A total of 72 plant specimens ( Supplementary Table 1 ), including C. oppositifolia, were collected from two locations in Himachal Pradesh, India: Solan (altitude 1350 m, temperature 20-30°C, humidity 55-68%) and Shimla (altitude 2202 m, temperature 12-25°C, humidity 62-80%). The specimens, comprising leaves, buds, flowers, and tubers, were identified and verified using the herbarium at Dr Y.S. Parmar University of Horticulture and Forestry, Nauni, Himachal Pradesh. The collected specimens were washed with water and surface-sterilized using 1% hydrogen peroxide (H₂O₂), followed by rinsing with autoclaved distilled water to remove any residual H₂O₂, as per an established protocol in our laboratory. The surface-sterilised plant material was then dried in an oven at 40°C until completely desiccated. The dried material was ground into a fine powder using an electronic mixer grinder (Sharma and Mishra, 2019). Among the specimens, C. oppositifolia extract exhibited significantly higher antimicrobial activity, as shown in our published data. Phytochemical analysis of the extracts revealed the presence of various bioactive compounds, including phenols, terpenoids, flavonoids, tannins, coumarins, glycosides, and alkaloids. EOs were subsequently extracted from C. oppositifolia and further investigated for this study.

The EOs were extracted using an environmentally friendly hydro-distillation method (Abbas et al., 2022) and calculated by Extract Yield (%)= (Weight of starting material/Weight of extract obtained)×100. Fresh leaves and inflorescence of C. oppositifolia were subjected to hydro-distillation using a Clevenger apparatus. Two hundred grams of chopped plant material were placed in a round-bottom flask, and 400 mL of distilled water was added. The mixture underwent hydro-distillation for 3 hours. Afterwards, the organic phase was dried over sodium sulphate, and filtered, and the solvent was evaporated to dryness. The volatile oil was then stored at 4°C until analysis. This method combines extraction and steam distillation into a single process, which reduces trial time, saves solvents, simplifies equipment, and facilitates operation. While the extraction rate is relatively low, the essential oil obtained is of high purity. However, the process involves high temperatures, which may lead to the volatilization or oxidation of certain components in the EOs. The method primarily collects insoluble EOs, resulting in the loss of water-soluble components and, consequently, a lower extraction yield. The extracted oil is still a multi-component composite essential oil, making it challenging to achieve selective extraction and complete purity.

The EOs were further processed to isolate terpenoids using TLC (Jiang et al., 2016). Initially, a small quantity of the essential oil was dissolved in a minimal amount of an extraction solvent, typically a mixture of hexane and ethyl acetate in a 7:3 ratio. This solution was then placed onto a pre-coated silica gel TLC plate, which had been activated by heating at 110°C for 30 minutes. The TLC plate was placed in a development chamber containing a mobile phase of hexane and ethyl acetate (7:3 ratio), allowing the solvent front to rise to about 3/4th of the plate’s height. After development, the TLC plate was removed from the chamber and air-dried. Visualization of the terpenoids was achieved by observing the plate under UV light at 254 nm and 365 nm, where terpenoids typically have fluorescence. For further visualization, the plate was sprayed with anisaldehyde-sulfuric acid reagent and heated at 110°C for 10 minutes, revealing distinct colored bands corresponding to the terpenoids. The retention factor (Rf) values of these bands were calculated and compared with known standards to identify the specific terpenoids present in the essential oil α-pinene, δ-carene, and caryophyllene. The results were documented by photographing the TLC plates and noting the colors and Rf values of the terpenoid spots.

FTIR spectroscopy was employed to investigate the molecular interactions between terpenoids (α-pinene, δ-carene, and caryophyllene) and antibiotics-amoxicillin and erythromycin. Terpenoids and antibiotics were prepared in solution, and FTIR spectra were recorded over the range of 4000–400 cm⁻¹ using an ATR accessory for liquid samples and KBr pellets for solid samples. The spectra were analyzed for characteristic peaks and shifts to identify functional groups and potential interactions. Comparison of the spectra of individual terpenoids with their combinations with antibiotics revealed changes in peak positions or intensities, providing insights into molecular interactions and potential synergistic effects.

UV spectrophotometry was utilized to analyze the absorbance characteristics of terpenoids-α-pinene, δ-carene, and caryophyllene and their combinations with antibiotics-amoxicillin and erythromycin. Solutions of each terpenoid and antibiotic were prepared at a concentration of 0.1 mg/mL in methanol or acetonitrile. Combined samples were also prepared by mixing equal volumes of the terpenoid and antibiotic solutions. Using a UV-visible spectrophotometer, spectra were recorded over the range of 200–400 nm. The baseline spectrum of the solvent was first recorded to correct for any background absorbance. Each sample was then analyzed to identify characteristic absorbance peaks and any shifts or changes when terpenoids were combined with antibiotics. These changes provided insights into the interactions between the compounds and their potential synergistic effects.

The chemical structures of the selected terpenoids α-pinene, δ-carene, and caryophyllene and antibiotics-erythromycin and amoxicillin were first retrieved from the PubChem database- https://pubchem.ncbi.nlm.nih.gov/in.sdf format and converted to.pdb format using OpenBabel. These ligands were then subjected to energy minimization using the MMFF94 force field to obtain their most stable conformations, with protonation states adjusted and any missing hydrogens added.

The TLC analysis of the EO from C. oppositifolia revealed the presence of several terpenoids, including monoterpenes- α-pinene, δ-carene, and sesquiterpene-caryophyllene ( Figure 1 ). These terpenoids were identified based on their characteristic retention factor (Rf) values compared to known standards. The terpenoids were tested in combination with two antibiotics, amoxicillin and erythromycin ( Figure 1 ).

