2.1 Essential oil extraction

Fresh plants of Catharanthus roseus that were gathered from nearby local nurseries. At Agriculture University Faisalabad, a voucher specimen was deposited (181–1-23). After being air-dried at 25 to 28 °C, fresh plant material was ground into a powder. A laboratory scale Clevenger-style hydro-distillation system was used for hydrodistillation of essential oil conducted at 100 °C for four to six hours. 2.5 L of water was added as solvent to dried plant powder weighing 150 g. After the dried plant was hydro-distilled, the yield of essential oil obtained was calculated. For GC–MS analysis and phytocompound isolation, the extracted essential oil was stored in a sealed glass vial at 4 °C. (Conde-Hernández et al. 2017).

2.2 GC/MS analysis

A gas chromatography approach combined with a mass spectrophotometer was used to accomplish the GC/MS analysis of EO. Helium was used as the carrier gas, and 1:100 split ratio was used together with a direct injection of the essential oil (0.1 μL) (Furuhashi and Okuda 2017). The oven was preheated to 50 °C for one minute, and then at 25 °C/min temperature gradient. The temperature was maintained constant for 20 min after it hit 160 °C. After that, the oven temperature was raised by 5.0 °C every minute for 15 min, to reach 250 °C. One milliliter per minute of helium was employed as the carrier gas. The temperature of the injector and transfer line were both set to 250 °C. Setting the electron energy to 70 eV allowed the mass spectrometer to function in the electron impact mode. The conventional procedure of identifying volatiles involved comparing mass spectra and Kováts Indices using the National Institute of Standard and Technology (NIST) library's database (Spyridopoulou et al. 2019).

2.3 Isolation of terpene

Column chromatography was used as a subsequent purification step to isolate the compound from C. roseus essential oil. Despite using a mobile phase of solvents consisting of methanol and ethyl acetate (7.6:2.4) in column chromatography, E. Merck's silica gel 60 (mesh size 70–230) was utilized. To conduct column chromatography, the C. roseus essential oil was diluted in solvents and triturated using silica. A glass column was filled with a silica gel and solvent slurry. After a few hours, the sample was put to the top of the packed column using the wet-packing technique. When the column's valve was opened, 10 to 20 ml of solvent that was released and were collected in test tubes. Samples separated from column fractions were examined using thin layer chromatography (TLC) plates in acetone: ethyl acetate (4:2, v/v) solvent systems to find the retention factor (Rf) of components that appeared as spots. Following solvent separation, the proportion of separated components was computed (Ngo and Chua 2019).

2.4 Characterization of terpene

The ¹H NMR and ¹³C NMR spectra were measured at 500 and 125 MHz, respectively, using a JEOL JNM-ECA 500 Spectrometer for NMR spectroscopy. The methodology employed was, with minor adjustments, that of Kim et al (Kim et al. 2023). The sample was put into a different, 2-milliliter microtube and added 0.5 ml of CD₃OD and 0.5 ml of KH₂PO₄ buffer (pH 6.0) in D₂O. To generate a clear supernatant, the mixture was centrifuged (Centrifuge MPW-260, Instruments, Poland) at 13,000 rpm for 10 about minutes after being vortexed (Vortex Mixer, Thermo Fisher Scientific, USA) for 1 min and ultrasonicated for 20 min. A volume of 0.8 ml supernatant was transferred to an NMR tube promptly. Sample was subjected to ¹H NMR using a preset setting after about 0.8 mL of the supernatant was transferred NMR tube. A 500 MHz NMR Spectrometer (JEOL ECZR, JEOL Ltd., Tokyo, Japan) was used to perform the ¹H NMR experiments (Rohman et al. 2020). Based on available database of NMR absorptions and proposed structure provided by the NIST library, https://www.nist.gov/nist-research-library each ¹H NMR and ¹³C NMR observed were determined (Efdi et al. 2010).

2.5 In silico prediction and computational analysis

2.5.1

2.5.1 Docking tools and protein selection

For computational in silico study, software used are Python, the Desmond simulation package of Schrodinger and Discovery Studio 4.0, ChemDraw 3D Pro 12.04v, Auto dock tools in Auto-dock 4.6 program and PubChem database. The three-dimensional protein structures of the human estrogen receptor, human progesterone receptor and HER2 receptor (PDB id: 2iok, 1e3k and 3 pp0 respectively) were downloaded with the protein databank (https://www.rcsb.org/pdb/ home/home.do) (Fig. 1). In order to advance docking, the structure was arranged and refined, every receptor's binding site was examined, and protein files were created using the AutoDock4 application and saved in PDBQT format.

