INTRODUCTION:

Breast cancer ranks as the most prevalent form of cancer among women and results in a significant number of deaths worldwide. By the end of 2020, breast cancer had been diagnosed in 7.8 million women, with a significant number of them unfortunately passing away due to the illness¹. Breast cancer occurs in every country, with varying incidence rates. The number of newly reported instances of breast cancer is projected to continue rising by more than 3 million each year until 2040². Current breast cancer treatments involve multidisciplinary collaboration, focusing on the patient characteristics and tumor type and seeking patient preferences by considering the advantages and disadvantages of various treatment options.

While surgery, chemotherapy, and radiotherapy are quite effective, they could be detrimental to women, considering the side effects of each treatment option³. Therefore, it is crucial to discover and develop substitute drugs made from natural ingredients as therapeutic options for patients.

Currently, many anticancer drugs from natural ingredients are being developed⁴. Many studies have used various models with cancer cell lines to determine their pathobiology and efficacy as new drugs. Cell lines play an important role in understanding disease prognosis and treatment mechanisms. They provide a reliable and reproducible model system to study the phenotypic and molecular characteristics of diseases⁵. The advantages of conducting research by using the cell lines are easy handling, cellular homogeneity, and unlimited self-replication⁶. MCF-7 and T47D cell lines in xenograft tumor models are particularly advantageous due to their key characteristics specific to the mammary epithelium. These cell lines exhibit hormone receptor expression and respond to hormonal stimulation, making them suitable for studying the impact of endocrine therapies on breast cancer. Both cell lines depend on estrogen-sensitive hormones via receptor alpha (ERα-positive). Although the phenotypic and molecular characteristics of both cell types are similar to the luminal-A subtype of breast cancer, the T47D cell line serves as an excellent in vitro model for studying the biology and potential therapeutic targets of this subtype. This cell line is commonly used to investigate the response of luminal-A breast cancer cells to hormonal therapies, such as endocrine therapy⁷.

The use of natural ingredients in the search for anti-cancer compounds has gained significant attention due to their potential therapeutic benefits and reduced side effects when compared to chemotherapeutic drugs discovery . Furthermore, the discovery of agents that can induce cancer cell apoptosis is believed to be a tumor suppressor agent in cancer chemotherapy expected. Many active compounds have shown effectiveness in inducing apoptosis in various cancer cell lines¹⁰. C. martinii belongs to the Poaceae family and has great anti-cancer benefits due to its numerous pharmacological properties¹¹. The essential oil from this plant commonly called Palmarosa essential oil (PEO), is proven to have cytotoxic effects on the MCF-7 cell line, demonstrating an IC₅₀ value of 6.29±0.56¹². However, the effects of its cytotoxic activity on T-47D cells have not been proven. The proliferation of breast cancer cells is inhibited by the metabolite compounds present in PEO. The main compound in PEO is geraniol 83.75±2.07%¹³, which considered to have the potential to inhibit various cancer cells, including breast cancer cells¹⁴. Geraniol is reported to have cytotoxic ability against the MCF-7 cell line with an IC₅₀ of 138.9µg/mL¹⁵. The IC₅₀ value demonstrated that the cytotoxic ability of the PEO compound was more active in inhibiting the proliferation of breast cancer cell lines than geraniol as a single compound. Thus far, the main biological marker compound related to the cytotoxic activity of PEO on breast cancer and its inhibitory mechanism remains unknown. Therefore, in this study, PEO was extracted from various parts of C. martinii, and a cytotoxicity test was followed by an apoptosis induction test and molecular docking with T-47D to predict its binding potential.

MATERIALS AND METHODS:

Materials:

The materials were C. martinii harvested from Rumah Minyak Atsiri Indonesia, Tawangmangu, Indonesia, RPMI (Gibco), DMSO (Gibco), MTT (50 mg MTT and 10 mL PBS), DMEM, T47D cells and Vero cells collected from the Parasitology Laboratory (Gadjah Mada University), Foetal Bovie Serum 10% (Gibco), SDS stopper 10% (Sigma), and 0.1 HCl (Merck). All remaining chemicals were of analytical grade.

Sample Preparation:

The C.martinii has been determined at the Functional Services Unit of Tawangmangu Yankestrad, Indonesia. Palmarosa was harvested and sorted, separated into 3 parts: leaves, stems, and all parts of the plant. Each part was dried at room temperature for 24hours¹⁶ and distilled.

