-specific modulation of basil phytochemistry.