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Fig. 1

Breast cancer receptors used for In silico study.

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3D structure of 2-carene was attained from open-source PubChem (https://pubchem.ncbi.nlm.nih.gov/) and transformed into PDB files using Open Babel (Boyles, Deane, and Morris 2020).

2.6 Nanoemulsion formulation

2-carene mediated nanoemulsion was prepared following (Periasamy, Athinarayanan, and Alshatwi 2016) with adjustments. Nano-emulsion was formulated using Tween 80 (12.2 % v/v) and Tween 20 (8 % v/v) to formulate nano-sized droplets and to preserve particle’s stability. A homogeneous aqueous phase was produced by swirling mixture of double-distilled water (69.8 % v/v) and surfactant for 50 min and then 2-carene (10 % v/v) was added dropwise and stirred gently for 20 min at 8000 RPM. It was thought that sonication and the self-emulsifying process could work together to improve the stability of the nanoemulsion. As it was high energy emulsification process so the procedure was conducted in an ice bath and the emulsion was sonicated for ten minutes using a 20 kHz sonicator (Ultrasonic Processor, GEX 750, USA) with power output of 750 Watt (Navaei Shoorvarzi et al. 2020). After ensuring that all of the solvent had been removed from the emulsion, it was centrifuged at 4000 rpm for four hours at room temperature. The nanoemulsion was subsequently employed for additional characterization after being filtered using a membrane syringe filter to remove contaminants and to separate extra amount of surfactant concentration. The entire process of creating the 2-carene-mediated nanoemulsion was carried out in the dark and at a regulated temperature because terpene chemicals are volatile by nature and sensitive to light.

2.7 DPPH antioxidant activity

The antioxidant potential of natural phytocompounds and nano-formulations was assessed using the DPPH antioxidant test. 5 mL of methanolic DPPH (0.2 mmol/L) solution was mixed with 3 mL of a selected concentration of 2-carene and nanoemulsion at different concentrations (0.25, 0.5, and 1 mg/ml), using ascorbic acid as a standard reference. The content was incubated at room temperature in the dark for thirty minutes. Using a UV wavelength of 516 nm, the sample's absorption activity was measured in triplicate. The inhibitory action was ascertained as the percentage of DPPH decreased relative to the control. To determine the inhibitory action, the percentage of DPPH that decreased relative to the control was utilized (Valko et al. 2007).

2.8 Cytotoxicity analysis (MTT assay)

Anti-proliferative activity and cell viability of 2-carene and 2-carene induced nanoemulsion were assessed using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test,. The University of Lahore's Institute of Molecular Biology and Biotechnology department provided the MDA-MB-231 breast cell lines and the HMEpC healthy epithelial cells. A density of 6 x 103 cancer cells were injected into microtiter plate wells using 100 μL of cell media per well. The plate is then incubated in a CO₂ incubator for an extra night to promote cell adherence. Separate additions of test complexes (2-carene and varying nanoemulsion concentrations) were made to every well that contained cells. The plates were housed in an incubator at 37 °C for 72 h. After four hours, each well was re-incubated with 20 μl of MTT reagent. The plates were then scanned using a traditional ELISA microplate reader at 570 and 620 nm wavelengths (Luis et al. 2019).