Steam - Water Distillation:

The bottom of the perforated filter rack in the distillator was filled with 10 L of water, and the sample put on the rack. The distillator was heated for 45 minutes and then the water flowed through a hose to the condenser. The distillation time from the first drop in the condenser to the last drop was calculated, and the essential oil was taken from the separator. Each PEO was stored in closed containers away from light. The distillation process was carried out for each sample part of C. martinii.

Chemical Composition Analysis of Palmarosa Essential Oil:

Analysis composition of each PEO using GC-MS techniques. GC-MS was used with an HP-5 chromatography column (length 30 meters, film thickness 0.32mm, internal diameter 0.25μm) filled with 5.5% phenylmethylsiloxane was employed with GC-MS. The interface temperature was fixed at 280°C, and the injection port was kept at 230°C. Helium was used as the carrier gas, and the temperature was progressively raised from 50°C to 280°C, rising by 10°C every minute. The PEO sample was injected into the injector and subsequently vaporized. The vaporized sample was transported by the carrier gas to the column for the separation procedure. Upon being separated, every part underwent ionization in an ionizing chamber and was bombarded by electrons. The detector captured the ion fragments produced, resulting in the creation of a mass spectrum¹⁷. Every PEO's chemical composition was examined by comparing it to the database in WILLEY.LIB.

Cytotoxic activity Test on T-47D and Vero cell line:

The MTT (3 (4, 5-Dimethyllithiazol-2-il)-2,5 diphenyltetrazolium bromide) assay technique was used to assess the cytotoxic effect of PEO on T-47D and Vero cell lines. The 96-plate cells were cultured in a CO2 incubator for 24hours¹⁸. Each concentration series of 100µL was transferred to a 96 plate containing cells for 3 replications and then incubated for 48 hours. The test solution's concentration on T-47D cells consists of various levels from 15.625μg/mL to 500μg/mL, while the Vero cell cultures were tested to concentrations ranging from 31.25μg/mL to 1000μg/mL¹⁹. Afterward, 20μL of MTT solution at a concentration of 5 mg/mL was added to each wellplate and left to incubate for an additional three days. The formazan crystals were dissolved by adding 100μL of DMSO following the removal of the medium. At a wavelength of 570nm, the formazan crystals were measured with an ELISA reader (BioTekELx800, USA). The cytotoxicity interpretation data in this study were processed using probit analysis to determine the IC₅₀, it was presented in a graph of log concentration versus percentage of cell viability.

Live cells

Cell viability (%) = ------------------------------------ x 100

Total number of cells²⁰.

The National Cancer Institute (NCI) was evaluated and its cytotoxicity research was carried out using IC₅₀ values to categorize activity levels: IC₅₀ ≤ 20µg/mL as highly active, IC₅₀ 21-200µg/mL as moderately active, IC₅₀ 201-500µg/mL as weakly active, and IC₅₀ > 501 µg/mL as inactive²¹. The selectivity of essential oil was determined from the Selectivity Index (SI) value obtained by comparing the IC₅₀ of Vero cells to the T-47D cells. SI > 3 high selectivity and were classified as prospective anti-cancer samples²². The PEO that had the most active cytotoxic effects were treated with apoptosis induction assay.

Apoptosis Assay Using Double Staining Method:

The cover slip (Thermanoxâ Plastic Coverslips) was placed into a 24-well plate (Nunc), then 5x10⁴ cells were planted on it and left for 30 minutes. After that, 500µl of media was given to the cells, and they were allowed to incubate overnight at 37˚C in an incubator with 5% CO₂. Following the removal of the medium, the well was added with 1000µl of the test solution, and it was kept in an incubator for 10hours. In the meantime, the other well is solely filled with DMSO solvent and medium for solvent control and cell control. Following the incubation period, the coverslip was taken off and set on a glass surface, and the media was disposed of. After adding 10µl of 1X Working Solution acridine orange-ethidium bromide to the coverslip, it was promptly examined using a fluorescence microscope (Axiolab)²³.

Molecular Docking Study of PEO:

Protein and Ligand Preparation:

In this study, PEO oil was identified 12 compounds by using GC-MS. The analysis used energy minimization by using the MM2 force field to reach stable conformations for docking studies²⁴. The target proteins were receptors expressed on T-47D cells, such as estrogen receptor alpha (ER-α) and progesterone receptor (PR). These two proteins were involved in the development and proliferation of breast cancer cells^25;26 (Clusan et al., 2023; Trabert et al., 2020). The protein targets were chosen for molecular docking based on their presence and role in the life cycle of breast cancer cells. The PDB database RCSB (www.rcsb.org) provided the three-dimensional structures of the ER-α (PDB ID: 1XP1) and PR (PDB ID: 4OAR) proteins and downloaded in the PDB file. Each ER-alpha and PR receptor PDB ID was obtained based on previous studies that tested the validity of these receptors by redocking the native ligand in each tested using the native ligand of each protein to obtain an RMSD value of less than 2 Å^7;
27. Proteins were prepared using PyMOL 2.5.7 to remove native ligands and water molecules to minimize the effect of water on compound interactions with target proteins²⁸.

