1. INTRODUCTION:

For centuries, cloves have been a prized medicinal spice, with their primary active compound, eugenol, being utilized for its diverse therapeutic properties^1,2. Eugenol has been employed as a topical anesthetic, dental adjunct, and has recently garnered attention for its potential in treating various ailments. The essential oil of cloves, extracted through steam distillation of the dried flower buds of Syzygium aromaticum L., an evergreen tree, serves as the primary source of eugenol^3,4. Further analysis of the essential oil reveals a secondary constituent, acetyl eugenol (eugenol acetate), and has been shown to possess anti-inflammatory, antimicrobial, and antioxidant properties, making it a valuable compound in traditional medicine⁵.

It has been reported to possess anti-inflammatory and antimicrobial properties, contributing to the oil's therapeutic effects^6,7. The synergistic interactions between eugenol, acetyl eugenol, and terpenes may enhance the medicinal benefits of clove essential oil, making it a valuable adjunct therapy in various healthcare applications⁵.

Eugenol and Sanguinarine have shown remarkable anticancer potential through various mechanisms. Clove's primary constituent, Eugenol, exhibits antiproliferative effects, inhibiting cancer cell growth and inducing apoptosis, while also showcasing antioxidant and anti-inflammatory properties^2,8. Both clove and Sanguinarine share common mechanisms, inhibiting NF-κB and modulating the PI3K/Akt signaling pathway, which regulates cell survival and proliferation. Additionally, they exhibit antimicrobial properties, potentially preventing cancer-promoting infections^9–11. While these findings are promising, further research is needed to fully understand their anticancer potential and explore possible applications in cancer prevention and treatment^12,13. In this research, we have evaluated anticancer potential of eugenol in combination with Sanguinarine.

2. MATERIAL AND METHODS:

Clove buds, obtained in their dried form from a local supermarket, were processed into a fine powder using a coffee bean grinder. The necessary solvents, including isopropanol, ethyl acetate, hexane, and chloroform, were sourced from GLR Innovations, a reputable supplier of laboratory chemicals. Additionally, MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) dye and DMSO (Dimethyl sulfoxide) were also acquired from GLR Innovations for use in the bioactivity assays. Sanguinarine, a flavonoid compound, was generously provided as a gifted sample by Edudap Chemicals Suppliers. This enabled the investigation of its potential bioactive properties in conjunction with the clove extracts.

2.1 Extraction:

Extracting eugenol from cloves involves a multi-step process beginning with grinding the cloves into a fine powder to increase the surface area and facilitate efficient extraction^14,15. Next, the powdered cloves are mixed with water and subjected to distillation, where the mixture is heated, causing the water and eugenol to vaporize, and then condensed and collected in a separate container, repeating the process until approximately 25 mL of distillate is obtained^16,17. The distillate is then transferred to a separation funnel, where it is mixed with diethyl ether, allowing the ether to extract the eugenol from the water, resulting in two distinct layers, with the top layer containing the eugenol and diethyl ether being separated from the bottom layer^18–20. The top layer is then transferred to an Erlenmeyer flask, where small portions of magnesium sulfate are added to absorb any remaining water, followed by filtration to remove the magnesium sulfate, yielding a dry and clear solution^21,22. Finally, the filtered liquid is placed in a beaker and subjected to a steam bath, where the heat causes the diethyl ether to evaporate, leaving behind a concentrated solution of eugenol, with the steam bath continued until only 1-2mL of solution remains, resulting in a highly concentrated eugenol extract, which can then be further purified and analyzed for its chemical composition and potential applications^23,24. Throughout this process, careful attention is paid to temperature, solvent ratios, and extraction times to optimize the yield and characterized using IR spectroscopy as depicted in figure 3 and table 1.