2.9 Cancer induced experimental study

Weighting between 150–170 g, adult female SD rats were attained from the Institute of Molecular Biology and Biotechnology, University of Lahore's research animal facility. Institutional animal ethics strictly followed the guidelines for monitoring and controlling experimental animals. A climate-controlled chamber with a 12-hour light/dark cycle, 50 % humidity, and a 24–27 °C temperature range was employed to house the rats. After a certain amount of days for acclimation, rats were divided into six groups having seven rats into each group. To promote breast cancer in rats, 25 mg/kg DMBA (7, 12-dimethylbenz[a]anthracene), (product number D807576-100 mg MACKLIN, China.) was administered (Arroyo-Acevedo et al. 2015). Rats in the control group were not exposed to DMBA or treated with nanoemulsion, rats in the positive control group were given only 50 mg/kg of nano-emulsion, and rats in the DMBA group were given 25 mg/kg of DMBA without being treated with 2-carene or nanoemulsion. Rats in 2-carene treated group were given 25 mg/kg of cancer drug and 25 mg/kg of 2-carene terpene compound, rats in the nano-emulsion treatment group were given 50 mg/kg of nanoemulsion after cancer induction and rats in the EO group were exposed to 25 mg/kg of DMBA drug and then 25 mg/kg of EO. Following four to five weeks of intraperitoneal injection of a single dosage of DMBA, cancer was generated and tumor palpation was noted in animals administered with DMBA. Subsequently the tumor was induced, EO, 2-carene, and synthesized nanoemulsion therapies were given intra-gastrically for four weeks at predetermined times on different days. To take blood samples, a cardiac puncture was performed with a sterile needle. Following the separation of the blood sample into two fractions, the first was transferred to plain sample tubes, and the second, including complete blood samples, was transferred to EDTA sample collecting tubes. In order to extract serum, blood was spun at 3000 rpm to separate the serum from the plasma. Following centrifugation, serum samples were stored at −20 °C until their cytokine cancer and stress indicators were examined. To prevent cell hemolysis, the anticoagulant was able to mix with the blood samples in the EDTA vials by gently shaking them. Immediately upon the collection of blood samples, the hematological and biochemical tests were performed.

2.10 Statistical investigation

Every experiment's levels of examination were compared to those of the vehicle control group, nanoemulsion control group and the DMBA-induced group (which was not given any treatment). Versions 8.5. of the graph pad prism program and SPSS statistics software (SPSS Inc., USA) were used for the statistical analysis. ANOVA was applied to look for differences between each sample's means, and Dunnett analysis was accomplished. 0.05, 0.001, and 0.0001p value was employed to assess significant values.

3.1 GC–MS analysis of essential oil

Phytocompounds present in essential oils act as secondary metabolites, which are intricate blends of volatile chemical compounds. The essential oil was extracted with 6.41 ± 0.21 % concentration, indicating that hydro distillation was used to extract a considerable amount of essential oil from C. roseus. (Supplementary figure S1). (Percent compositional data is also given in (Supplementary data table T1). The isolated chemical, 2-carene, was determined with RT (time taken for solute to pass through a chromatography column) 15.772. The compound's computed retention index (RI, which is retention time interpolated between neighboring n-alkanes) was 1171, while the RI reported in the literature is 1186. (This compound previously been reported to occur in Fagonia longispina (Ourzeddine et al. 2017).

3.2 FT-IR and NMR spectroscopy analysis

The GC–MS analysis showed retention time of 2-carene at 15.772 min and the molecular ion peak for this sample appeared at 136.2 amu. The prominent fragmentation pattern involved loss of methyl group and the FT-IR spectrum of 2-carene reflected absence of any prominent functional group. There was no indication of presence of hydroxyl or amine functionality, carbonyl moiety was also not indicated from IR spectrum. However, the FTIR spectrum did indicate presence of C=C which appeared around 1446 cm⁻¹. Stretching corresponding to sp³ hybridized C–C-H were also prominent this indicating the isolated compound to be a hydrocarbon with some degree of unsaturation (Supplementary figure S2).

NMR analysis of compound was obtained by proton (¹H) and Carbon (¹³C) NMR to integrate selected compound. The ¹H NMR of compound indicated presence of 3 methyl groups; two methyl groups appeared at 1.06 δ (ppm) as a single of 6 proton integration and one methyl group appeared at 1.69 ppm. 2 protons appeared as a multiplet from 2.21 to 2.34 δ (ppm), 4 protons appeared as multiplet from 2.59 to 2.66 δ (ppm) while a single proton appeared as a doublet at 5.46 δ (ppm). The signal at 5.46 δ (ppm) indicated presence of a single olefinic proton. The ¹³C NMR indicated presence of 6 aliphatic carbons and two olefinic carbons. The aliphatic carbons appeared at 21.2, 22.9, 24.0, 27.5, 31.6 and 34.6 δ (ppm) while the olefinic carbons appeared at 115.9 and 118.9 δ (ppm). From this spectroscopic, IR spectrum and spectrometric data, the isolated terpene compound was identified as 2-carene and NMR spectral data of proton assigned and carbon assigned was shown in Supplementary figure S3 and Supplementary figure S4.