Molecular Docking Analysis:

The molecular docking study in this research used Autodock Vina in the PyRx 0.8 interface²⁹. Determination of the Root-mean-square deviation (RMSD) and energy binding affinity (kcal/mol) values needed to assess the various docking orientations based on the position of the identical ligand molecule, molecular docking was performed on the natural ligand for each protein³⁰. The RMSD values resulting from the re-docking of native ligands were calculated using DockRMSD³¹. The docking algorithm in Autodock Vina is used to search for docking conformations between ligands and proteins. PyMOL 2.5.7 and Biovia Discovery Studio Visualizer v21.1.0.20298 were used to examine interactions between the target protein and the ligand, resulting in the optimal conformation with the lowest binding affinity energy.

RESULT:

Chemical composition of the PEO:

The results of GC-MS analysis of PEOs extracted from leaves, stems, and dried herbs are presented in Table 1 based on WILEY.LIBRARY. The yield of leaves was 1.835% (10 compounds were identified), stems were 0,065% (11 compounds were identified), and dried herbs were 1,515% (9 compounds were identified). The part of the plant affects the chemical composition. The main compounds in the leaves PEO were Geraniol (85.67%), Geranyl acetate (10,64%), β-Caryophyllene (1.19%), Linalool (1.58%); in stems PEO were Geraniol (78.96%), Geranyl acetate (17.37%), Linalool (1.49%), β-Caryophyllene (1.06%) and those in dried herbs PEO were Geraniol (86.73%), Geranyl acetate (9.29%), Linalool (2.06%), Trans(beta)-caryophyllene (0.89%).

Table 1. Chemical composition of the Palmarosa Essential oil

Compounds	Leaves % Area	Stems % Area	Herbs % Area
Limonene	0.05	0.05	0.06
Trans-α-Ocimene	0.1	0.13	0.15
Trans-β-Ocimene	0.52	0.57	0.72
Linalool	1.58	1.49	2.06
Neral	-	0.07	-
Geraniol	85.67	78.96	86.73
β -Geranial	0.15	0.25	0.69
Nerol	-	0.01	0.01
Geranyl acetate	10.64	17.37	9.92
β-Caryophyllene	1.19	1.06	0.89
α-Humulene	0.05	0.05	-
4,11-Selinadiene	0.05	-	-

Cytotoxic activity:

The cytotoxic activity showed the antiproliferative activity in vitro of essential oils by using the MTT Assay method on T-47D cancer cells. Proliferative activity in all parts of PEO showed a relatively dose-dependent inhibition of T-47D cells (Fig. 1). The results of cytotoxic activity of PEO are shown in Table 2. It showed that the essential oil from the leaves has the highest inhibitory effect after 48 hours of incubation compared to the stems and herbs. However, the inhibitory effect was stronger than the Vero cell line (IC₅₀ 493.86µg/mL) with a Selectivity index of 5.92.

Figure 1. Cytotoxic activity of the PEO on T-47D Cell lines

Table 2. The inhibitory concentration 50% (IC₅₀) of PEO on T-47D Cell lines (Mean±SD)

Sample test	IC₅₀(µg/mL)
Leaves PEO	83.41+3.24
Stems PEO	95.53+3.28
Herbs PEO	141,63+1,63

Figure 2. Morphology of control T-47D cells after 48h incubation. Cells without treatment (A) cells treated with Leaves PEO (B). Live cells appear in the form of leaves or bright dots; meanwhile, dead cells are round, appear cloudy, and float. It can be seen that dead cells were found when treated with Palmarosa essential oil. Description: (1) living cells, (2) dead cells.

Essential oil-induced apoptosis of T-47D:

The indication for chemoprevention, the process of cell death through the important mechanism of apoptosis was used. Induction of apoptosis was carried out by using staining to determine possible biological effects on T47D cells. This method is used for the rapid detection of apoptosis. The results showed that the media control in Fig. 3A. was green. The green color comes from orange acridine penetrating all parts of living cells that have intact membranes, including the nucleus.

