The aerial parts of C. martinii were gathered at three distinct growth stages: (i) July (Vegetative stage), (ii) October (Reproductive stage), and (iii) January (Post-reproductive stage) from Kailashpuri, a hilly area in Udaipur, Rajasthan, India, across the years 2021 and 2022. The plant sample was submitted to the University of Rajasthan, Jaipur, India, for proper identification (Voucher number: RUBL21430). The collection site is located at 24° 44’ 46” N and 73° 43’ 20” E, with recorded environmental conditions: relative humidity between 33–72%, temperature ranging from 24–37°C, and precipitation varying from 76.8–235.6 mm.

All chemicals and reagents utilized in this study were of analytical grade, ensuring high purity for accurate and reliable results. Methanol (Merck, 34860, ≥99.9%) was employed as the solvent for gas chromatography-mass spectrometry (GC-MS) analysis. For the assessment of antioxidant activity, the following reagents were used: 2,2-diphenyl-1-picrylhydrazyl (DPPH) (Merck, 102839, ≥95%), ascorbic acid (Merck, A92902, ≥99%), ABTS (2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt) (Merck, A1888, ≥98%), potassium persulfate (Merck, 379824, ≥99%), β-Carotene (Merck, 22040, ≥97%), linoleic acid (Merck, L1376, ≥99%), and Tween 40 (Polysorbate 40) (Merck, P1504, ≥99%). These high-purity reagents were selected to ensure the reliability and consistency of the experimental results.

The CMEOs was extracted following the method outlined by Rathore et al. (2022). Aerial parts of C. martinii, including leaves, stems, and flowers were collected at different intervals across the three growth stages: vegetative, reproductive, and post-reproductive. For each extraction, 500 g of fresh plant material was placed in a round-bottom flask (3 L capacity) with 1 L of water and subjected to hydro distillation for 4 hours using a Clevenger-type apparatus, suitable for extracting EOs lighter than water. The extracted oils were then dried over anhydrous sodium sulfate and stored in amber glass vials at 4°C until analysis. The essential oil yield at each growth stage was determined as a percentage of the fresh plant material weight, using the Equation (1).

The chemical profiling of the CMEOs was conducted with a Thermo Fisher Scientific gas chromatograph (TRACE™ 1300), coupled to a Thermo Fisher Scientific triple quadrupole mass spectrometer (TSQ 9000). The separation process utilized two columns: a TG-SQC front column (0.25 µm thickness, 0.25 mm ID, 15 m length) with 5% phenyl methyl polysiloxane and a TG-1MS back column (0.25 µm film thickness, 0.25 mm ID, 30 m length) made of 100% dimethyl polysiloxane. The oven temperature started at 60°C for an initial hold of 10 minutes. For analysis, 1 μL of diluted samples (1/100 in GC-MS grade methanol, v/v) was injected in split mode (split ratio 1/50) via an autosampler (AI 1310). Injection and ion source temperatures were set to 250°C and 300°C, respectively, with helium as the carrier gas at a flow rate of 1.0 mL/min. The column temperature was maintained at 60°C for 5 minutes, then increased to 250°C and held there for 15 minutes. Ionization was set at 70 eV with a 0.7 kV ionization current. The essential oil components were identified based on MS response, retention time, peak area, and relative peak percentage, with mass spectra matched to the Wiley 7 library. Retention indices of n-alkanes (C9-C40) were also compared with NIST database entries and relevant literature (de Alencar Filho et al., 2017). Component percentages were averaged from GC and GC-MS peak areas, with data processed using the Thermo Scientific™ Dionex™ Chromeleon™ 7 software, version 7.3.