3.3 Molecular docking study

Docking studies were conducted using the modeled 3D structures of ER, PR, and HER2 in order to better understand the interaction between 2-carene and breast cancer receptors. Clear observations of the hydrogen bonding molecules and free binding energy were made; the binding's negative least free energy value indicates a strong, advantageous link between the estrogen and HER2 receptor, with 2-carene in the most advantageous conformations. 2-carene showed better interaction with ER protein and showing hydrogen interaction with amino acid residues of ALA 351, TRP 383and GLU 353 and showed binding score of −6.3 kcal/mol. 2-carene compound when docked with progesterone receptor, showed Van Der waals interaction with amino acid residues of LEU 718 and THR 894 with binding score of −6.3 kcal/mol. Isolated compound 2-carene show Van Der waals interaction with amino acid residues SER 783, THR 798, ASP 863, LYS 753 and showed binding score of −6.6 kcal/mol with HER2 receptor. The results are displayed in Table 1. Ligand-protein visualization was shown in Figs. 2 and 3D interaction of 2-carene with BC targets displayed in Fig. 3. From binding affinity score, it was detected that 2-carene docked fine with HER2 protein with high docking value −6.6 kcal/mol and multiple hydrophobic interactions.

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Fig. 2

2D visualization of the ligand–protein complex with (A) Estrogen receptor (B) Progesterone receptor (C) HER2 receptor.

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Fig. 3

3D images of 2-carene with residual amino acids.

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3.3.3

3.3.3 Molecular dynamic simulation

MD simulation studies substantially aid in understanding the link between the atomic-level ligand residues and the dynamics of protein structure (De Vivo et al. 2016). After the ligand–protein complex were virtually screened, the stability and receptor flexibility was evaluated using MD simulations running at nanosecond time scales.

To assess the accuracy and stability of the systems, 100 ns of MD simulation trajectories were analyzed. Fig. 4 shows the RMSD of 2-carene-HER2 complexes. Cα-RMSD rises up to 32 ns, and then it evidently increases once more at around 45 ns. The protein attached to the ligand stabilizes at 34 ns with a tolerable average RMSD of 2.5 ± 0.03 Å throughout the simulation. The ligand is maintained in the active region of the protein during the simulation, and the tolerable range for the RMSD of 2-carene in contact with protein is 0.3–1.2 Å until 20 ns, at which point it gradually rises to 2.8 Å. Fig. 5 shows the protein–ligand 2-carene-HER2 complex RMSF. From an atomic perspective, RMSF is between 0.5 and 4.0 Å. The average residue-wise RMSF remains between 2.0 and 2.5 Å, with larger peaks at the beginning (between 15 and 35 residues) and middle (between 150 and 200 residues). In Fig. 6, atom-by-atom RMSF of the protein in interaction with the ligand was displayed. Higher peaks, or loop regions, are flexible and reach a maximum of 4.0 Å. A crystal structure with some degree of thermal mobility is suggested by an average experimental (crystallographic) B factor of 3.52 Å. Protein structural mobility is revealed by the values of the B factors and RMSF at the residue level. The 2-carene-HER2 complex was shown to have a high affinity for protein binding in terms of binding energy, according to these results. In Fig. 7, interactions between proteins and ligands are compiled during the course of a 100 ns simulation. Hydrophobic contacts are formed by positively charged TYR-803 and LEU-852, and residues, including H-bonds, engage with ARG-811, THR-862, ASP-863, LEU-726, SER-728, and THR-811. Water bridges are formed by the residues ASN 850, LEU-807, GLY-804, and ASP-863, whereas negatively charged SER-728 and ARG-811 form ionic connections with 2-carene. Stability in simulation is represented by the interaction of 2-carene-HER2 complex complexes with the active site residues. Over 30.0 % of the simulation run time was devoted to protein–ligand interaction during MD simulation.

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Fig. 4

RMSD of ligand 2-carene with HER2′s C-atoms. The left Y-axis (blue line) displays the deviation in protein RMSD values, whereas right Y-axis (red lines) displays variance in ligand during the simulation. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Fig. 5

Residue-wise RMSF of 2-carene with target protein HER2.

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Fig. 6

Atom-wise RMSF of protein in contact with 2-carene.

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Fig. 7

Histogram characterize contacts between protein–ligand interaction during MD simulation.