The antimicrobial screening results for S. aureus ATCC-43300 and E. coli MTCC-739 demonstrated varying degrees of efficacy for the essential oil terpenoids and their combinations with antibiotics ( Table 1 ). For S. aureus ATCC-43300, erythromycin alone yielded a zone of inhibition of 7 ± 0.2 mm. When tested individually, α-pinene also showed a zone of 7 ± 0.2 mm, while δ-carene and caryophyllene produced zones of 10 ± 0.2 mm and 8 ± 0.2 mm, respectively. The combination of terpenoids with erythromycin enhanced the inhibitory effect, with α-pinene + erythromycin producing a zone of 10 ± 0.2 mm, δ-carene + erythromycin showing the largest zone of 11 ± 0.2 mm, and caryophyllene + erythromycin yielding 8 ± 0.2 mm. Among these, δ-carene exhibited the most significant synergistic effect with erythromycin against S. aureus. For E. coli MTCC-739, amoxicillin alone resulted in a zone of inhibition of 4 ± 0.2 mm. The individual terpenoids showed improved activity, with α-pinene, δ-carene, and caryophyllene producing zones of 9 ± 0.2 mm, 11 ± 0.2 mm, and 7 ± 0.2 mm, respectively. Combinations of terpenoids with amoxicillin led to further enhanced inhibition: α-pinene + amoxicillin resulted in a zone of 12 ± 0.2 mm, δ-carene + amoxicillin showed a zone of 11 ± 0.2 mm, and caryophyllene + amoxicillin achieved 8 ± 0.2 mm. The combination of α-pinene with amoxicillin exhibited the most significant synergistic effect against E. coli.

The FTIR spectra analysis of antibiotics and terpenoids ( Supplementary Figure 1 ) revealed significant modifications in the functional groups when combined, suggesting potential synergistic interactions. The overlay spectra for the combinations were analyzed. For the amoxicillin + α-Pinene combination, distinct peak shifts were observed at 1599, 1774, and 2259 cm⁻¹ ( Figure 3A ). Similarly, the combination of α-Pinene with Amoxicillin showed a peak shift at 1715 cm⁻¹ ( Figure 3B ). The combination of amoxicillin with caryophyllene exhibited peak shifts at 1599 and 1774 cm⁻¹ ( Figure 3C ). Notably, the overlay of caryophyllene with amoxicillin resulted in similar peak modifications as seen with carene, at 1599 and 1774 cm⁻¹ ( Figure 3D ). In contrast, the combination of δ-carene with erythromycin generated new peaks at 1648 and 1287 cm⁻¹, indicating a different interaction pattern ( Figure 3E ). Furthermore, the overlay of caryophyllene with amoxicillin led to the disappearance of the amoxicillin peaks at 1599 and 1774 cm⁻¹, while no modifications were observed in the caryophyllene and erythromycin combination ( Figure 3F ). These spectral changes suggest that the combination of terpenoids with antibiotics results in functional group modifications, which could be responsible for the observed synergistic effects in antimicrobial activity.

Notable changes in the UV spectra were observed when terpenoids were mixed with antibiotics, suggesting possible interactions or alterations in the molecular structure of the components. In the combination of amoxicillin with α-Pinene ( Figure 4A ), significant shifts in the absorption peaks were noted, indicating potential interactions that may alter the electronic environment of the molecules. These shifts could be indicative of changes in the conjugation of the antibiotic’s chromophore when combined with the terpenoid. Similarly, the combination of erythromycin with α-Pinene ( Figure 4B ) also showed noticeable peak shifts, further supporting the possibility of complex formation between these molecules. When amoxicillin was combined with δ-carene ( Figure 4C ), distinct modifications in the absorption spectra were observed, highlighting potential changes in the molecular interactions. Caryophyllene, when mixed with amoxicillin ( Figure 4D ), showed similar alterations in the UV spectra, suggesting that the interaction between these molecules may involve modifications in their electronic structures. The combination of δ-carene and erythromycin ( Figure 4E ) revealed the formation of new absorption peaks, indicating the possible creation of new molecular entities or complexes. Finally, the spectra for caryophyllene and erythromycin ( Figure 4F ) showed minimal modifications, suggesting that while interactions may occur, they might be less pronounced than with other combinations. Overall, these results suggest that the mixing of terpenoids and antibiotics leads to changes in their UV absorption spectra, which could indicate complex formation or alterations in their molecular structures. These findings provide valuable insights into the potential synergistic effects observed in combination therapies involving these compounds.

The docking results suggested the interaction energy values were analyzed to determine the strength of the interactions. Lower (more negative) values of interaction energy indicate stronger interactions. The docking structure for terpenoids- α-pinene, δ-carene and antibiotics- erythromycin, amoxicillin adopted, we have not included caryophyllene for the docking study ( Supplementary Figure 2 ). The highest interaction was observed between δ-carene and erythromycin, with a total interaction energy value of -96.10 kcal/mol, suggesting a strong binding affinity between these two compounds ( Figure 5A ). This was followed by δ-carene with amoxicillin, which had an interaction energy value of -87.75 kcal/mol, indicating a significant interaction as well ( Figure 5B ). The α-pinene with erythromycin demonstrated an interaction energy of -83.58 kcal/mol, also reflecting a strong interaction but with amoxicillin had a slightly lower interaction energy value of -74.26 kcal/mol ( Figures 5C, D ). Lastly, the combination of α-pinene with δ-carene displayed an interaction energy of -57.02 kcal/mol, which, although lower compared to the other pairs ( Figure 5E ). These docking results suggest that δ-carene exhibits the highest potential for interaction with both erythromycin and amoxicillin, which could imply enhanced efficacy or synergistic effects in therapeutic applications involving these combinations.