Figure 3. Morphology of T-47D cells after staining using double staining acridine orange-ethidium bromide. Control cells (A), solvent control (B), and cells produced by Leaves PEO treatment (C). Observations were carried out under a fluorescence microscope. The living cells were leaf-shaped or circles with an even green color. Apoptotic cells have an irregular shape with an uneven orange color. In the treatment group, cells appeared to die due to apoptosis. Description: 1. Living cells, 2. Early Apoptosis 3. Apoptosis.

Molecular docking:

The compounds in PEO were treated by molecular docking with progesterone receptor (PR) and estrogen receptor alpha (ER-α). The compounds CHEMBL 182980 and ulipristal acetate were used as native ligands to validate the capability of the docking method. For both proteins, RMSD 0.722 (ER-α) and 0.510 (PR) refer to optimal results when they have RMSD ≤ 2.0Å (Ramírez and Caballero, 2018). This RMSD value indicates that the native ligand has a similar pose and conformation between the docking results and the pose and conformation of the crystallized protein. The molecular docking results (Table 3) of the native ligand CHEMBL 1828980 with ER-α protein showed a strong binding affinity (-12.4kcal/mol) due to many intermolecular interactions, including hydrogen bond (conventional and carbon), π-α interaction, π-sulfur interaction, π- π T shaped, alkyl/π-alkyl interaction, and van der Waals interaction. One of the interactions is the hydrogen bond between the ligand and residues Glu353, His524, Arg394, and Gly521 (Fig. 4A.). Molecular docking of compounds from PEO on ER-α, has a binding affinity that varies, the more negative the binding affinity value, the higher the affinity and the more stable the bond or interaction formed. As presented in Table 3 and Fig. 4B, The best binding affinity among all PEO compounds is β-Caryophyllene (-8.4 kcal/mol). The presence of 12 alkyl/π-alkyl interactions at 11 amino acid residues, Met343, Leu346, Ala350, Leu384, Leu387, Met388, Phe404, Ile424, Leu428, Phe425, Leu525, which caused the best affinity among other PEO compounds. Despite its binding affinity being relatively strong compared to other compounds, the β-Caryophyllene compound was not stronger than the native ligand due to intermolecular interactions dominated by van der Waals binding. In contrast, the native ligand has more than six molecular interactions that are responsible for the high binding affinity to the receptor.

The molecular docking results of the native ligand Ulipristal acetate with PR protein showed a good binding affinity (-10.8kcal/mol) due to many intermolecular interactions, including conventional hydrogen bond, carbon/π donor hydrogen bond, π-α interaction, π- π T shaped/amide-π stacked, alkyl/π-alkyl interaction, and van der Waals interaction. One of the interactions is the hydrogen bond between the ligand and residues Gln725, Arg766, Cys891, and Thr894 (Fig. 4C.). Based on the binding affinity from molecular docking in PR protein, 4,11-selinadiene had a higher binding affinity value (-6.8kcal/mol) when compared with other PEO compounds, which due to 13alkyl/π-alkyl interaction ligand with eight amino acid residues such as Leu718, Met756, Met759, Val760, Leu763, Phe778, Leu797, Met801. Although it has good affinity compared to other PEO compounds, 4,11-Selinadiene in the PR protein shows a lower affinity than the native ligand. This is because the interactions between this compound and PR are only two types of interactions in the form of alkyl/π-alkyl interaction and van der Waals only (Fig. 4D.).

(A) estrogen receptor-α and native ligand; (B) ER-α and β-Caryophyllene

(C) progesteron receptor and native ligand (D) progesteron receptor and 4,11-Selinadiene

Figure 4. 3D and 2D molecular interaction between protein and ligand

Table 3. Binding affinity of breast cancer-related protein (ER-α and PR) and the PEO compound as ligands