2.2 Free radical scavenging Activity:

The DPPH (2,2-diphenyl-1-picrylhydrazyl) solution was prepared by dissolving 2.4 milligrams of DPPH in 100 milliliters of methanol, creating a solution containing oxidative moieties^25,26. This solution serves as a reference for measuring the antioxidant activity of the sample extract²⁷. To assess the antioxidant potential, 4 milliliters of the DPPH solution was combined with 5 microliters of the sample extract in a cuvette^28,29. The mixture was vigorously agitated for 10 minutes to ensure thorough mixing, followed by incubation in the dark for 30 minutes to allow the reaction to reach completion^30,31. After incubation, the absorption of the resulting solution was measured using UV spectrophotometry at a wavelength of 550 nanometers³².

To prepare a 40mM hydrogen peroxide solution, a precise mixture of hydrogen peroxide and phosphate buffer solution was required. Specifically, 4.09 milliliters of hydrogen peroxide was added to 1000 milliliters of phosphate buffer solution, which had been previously adjusted to a pH of 7.4³³. This ensured that the resulting solution had the desired concentration and pH for the subsequent assay^34–37. Next, 0.24 milliliters of the drug extract solution was added to 2.4 milliliters of the freshly prepared 40mM hydrogen peroxide solution^38,39. Following mixing, the solution was placed in a dark environment for 20 minutes to allow the reaction to proceed without any external influences^40,41. Finally, the absorbance of both the mixture and a control solution (hydrogen peroxide solution without drug extract) was measured at a wavelength of 560 nanometers using UV spectrophotometry^42,43.

2.3 Cell Line Maintenance:

MCF-7 cell line was Procured from NCCS Pune. The cells were harvested from flask at 90% confluency and seeded in 96 well plates at appropriate density in DMEM medium supplemented with 10% FBS and 1% antibiotic solution at 37°C with 5% CO₂^44,45. 10% of the total medium (5 to 50µl) of Treatment dilutions (of different concentrations) were added to the defined wells and treated plates were incubated (Heal Force-Smart cell CO₂ Incubator-Hf-90) for 24 h^46,47.

2.4 Cytotoxicity Assay:

Cytotoxicity of the provided samples on MCF-7 (Procured from NCCS Pune) cell line was determined by MTT Assay^48,49. The cells (10000 cells/well) were cultured in 96 well plate for 24 h in DMEM medium supplemented with 10% FBS and 1% antibiotic solution at 37°C with 5% CO₂^50,51. Next day cells were treated from (as per mention in the excel sheet) of the formulations (different concentrations were prepared in incomplete medium)⁵². After incubation for 24 hours, MTT Solution (a final concentration of 250µg/ml) was added to cell culture and further incubated for 2h^53,54. At the end of the experiment, culture supernatant was removed and cell layer matrix was dissolved in 100 µl Dimethyl Sulfoxide (DMSO) and read in an Elisa plate reader (iMark, Biorad, USA) at 540nm and 660nm. IC₅₀was calculated by using software Graph Pad Prism -6. Images were captured under inverted microscope (Olympus ek2) using Camera (AmScope digital camera 10 MP Aptima CMOS)^55,56.

3. RESULTS:

3.1. Free radical scavenging activity:

DPPH % scavenging activity of the novel phytochemical combination was performed against quercetin (As a standard). The DPPH radical scavenging potentiality of combination and quercetin were approximately equal in 200 μg/mL concentration. But 600 μg/mL solution of quercetin shows the 57% inhibition against DPPH while combination have shown 51% inhibition against DPPH as given in figure 1.

Figure 1: DPPH % Scavenging Activity of Novel Phytochemical Combination

The results for the percentage H₂O₂ scavenging activity have been discussed as the percentage H₂O₂ scavenging activity in Figure no. Quercetin was used as the standard. In the starting there was a difference in the % inhibition of Hydrogen peroxide at 200 μg/mL while at 600μg/mL solution of combination have shown the same % inhibition as the standard quercetin shows as depicted in figure 2.

Figure 2: H2O2 % Scavenging Activity of Novel Phytochemical Combination

3.2 Infra-red Spectroscopy of Eugenol:

Figure 3: Infra-Red Spectroscopy of Eugenol extracted from clove buds

Table 1: depicts IR interpretation of eugenol.