The antimicrobial activity of CMEOs was evaluated against a range of clinical bacterial pathogens, including Escherichia coli (ATCC 25922), Salmonella typhi (MTCC 3216), and Staphylococcus aureus (ATCC 25923). Post-harvested fungal pathogens isolated from decayed fruits included Alternaria alstroemeriae (BG3; PP594937, Source: Black grapes), Fusarium fujikuroi (BN1; PP594939, Source: Banana), and Colletotrichum queenslandicum (PY1; PP594922, Source: Papaya). Bacterial and fungal cultures were maintained at 4°C and sub-cultured monthly to ensure viability. Prior to exposure to the essential oils, bacterial strains were incubated on Mueller-Hinton agar (MHA) at 37°C for 12–18 hours, while fungal strains were cultivated on Potato Dextrose Agar (PDA) at 26°C.

OriginPro software was utilized for statistical analysis. Descriptive statistics, including the calculation of means (n=3) and standard deviations (SD), were reported as mean ± SD for each treatment group. To evaluate statistical significance among groups, both one-way and two-way ANOVA were applied. One-way ANOVA was used to compare the means among the different essential oil treatments, while two-way ANOVA examined interaction effects between growth stages and variations in bioactivity. Following ANOVA, the Tukey test was performed to pinpoint significant differences between group means. Principal Component Analysis (PCA) was conducted to demonstrate how various growth stages of the plant influenced the bioactivity of the essential oils. All experimental procedures were conducted in triplicate to enhance reliability and reproducibility. The normal distribution of the data was checked before analysis, and statistical significance was defined at a p-value of less than 0.05 for all tests. IC₅₀ values were determined using non-linear regression analysis with a sigmoidal dose-response model.

As shown in Table 1 and Figure 1 , the essential oils (EOs) isolated from C. martinii at different growth stages, vegetative, reproductive, and post-reproductive, exhibited significant variations in yield, composition, and appearance. CMEOs color transitioned from olive (vegetative) to pale yellow (reproductive) and dark (post-reproductive) as seen in Supplementary Figure S1 . Oil yield was highest during the reproductive stage (0.73%), compared to the vegetative (0.35%) and post-reproductive (0.53%) stages. GC-MS analysis identified a total of 59 compounds across all stages. The reproductive phase had the highest number of constituents (49; 97.86%), followed by the post-reproductive (42; 98.70%) and vegetative (30; 99.77%) stages. In the vegetative stage, major compounds included carveol (20.87%), trans-p-mentha-1(7),8-dien-2-ol (12.9%), cis-p-mentha-1(7),8-dien-2-ol (8.77%), trans-carveyl acetate (7.42%), D-limonene (6.2%), and β-myrcene (3.38%). During the reproductive stage, the dominant constituents shifted to cis-piperitol (15.27%), cis-p-mentha-1(7),8-dien-2-ol (15.52%), carveol (9.86%), D-limonene (6.83%), trans-carveyl acetate (6.6%), and carvone (3.31%). In the post-reproductive stage, trans-p-mentha-1(7),8-dien-2-ol (19.58%), cis-p-mentha-1(7),8-dien-2-ol (17.59%), cis-piperitol (13.29%), carveol (11.32%), trans-carveyl acetate (7.93%), and carvone (3.41%) predominated. Across all stages, oxygenated monoterpenes constituted the majority (75–80%), followed by monoterpene hydrocarbons (15–20%), and a minor fraction (1–2%) of non-terpenoid compounds, including aromatic compounds, ethers, esters, alkane derivatives, and chalcones. Stage-specific variation in compound abundance and distribution was effectively visualized using a heat map ( Supplementary Figure S2 ), clearly illustrating the dynamic changes in EO composition during plant development.