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3.4 Nanoformulation characterization

3.4.3

3.4.3 UV–visible and FT-IR analysis

The UV–visible spectra of 2-carene, 2-carene with nanoemulsion, and EO revealed large absorption peaks along with a significant drop in intensity that occurred when the concentration of nanoemulsions in the sample was decreased. Absorption was also shifted to longer wavelengths. When the 2-carene curve was finally examined, the wavelength with the greatest peak, 482.6 nm, had an absorbance of 2.43. As the observed wavelengths comes within specified range so, confirmative to the formation of terpenes-based nano-emulsion formulation. The sharp maximum absorption peak at 530 nm (0.5 mg/ml of NE) and 505.6 nm (1 mg/ml of NE) was an indicative of spherical shaped NPs respectively (Fig. 8).

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Fig. 8

UV–visible absorption spectrum.

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The FTIR spectroscopic study of the nanoparticles was used to recognize functional groups that were likely in charge of reduction of different ions present in formulation. FTIR of nanoemulsion had been shown in Fig. 9 where values of wavenumber cm⁻¹ had been plotted versus transmittance and spectral peaks were observed for NEs. There were numerous characteristic peaks observed at 3336.0 cm⁻¹, 2922.2 cm⁻¹, 1636.3 cm⁻¹, 1457.4 cm⁻¹, 1349.3 cm⁻¹ 1086.5 cm⁻¹ and 944.9 cm⁻¹ as evident from FTIR plot of nanoemulsion of 2-carene. The vibrational peak around 3336.0 assigned to O–H that could possibly derive from carbohydrate or phenolics. The peak at 2922 cm⁻¹ is ascribed to C–H of the alkyl group, the peak at 1636.3 cm ^-1 suggest C=O from carboxyl group or esters, the peak at 1086.5 cm ^-1 is likely due to C-O from phenols. The peak at 944.9 cm⁻¹ possibly deviate from ether like groups and shown C-O-C stretching. The tentative assignment of the peak is supported by published literature reports.

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Fig. 9

FT-IR spectra of 2-carene mediated Nanoemulsion.

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3.4.4

3.4.4 SEM and EDX analysis

SEM examination was performed on the samples to ascertain the size distribution and morphology of the particles. The nanoemulsion imaging demonstrated that these were tiny globular shaped molecules, with an average diameter ranging from 180 to 270 nm. SEM scans showed that the 2-carene based nanoemulsion had randomly distributed, interconnecting nanoparticles. Because it allows for adequate medicine loading, the porosity structure of formulations is caused by the aromatic ring present in the monoterpene structure of 2-carene (Fig. 10). The emulsifying agents that were used during the formulation process may have caused the nanoemulsion droplets to coalesce at a certain point, leading to the development of particles, as supported by previous studies (Giunti et al. 2019).

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Fig. 10

SEM representation of 2-carene based Nanoemulsion.

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Using an EDX investigation, typical concentrations of C, O, S, Zn, and AU were found to be around 29.23, 21.55, 1.49, 11.15, and 1.2, respectively (Supplementary Figure S10). The spectral signals of sulfur (S) and carbon (C) also demonstrated the presence of biomolecules adsorbed on nanoemulsions. The presence of the OH group may also be indicated by the oxygen peaks as demonstrated by longer peaks observed from EDX of nanoemulsion.

3.5 DPPH antioxidant activity

The in vitro antioxidant activity of 2-carene and its nanoemulsion at different doses was evaluated using the DPPH technique (Fig. 11). 5 mL of methanolic solution (containing 0.2 mM of DPPH radicals) were mixed with 2-carene and varied doses of 2-carene mediated nanoemulsion. Terpene compound exhibited higher antioxidant activity compared to the nanoemulsion formulation. 2-carene demonstrated scavenging activity of 43 % to 57 % at concentrations ranging from 0.25 to 1 mg/mL, while nanoemulsion demonstrated scavenging activity of 24 % to 56 % with an average IC50 value of 0.72 ± 0.02. The DPPH activity of nanoemulsion increased in a dose-dependent manner, reaching its maximum at a concentration of 54.29 % at 1 mg/ml of nanoemulsion. However, the supreme antioxidant activity of 2-carene nanoemulsion was less than that ascorbic acid (used as standard), which was approximately 66 %. 1.5 mg/ml of nanoemulsion produced considerably stronger DPPH radical scavenging capabilities than all other all examined doses of nanoemulsions (p < 0.05).

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Fig. 11

DPPH radical scavenging activity of 2-carene and nanoemulsion. (The data represents statistical significance where *p < 0.05, ** p < 0.001, and *** p < 0.0001.