Wavenumber	Nature of Peak	Proposed Structure
3257-3400 cm^-1	Broad medium	Alcoholic and methoxy group
2835 cm^-1	Single peak with W shape	Methyl group
1638 cm^-1	Single, small intense	C=C stretching (Aromatics)
1056 cm^-1	Medium Intense	C-O stretching

3.3 Cell Culture Maintenance:

MCF-7 cells were grown in DMEM medium; evidentiary images have been shown in following figure 4.

Figure 4: MCF-7 Cell lines growing in DMEM medium with the aid of FBS and PenStrep Solution

3.4 Cytotoxicity Assay:

Based on the results obtained from the MTT assay, it was observed that when the MCF-7 cell line was exposed to different concentrations of the sample, moderate cytotoxic activity was observed with the administration of novel phytochemical combination (IC₅₀= 147.2±0.098µM) as given in figure 5. The IC₅₀ is the concentration of an inhibitor/sample/ formulation at which the viable cells reduced by half.

Figure 5: MTT Data analysis of novel phytochemical combination.

4. DISCUSSION:

The present study successfully extracted eugenol from cloves and evaluated its antioxidant potential in combination with Sanguinarine. The IR spectroscopy analysis confirmed the presence of eugenol, which was then combined with Sanguinarine to create a novel antioxidant mixture. The DPPH and H2O2 scavenging assays revealed that this combination exhibited potent antioxidant activity, comparable to the standard antioxidant quercetin, at a concentration of 600μg/mL. This suggests that the synergistic interaction between eugenol and Sanguinarine enhances their individual antioxidant properties.

The MTT assay revealed an IC50 value of 147.2μM, indicating moderate cytotoxicity against MCF-7 cell lines. This suggests that the novel combination may have potential anti-cancer properties, although further studies are needed to confirm this. The antioxidant and cytotoxicity results suggest that the eugenol-Sanguinarine combination may have potential therapeutic applications, particularly in the prevention and treatment of oxidative stress-related diseases and cancer. However, further studies are needed to fully elucidate the mechanisms of action and optimal dosing regimens. Overall, this study demonstrates the potential of combining natural compounds to create novel antioxidant and anti-cancer agents, and highlights the importance of continued research into the therapeutic properties of plant-derived compounds.

5. CONCLUSION:

The study underscores the remarkable anticancer and antioxidant potential of the synergistic combination of eugenol and Sanguinarine, showcasing moderate cytotoxicity against MCF-7 cell lines and pronounced free radical scavenging activity. The observed bioactivity of this novel phytochemical combination is a testament to the potential benefits of integrating natural compounds in the development of innovative therapeutic strategies.

The moderate cytotoxicity exhibited by the eugenol-Sanguinarine combination against MCF-7 cell lines suggests that this phytochemical duo may be a valuable adjunct in the treatment of certain types of cancer, particularly those characterized by oxidative stress and inflammation. Moreover, the significant free radical scavenging activity demonstrated by this combination highlights its potential in mitigating oxidative stress-related diseases, such as neurodegenerative disorders and cardiovascular disease. The findings of this study suggest further investigation to fully elucidate the mechanisms of action underlying the anticancer and antioxidant effects of the eugenol-Sanguinarine combination. Additionally, further research is needed to optimize the dosing regimens, evaluate the efficacy of this combination in vivo, and explore its potential applications in the prevention and treatment of various diseases. Nevertheless, the present study provides a promising foundation for the development of novel phytochemical-based therapeutic approaches, underscoring the importance of continued research into the medicinal properties of plant-derived compounds.

6. CONFLICT OF INTEREST:

Authors declare no conflict of interest.

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Received on 09.09.2024 Revised on 25.01.2025

Accepted on 16.04.2025 Published on 05.09.2025

Available online from September 08, 2025

Research J. Pharmacy and Technology. 2025;18(9):4409-4414.

DOI: 10.52711/0974-360X.2025.00632

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.