The antibacterial activity of CMEOs from vegetative, reproductive, and post-reproductive stages was evaluated against S. aureus, E. coli, and S. typhi. Significant differences (P < 0.0001) were observed across growth stages and bacterial strains ( Supplementary Figure S3 ), reflecting the variation in CMEOs chemical profiles. Reproductive-stage CMEO exhibited the highest antibacterial efficacy, with the largest ZI and lowest MIC and MBC values ( Table 2 ). Post-reproductive CMEO showed moderate activity, while vegetative-stage CMEO was least effective. For E. coli, ZI was 27.7 mm in the reproductive stage, compared to 17.7 mm and 15.3 mm in post-reproductive and vegetative stages, respectively. S. typhi showed ZIs of 35.7 mm, 24.3 mm, and 19.3 mm across the respective stages. S. aureus followed the same trend: 33.7 mm (reproductive), 22.3 mm (post-reproductive), and 14.7 mm (vegetative) ( Figure 2a ). MIC and MBC values supported these findings ( Figures 2b, c ). S. aureus was the most sensitive strain across all stages, with an MIC of 383.3 µg/mL and MBC of 638.3 µg/mL during the reproductive stage. S. typhi and E. coli exhibited higher MICs (483.3 and 533.3 µg/mL) and MBCs (875.0 and 891.7 µg/mL, respectively). This pattern persisted in the other two growth stages, with E. coli consistently showing the lowest susceptibility. Although the positive control exhibited stronger antibacterial effects, CMEOs demonstrated considerable efficacy. Correlation analysis ( Supplementary Figure S4 ) revealed a negative relationship between MIC values and key compounds such as trans-p-mentha-2,8-dien-1-ol, carvone, and D-limonene, suggesting their contribution to antibacterial activity.

The antifungal activity of CMEOs from vegetative, reproductive, and post-reproductive stages was evaluated against C. queenslandicum, F. fujikuroi, and A. alstroemeriae ( Supplementary Figure S5 ). As shown in Table 3 and Figure 3 , variations in CMEO composition significantly influenced fungal growth inhibition, MIC, and MFC (P < 0.0001). Among the tested strains, A. alstroemeriae was the most sensitive. Inhibition increased from 31.36% (vegetative) to 53.70% (reproductive), before declining to 45.72% (post-reproductive). MIC values followed the same trend: 850 µg/mL (vegetative), 660 µg/mL (reproductive), and 810 µg/mL (post-reproductive). Corresponding MFC values were 1270, 930, and 1200 µg/mL, respectively. F. fujikuroi showed moderate sensitivity. The lowest inhibition (25.91%) and highest MIC (600 µg/mL) and MFC (1010 µg/mL) were recorded in the vegetative stage. The reproductive-stage CMEO exhibited the highest efficacy (52.50% inhibition, 500 µg/mL MIC, and 960 µg/mL MFC), while post-reproductive EO showed moderate improvements over the vegetative stage (39.21% inhibition, 500 µg/mL MIC, and 960 µg/mL MFC). C. queenslandicum was the least sensitive. Inhibition was 27.26% in the vegetative stage, peaking at 52.52% in the reproductive stage, then dropping to 33.32% in the post-reproductive stage. MIC and MFC values showed a similar pattern: highest in the vegetative stage (950 and 1270 µg/mL), lowest in the reproductive stage (800 and 1170 µg/mL), and intermediate in the post-reproductive stage (850 and 1250 µg/mL). Although the positive control showed substantially higher antifungal activity ( Figure 3 ), CMEOs demonstrated meaningful inhibitory effects. Correlation analysis ( Supplementary Figure S4 ) indicated strong negative correlations between MFC values and compounds such as (S)-perillyl alcohol, α-methylcinnamaldehyde, and isoeugenol, suggesting their potential contribution to antifungal activity against A. alstroemeriae, F. fujikuroi, and C. queenslandicum.