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3.6 Cytotoxicity assay

Using varying concentrations of tested substances (0.10 µg/ml, 0.25 µg/ml, 0.5 µg/ml, and 1 µg/ml) at multiple time intervals, the cytotoxicity of 2-carene and its nanoemulsion was examined. When applying 2-carene and biosynthesized nanoemulsion to MDA-MB-231 cancer cell lines and HMEpC healthy epithelial cells, the antiproliferative effect is contingent upon the duration and concentration of application. When compared to 51.62 % viability observed with nanoemulsion at a concentration of 1.0 µg/ml it was seen, cells that were treated with 2-carene shown highly significant p value (p < 0.0001). While pure 2-carene at 0.5 µg/ml showed significant cytotoxicity at p < 0.0001, the observed cytotoxicity was reduced at 0.25 µg/ml (p = 0.0008) (Fig. 12A). While nanoemulsion decreases cancer cell viability, cytotoxicity was found at concentrations of 1 µg/ml (p = 0.0013) and 0.5 µg/ml (p = 0.005) as compared to MDA-MB-231 control cell lines (Fig. 12B). With nanoemulsion, the maximum IC50 concentration with 1 µg/ml at 24 and 48 h, there was more than 50 % inhibition rate observed. As the concentration of nanoemulsion increased up to 1 µg/ml, the cytotoxicity was also raised substantially. Conversely, the HMEpC cells did not have any discernible impact on cell viability, with the exception of a p = 0.007 at a concentration of 1 µg/ml of nanoemulsion concentration.

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Fig. 12

MTT Cytotoxicity examination of (A) 2-carene and (B) nanoemulsion. The data displays the mean ± standard deviation, with * (p < 0.05), ** (p < 0.001), and *** (p < 0.0001) indicating statistical significance compared to vehicle group and DMBA induced group.

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In comparison to the viability of HMEpC cells, MDA-MB-231 cells treated with 2-carene and at various doses of the nanoemulsion via crystal violet assay (live cells detection), displayed less live cells (Fig. 13). Nanoemulsion shown considerable cytotoxicity response (p < 0.001) on cancer cells, while 2-carene showed minimal absorption (p < 0.001) in comparison to untreated MDA-MB-231 cells. The viability of healthy HMEpC cells was found to be p < 0.001 when treated with 2-carene and p > 0.005 when treated with nanoemulsion concentrations in comparison to untreated control cells.

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Fig. 13

Crystal violet cytotoxicity analysis of 2-carene and nanoemulsion. The data displays the mean ± standard deviation, with * (p < 0.05), ** (p < 0.001), and *** (p < 0.0001) indicating statistical significance compared to vehicle control group and DMBA induced group. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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3.7 Tumor growth rate

Effect of 2-carene and synthesized nanoemulsion was observed on tumor growth features. Average values of tumor length, tumor width and calculated tumor volume and tumor growth rate were mentioned by calculating tumor growth rate after administration of cancer and then treatment initiated with 2-carene and synthesized nanoemulsion in relevant groups. Rats in the DMBA control group had a higher tumor incidence than those in the vehicle control group (p < 0.001), despite not receiving any further nano-formulation treatment (Supplementary Table 5). In the groups treated with 2-carene (p < 0.001) and nanoemulsion (p < 0.05), there was a reduction in the incidence of tumors (Fig. 14). The groups treated with nanoemulsion showed a significant decrease in tumor size, with a tumor ratio of p < 0.0001 and a tumor ratio of p < 0.001 in the EO treated group, while the highest tumor ratio was found in the DMBA control group (p < 0.0001).

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Fig. 14

Pretreatment and posttreatment tumor volume and tumor ratio V/V0. The data displays the mean ± standard deviation, with * (p < 0.05), ** (p < 0.001), and *** (p < 0.0001) indicating statistical significance compared to vehicle control group and DMBA induced group.

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3.8 Apoptosis assay

Phytocompound’s anti-tumor efficacy is determined through apoptosis, a programmed death process in cells brought on by pharmacological stimuli. The vehicle control group's apoptosis rate was suggestively lower (p < 0.0001) than that of 2-carene and bio-synthesized nanoemulsion treatment groups (Fig. 15). The vehicle control group had an apoptosis rate of 5.31 ng/ml, the nanoemulsion control group 5.28 ng/ml, the DMBA induced group 10.23 ng/ml, the 2-carene group 11.1 ng/ml, the nanoemulsion group 12.54 ng/ml, and the EO group 7.31 ng/ml, respectively. Significant apoptotic activity was renowned in cancer induced animals (which was not given nanoemulsion treatment); p = 0.0008. 2-carene mediated nanoemulsion revealed a greater number of significant apoptotic events (p < 0.001) in comparison to the control and DMBA cancer group.

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Fig. 15

Apoptotic analysis after administration of 2-carene and nanoemulsion. The data displays the mean ± standard deviation, with * (p < 0.05), ** (p < 0.001), and *** (p < 0.0001) indicating statistical significance compared to vehicle control group and DMBA induced group.

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3.9 Inflammatory cytokine indicator analysis

Pro-inflammatory cytokines IL-1, IL-6, IL-23 and TNF-α were measured in DMBA induced cancer rats. A rise in the level of serum IL-1 (p < 0.0001) was observed in DMBA cancer induced rats as compared to vehicle control group. 2-carene treated group shown improvement in IL-1 level (25.21 pg/ml), where p < 0.001 and nanoemulsion treated group showed IL-1 level 36.22 pg/ml (p < 0.05) as compared to DMBA induced group which is statistically significant. 2-carene improve IL-6 level in DMBA induced rats as shown in Fig. 16. There was a significant difference (p = 0.031) in the levels of IL-6 between the nanoemulsion positive control group and the DMBA control group. Using NE treatment resulted in a substantial difference in IL-6 levels (p = 0.0014). When comparing DMBA-induced cancer-ridden rats to the vehicle control, a substantial increase in level of IL-6 was noted (p < 0.001). The DMBA-induced group treated with nanoemulsion showed a 56.14 ± 0.22 drop in IL-6 levels. The levels of IL-6 in animals treated with 2-carene and nanoemulsion significantly improve (p < 0.001). Additionally, the DMBA+EO treated group showed a significantly improved level (p < 0.001).

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Fig. 16

Effect of 2-carene and nanoemulsion on inflammatory marker level. The data displays the mean ± standard deviation, with * (p < 0.05), ** (p < 0.001), and *** (p < 0.0001) indicating statistical significance associated to vehicle control group and DMBA induced group.

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IL-23 was detected and a substantial rise in the level (p < 0.0001) was observed in DMBA cancer induced group in comparison to control groups. A substantial improvement was noticed in terpene compound treated group where p = 0.015 (p value was insignificant) as compared to DMBA untreated group, p < 0.001 in nanoemulsion treated group having IL-23 level (57.54 pg/ml) and a remarkable alteration was also observed in essential oil treated group (p = 0.014).

TNF-α levels were higher in the positive control group than in the vehicle control group (p < 0.05). The DMBA-induced group showed elevated serum TNF-α levels (95.67 pg/ml), which was significantly different from the control animals (p < 0.0001) and the positive control group treated with nanoemulsion (p < 0.001). There was a statistically significant difference in the serum level of TNF- α (74.75 pg/ml) between the groups treated with 50 mg/kg of nanoemulsion and the untreated group produced cancer with p < 0.001. TNF-α in serum samples was significantly lower in the cancer-induced terpene treatment group (62.11 pg/ml) than in the untreated DMBA induced group (p < 0.001).

3.10 Hematological analysis

Following four weeks of 2-carene and nanoemulsion therapy in both control and DMBA induced rats, hematological examination revealed certain alterations in the experimental animals' blood profiles. In contrast to the control group, the DMBA cancer induced group (not treated by terpene or nanoemulsion) exhibited higher levels of platelets, WBCs, and RBCs, while the nanoemulsion treated group showed a significant decrease in WBCs (p < 0.05). The DMBA group exhibited a statistically significant increase (p < 0.001) in WBC counts when compared to the normal group. When compared to the DMBA group, the administration of synthesized nanoemulsion in cancer-induced rats resulted in a significant (p < 0.001) decrease in red blood cells (RBCs) and platelets. While there was a decrease in hemoglobin levels in terpene treated and nanoemulsion treated groups but this difference was not statistically significant (p > 0.05). Compared to the DMBA-induced untreated group, 2-carene increased the platelet count (p = 0.05) and WBC count (p = 0.062). Neutrophils and monocytes were significantly higher in the terpene treated group and the nanoemulsion treated group compared to the control group (p < 0.001 and p < 0.05, respectively). According to Table 5 data, the rats' WBCs, TLC, and platelet profile were all considerably impacted by the 2-carene mediated nanoemulsion after they were given DMBA-induced cancer.