The antioxidant activity of CMEOs from vegetative, reproductive, and post-reproductive stages was evaluated using DPPH, ABTS, and β-carotene bleaching assays, with significant variation observed across stages (P < 0.0001) ( Figures 4 – 6 ). In the DPPH assay ( Figure 4 ), post-reproductive EO exhibited the highest scavenging activity (92.29% at 50 mg/mL), followed by reproductive (84.29%) and vegetative (79.77%) stages. IC₅₀ values followed the same trend: post-reproductive (26.44 mg/mL), reproductive (31.73 mg/mL), and vegetative (36.48 mg/mL). Ascorbic acid (standard) achieved 98.87% inhibition at 30 µg/mL with an IC₅₀ of 14.89 µg/mL ( Table 4 ). The ABTS assay ( Figure 5 ) also showed stage-dependent activity. Post-reproductive EO demonstrated the strongest inhibition (92.90% at 50 mg/mL, IC₅₀= 13.81 mg/mL), followed by reproductive (87.49%, IC₅₀ = 16.05 mg/mL) and vegetative (81.87%, IC₅₀ = 20.64 mg/mL). Trolox (standard) reached 92.90% inhibition at 20 µg/mL with an IC₅₀ of 4.01 µg/mL ( Table 5 ). In the β-carotene bleaching assay ( Figure 6 ), inhibition ranged from 76.6% to 87.26%. The post-reproductive EO showed the highest activity (87.26%), followed by reproductive (79.64%) and vegetative (76.6%) stages. IC₅₀ values were lowest in the post-reproductive EO (18.15 mg/mL), followed by reproductive (22.24 mg/mL) and vegetative (28.16 mg/mL) ( Table 6 ). Ascorbic acid again showed strong activity (IC₅₀ = 15.81 µg/mL). Kinetic monitoring of β-carotene degradation ( Figure 6c ) confirmed sustained inhibition over time, strongest in post-reproductive EO, followed by reproductive and vegetative stages. Overall, the antioxidant potential of CMEO increased progressively from vegetative to post-reproductive stages across all assays. Correlation analysis ( Supplementary Figure S4 ) revealed strong negative correlations between IC₅₀ values and compounds such as α-methylcinnamaldehyde, (S)-perillyl alcohol, and eugenol, indicating their key contribution to antioxidant activity.

Principal component analysis (PCA) was performed to assess the chemical variation and bioactivity profile of CMEO across different growth stages. The analysis included the relative abundance (% area) of identified chemical constituents along with corresponding biological parameters (MIC, MBC, MFC, IC₅₀). The PCA model demonstrated high statistical validity, accounting for 100% of the total variance. The first two principal components (PC1 and PC2) captured the full variability in the dataset, with PC1 explaining 56.07% and PC2 accounting for 43.93% of the total variance ( Supplementary Table S1 ). The 2D biplot ( Figure 7a ) effectively visualizes the grouping of CMEO samples across different stages and their relationship with both chemical and biological variables. The loading plot ( Figure 7b ) further illustrates the contribution of individual variables to each principal component, supporting the interpretation of stage-specific chemical signatures and bioactivities. The associated eigenvalues were 42.61 for PC1 and 33.39 for PC2, confirming their strong explanatory power. Detailed loading values for individual compounds and activities are provided in Supplementary Table S2 . Together, the PCA results underscore distinct compositional and bioactivity profiles of CMEO across growth stages, with clear separation and variable contributions effectively summarized by PC1 and PC2.

This study explored growth stage–dependent changes in the essential oil profile of wild-growing C. martinii and their relationship to antimicrobial and antioxidant activities. EO yield followed a bell-shaped trend, peaking during the reproductive stage, likely due to increased metabolic demand for volatiles involved in pollinator attraction, an observation consistent with other aromatic plants (Palá-Paúl et al., 2001). GC-MS analysis confirmed stage-specific quantitative variation in EO composition, particularly a broader activation of biosynthetic pathways during the reproductive phase. Such developmental modulation of EO output has been widely noted in aromatic species undergoing physiological transitions (Moghaddam and Mehdizadeh, 2017; Chrysargyris et al., 2021).

A developmental increase in oxygenated monoterpenes accompanied by a reduction in monoterpene hydrocarbons was observed, reflecting regulatory shifts in terpene metabolism. This pattern aligns with previous findings in C. martinii and other monoterpene-rich taxa (Rao et al., 2005; Verma et al., 2018), though past studies often overlooked stage-specific variation.