3.11 Oxidative stress marker analysis

It is essential to detect level of antioxidant enzymes developed due to DMBA induction and then evaluate effect of pure isolated compound 2-carene, EO and nano-emulsion on different stress markers. Increased level of SOD enzyme was observed in DMBA control group (p < 0.05) compared to the vehicle control group. Group treated with nanoemulsion displays p < 0.001. As it was demonstrated that, in comparison to DMBA-induced rats who were not treated, SOD concentration markedly increased in rats that had been given nanoemulsion treatment where p = 0.0012 and this differene was significant and group treated with 2-carene treatment shown decrease in SOD level (p > 0.05).

When comparing the 2-carene and EO treated groups to the untreated DMBA group (untreated group), the CAT level dropped in both cases (p < 0.05 and p < 0.001). The 2-carene treated group's CAT level, which was 21.02 ± 0.14 Unit/mg, improved. The untreated group induced with DMBA had the highest CAT level, at 28.41 ± 0.12 Units/mg. In comparison to the DMBA-induced cancer control group, CAT values in the nanoemulsion treated groups were found to be p = 0.041 (Fig. 17).

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Fig. 17

Effect of 2-carene and nanoemulsion on oxidative stress enzymes. The data displays the mean ± standard deviation, with * (p < 0.05), ** (p < 0.001), and *** (p < 0.0001) indicating statistical significance compared to vehicle control group and DMBA induced group.

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GPx activity was observed in the DMBA induced group (p = 0.0001) relative to the control group. In comparison to the control group, the nanoemulsion treatment group exhibits significant values (p < 0.05), but GPx value observed in the EO treated group was also statistically significant (p = 0.0014). As in contrast to the vehicle control group, the DMBA-induced and treated groups had higher GPx concentrations (p < 0.05) and rats with DMBA-induced cancer afterward treated with 2-carene and 2-carene mediated nanoemulsion dramatically raised GPx activity (p < 0.001) in comparison to vehicle control group but markedly shown improvement in GPx levels after nanoemulsion treatment suggesting that 2-carene based nanoformulation may have a positive impact on glutathione peroxidase levels.

GSH levels were monitered in rats in all experimental groups and DMBA induced animals exhibited a substantial (p < 0.0001) reduction in glutathione levels (18.11 ± 2.06 Units/g) compared to the vehicle control group of rats. In the DMBA-induced group, nanoemulsion administration raised the level of GSH (18.43 ± 2.06 Units/g) in contrast to DMBA induced untreated group, although this difference was not statistically significant (p > 0.05). Although rats treated with EO fter cancer induction shown a substantial improvement in GSH level where p < 0.05 (Fig. 17).

3.12 Histopathology of mammary cancer

Sections of the control group's mammary tissue that had been H&E stained showed normal morphology. Histopathological alterations were not observed in the emulsion control or vehicle control groups (Fig. 18 A and B). Histopathology determined that the tumor type in DMBA-treated groups was a differentiated adenocarcinoma. Cribriform adenocarcinomas, characterized by abnormal cell development, hyperplasia with dilated ducts, and a little amount of fatty tissue, were found in rats with mammary tumors in the DMBA induced group (who were not treated with terpenes or nanoemulsions). Adipose tissue invasion were present in the solid tumor in the DMBA group. Animals with cancer developed proliferative lesions and lobular hyperplasia. Comparing the DMBA group to the terpene 2-carene group, there was a minor decrease in the tubular structures and surrounding fibrosis. The DMBA+nanoemulsion group included a wide range of nuclei and exhibited very minimal ductular growth. But the group receiving nanoemulsion treatment also had some degree of fibrosarcoma (Fig. 18 E). Analyzing the mammary tumors in rats with nanoemulsion indicated the presence of an adenocarcinoma, corroborated by a slight damage of the parenchyma and mammary cells. Rats with breast tumors can have their hyperproliferation reduced by a particular dose of nanoemulsion administration, which is crucial for prolonging the survival of the rats.

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Fig. 18

Histology images of (hematoxylin and eosin) of mammary gland tissues. (Here A: control group; B: Nanoemulsion control; C: DMBA induced untreated group; D: 2-carene treatment; E: 2-carene mediated nanoemulsion treatment and F: Essential oil treatment group).

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