The transition from carveol dominance in the vegetative phase to cis-/trans-p-mentha-1(7),8-dien-2-ol in reproductive and post-reproductive stages suggests biochemical prioritization of compounds linked to reproductive signaling and defense, similar to trends reported in other species (Chu et al., 2013; Rathore et al., 2022).

Our results resemble the profile of C. martinii var. sofia reported from Rajasthan Jain and Sharma (2017), characterized by carveol-rich chemotypes rather than the geraniol-dominant var. motia (Raina et al., 2003; Rao et al., 2005). This chemotypic divergence likely reflects the influence of geography and wild-growing conditions on EO biosynthesis (Jangi et al., 2022).

Growth stage–specific enrichment of compounds reveals their probable ecological functions. Carveol and cis-piperitol were more abundant in the vegetative phase, supporting roles in constitutive defense. Their decline over time coincided with increased levels of trans-/cis-p-mentha-1(7),8-dien-2-ol, likely linked to reproductive signaling or adaptive protection under stress. Stable levels of trans-carveyl acetate suggest a constitutive protective function across development. Carvone, which increased in the post-reproductive stage, is a well-established antimicrobial agent and may serve in late-stage defense enhancement (Bakrim et al., 2022).

Key monoterpenes identified in C. martinii including carveol, carvone, D-limonene, and p-mentha-dien-2-ol isomers are synthesized via the plastidial MEP pathway from geranyl diphosphate (GPP), with downstream modifications by terpene synthases and cytochrome P450 monooxygenases (Dudareva et al., 2013; Vranová et al., 2013; Tholl, 2015). The increased abundance of oxygenated monoterpenes during later growth stages suggests developmentally regulated biosynthesis aligned with functions like reproductive signaling and oxidative stress mitigation. Although EO biosynthesis is influenced by environmental conditions such as rainfall and temperature, our sampling across identical phenological stages in two growing seasons minimized inter-annual variability. Averaged GC-MS data (mean ± SD) allowed a clearer focus on stage-specific metabolic trends. Future studies under controlled environments could further distinguish developmental effects from climatic factors.

Antibacterial potency varied with growth stage, paralleling chemical composition. Reproductive-stage EOs showed the strongest activity, attributed to elevated cis- and trans-p-mentha-1(7),8-dien-2-ol, which destabilize bacterial membranes and inhibit enzymatic systems (Alitonou et al., 2012; Ambrosio et al., 2020, 2021). Post-reproductive oils, though still active, showed slightly reduced efficacy, likely due to decreased D-limonene, β-myrcene, and eugenol compounds known for disrupting bacterial respiration and membranes (Nuñez and D’Aquino, 2012; Han et al., 2020; Połeć et al., 2020; Gupta et al., 2021; Ulanowska and Olas, 2021). Their partial replacement by carvone and trans-carvyl acetate, which act through quorum sensing and enzyme inhibition (Pina et al., 2022), may have altered overall potency. Vegetative-stage oils exhibited the weakest antibacterial action, dominated by carveol and cis-piperitol, which act on microbial enzymes but may lack strong membrane-disruptive properties (Roman et al., 2023). Additionally, limited synergism among constituents may contribute to reduced efficacy (Kamatou et al., 2008; Tamgue et al., 2011). Susceptibility varied by strain: S. aureus was most sensitive, likely due to its permeable Gram-positive cell wall (Oussalah et al., 2007; Galgano et al., 2022), while Gram-negative E. coli and S. typhi were more resistant due to their outer membranes (Longbottom et al., 2004; Dai et al., 2013; Rashid et al., 2013). Slightly higher sensitivity of S. typhi may relate to differences in membrane structure or EO interactions (Azad, 2021).

The antifungal potential of CMEOs showed strong dependence on plant growth stage, primarily linked to the abundance of oxygenated monoterpenes. Vegetative-phase oils exhibited the weakest activity, despite carveol’s known ability to disrupt fungal membranes (Soković et al., 2006; Abbaszadeh et al., 2014). The limited efficacy may reflect insufficient synergy with other active compounds or inhibitory interactions (Boni, 2017; James and Ikani, 2023). In contrast, reproductive-stage oils demonstrated the strongest antifungal effects, coinciding with maximum concentrations of cis- and trans-p-mentha-1(7),8-dien-2-ol compounds that disrupt fungal membranes and induce cell leakage (Kostik and Bauer, 2015; Brahmi et al., 2021). Their interaction with ergosterol alters membrane fluidity and integrity (Busato de Feiria et al., 2016; Benomari et al., 2018; Soliman et al., 2022), and may also interfere with ergosterol biosynthesis (Mohamed Ibrahim et al., 2018; Scalese et al., 2024). Post-reproductive EOs retained moderate antifungal activity, supported by elevated levels of carveol, trans-carveyl acetate, and p-mentha-1(7),8-dien-2-ol isomers. These compounds likely act through multiple mechanisms, including membrane destabilization, glycolytic inhibition, and morphological disruption (Yu et al., 2022; Xiong et al., 2023). The reduced presence of D-limonene, β-myrcene, and cis-piperitol may have contributed to the slight decline in activity. Sensitivity varied by fungal strain. A. alstroemeriae was most susceptible, likely due to its more permeable membrane structure. F. fujikuroi showed intermediate response, while C. queenslandicum was least sensitive, possibly due to inherent resistance mechanisms like enhanced sterol biosynthesis and stress adaptation (Moutassem et al., 2019; Vasileva, 2023; Erdoğan, 2024).

Antioxidant activity increased progressively from the vegetative to the post-reproductive stage, consistent across DPPH, ABTS, and β-carotene bleaching assays. Similar trends have been observed in other aromatic species such as Foeniculum vulgare and Origanum vulgare, where late-stage accumulation of active compounds enhances radical-scavenging potential (Salami et al., 2017; Morshedloo et al., 2018). Post-reproductive EO showed the highest antioxidant activity, attributed to elevated levels of cis- and trans-p-mentha-1(7),8-dien-2-ol, known for their radical stabilization and hydrogen-donating abilities (Sitzmann et al., 2014; Jordá Marín, 2018). Additional contributors included eugenol, D-limonene, 1,3,8-p-menthatriene, and carveol all recognized for their antioxidant efficacy (Niki, 2010; Gülçin, 2011; Mueller and Boehm, 2011; Serafim et al., 2021; Orlo et al., 2023). Trans-carveyl acetate may also enhance activity via hydrogen donation. In contrast, vegetative-stage EO showed the lowest antioxidant effect despite high carveol content, likely due to limited synergy with other active compounds. Reproductive-stage EO displayed moderate activity, supported by cis-piperitol and p-mentha-1(7),8-dien-2-ol, though lower levels of carvone and carveol may have reduced overall potency (Duarte-Almeida et al., 2006; Olszowy and Dawidowicz, 2018; Pombal et al., 2020). The antioxidant roles of β-myrcene and cis-piperitol remain underexplored and merit further study.

Principal component analysis (PCA) clearly differentiated EO samples by phenological stage, with vegetative, reproductive, and post-reproductive oils forming distinct clusters ( Figure 7a ). The loading plot ( Figure 7b ) revealed that antimicrobial compounds were strongly associated with reproductive-stage oils, while antioxidant-rich constituents clustered with post-reproductive samples. Vegetative EO appeared chemically intermediate.

The PCA model explained 100% of the variance (PC1 = 56.07%, PC2 = 43.93%) and was supported by eigenvalue and loading data ( Supplementary Tables S1, S2 ), confirming its robustness. These results highlight the importance of the growth stage in determining both EO composition and bioactivity, offering a framework for selecting optimal harvest times based on intended applications of C. martinii essential oil.