Compound Name	PDB ID : 1XP1				PDB ID : 4OAR
	RMSD	Binding Affinity (kcal/mol)	Molecular interaction	Amino acid residues	RMSD	Binding Affinity (kcal/mol)	Molecular interaction	Amino acid residues
CHEMBL182980	0.722	-12.4	Conventional hydrogen bond	His524, Glu353
			Carbon hydrogen bond	Asp351, Lys531
			Pi-Sigma	Trp383
			Pi Sulfur	Phe404, Met421
			Pi-Pi T-shaped	Phe404
			Alkyl/Pi-Alkyl	Leu346, Ala350, Leu354, Leu387, Leu391, Ile424, Leu525, Leu536
Ulipristal acetate					0.510	-10.8	Conventional hydrogen bond	Arg766, Thr894
							Carbon/pi donor hydrogen bond	Gln725, Cys891
							Pi-Sigma	Gly722
							Pi-Pi T-shaped / Amide-Pi Stacked	Phe778
							Alkyl/PI-Alkyl	Leu726, Trp755, Met759, Cys891, Leu2350
Limonene		-6.1	Alkyl/Pi-Alkyl	Leu354, Trp383, Lys531, Leu536, Leu539		-6.0	Pi-Sigma	Trp755
							Alkyl/PI-Alkyl	Leu726, Met759, Leu2350
trans-α-Ocimene		-5.3	Alkyl/Pi-Alkyl	Leu354, Trp383, Leu525, Cys530, Lys531, Leu536		-5.3	Alkyl/PI-Alkyl	Leu718, Leu721, Met759, Val760, Leu763, Phe778, Met801
trans-β-Ocimene		-5.5	Alkyl/Pi-Alkyl	Leu387, Met388, Leu391, Phe404, Ile424, Leu428, Leu525, His524		-5.4	Alkyl/PI-Alkyl	Met756, Met759, Val760, Leu763, Phe778, Leu797, Met801, Leu887
Linalool		-5.4	Pi-Sigma	Trp383		-5.5	Conventional hydrogen bond	Gln725
			Conventional hydrogen bond	Lys531			Alkyl/PI-Alkyl	Leu718, Met759, Leu763, Phe778, Leu797, Met801
			Alkyl/Pi-Alkyl	Leu354, Leu536.
Neral		-5.4	Conventional hydrogen bond	His524		-5.5	Alkyl/PI-Alkyl	Leu718, Leu721, Met759, Leu763, Phe778
			Carbon hydrogen bond	Gly521
			Alkyl/Pi-Alkyl	Leu346, Ala350, Leu387, Met388, Met421, Leu428, Phe404, Phe425
Geraniol		-5.7	Conventional hydrogen bond	His524		-5.8	Alkyl/PI-Alkyl	Leu718, Met759, Val760, Leu763, Phe778, Met801
			Alkyl/Pi-Alkyl	Leu346, Ala350, Met388, Phe404, Met421, Ile424, Phe425
β-Geranial		-5.6	Alkyl/Pi-Alkyl	Leu346, Ala350, Leu387, Met388, Leu391, Leu428, Phe404,		-5.1	Conventional hydrogen bond	Gln725
							Alkyl/PI-Alkyl	Met756, Met759, Val760, Leu763, Phe778, Leu797, Met801, Leu887
Nerol		-5.8	Alkyl/Pi-Alkyl	Leu346, Ala350, Trp383, Leu384, Leu387, Phe404, Leu525, Cys530		-5.7	Conventional hydrogen bond	Gln725
							Alkyl/PI-Alkyl	Leu718, Phe778, Met801
Geranyl acetate		-6.1	Alkyl/Pi-Alkyl	Leu346, Ala350, Trp383, Leu384, Leu387, Phe404, Leu525, Cys530		-5.6	Conventional hydrogen bond	Gln725
							Alkyl/PI-Alkyl	Leu718, Met756, Phe778, Leu797, Met801, Leu887
β-Caryophyllene		-8.4	Alkyl/Pi-Alkyl	Met343, Leu346, Ala350, Leu384, Leu387, Met388, Phe404, Ile424, Leu428, Phe425, Leu525		-6.7	Alkyl/PI-Alkyl	Leu718, Met756, Met759, Leu763, Phe778, Leu797, Met801, Leu887
α-Humulene		-8.0	Alkyl/Pi-Alkyl	Leu346, Ala350, Met388, Leu391, Phe404, Met421, Ile424, Phe425, His524, Leu525		-6.4	Alkyl/PI-Alkyl	Leu721, Leu718, Phe778
4,11-Selinadiene		-8.0	Alkyl/Pi-Alkyl	Leu346, Leu349, Ala350, Leu384, Leu387, Leu391 Met388, Phe404, Ile424, Leu428, Leu525		-6.8	Alkyl/Pi-Alkyl	Leu718, Met756, Met759, Val760, Leu763, Phe778, Leu797, Met801

DISCUSSION:

Essential oils from plants are believed to reduce the risk of cancer due to their antioxidant activity and toxicity. Essential oils could inhibit cancer growth through various mechanisms. Eugenol obtained from Eugenia caryophyllata has cytotoxicity against Hep G2 hepatoma cells and Caco-2 colon cells³². Moreover, eugenol can induce apoptosis by destroying the mitochondrial membrane and the production of Reactive Oxidative Species³³. Xanthorrhizol is one of the essential oil compounds in ginger rhizomes able to inhibit breast cancer cells, including hERα and T47D cells. This essential oil shows the ability to inhibit cancer cell growth by forming hydrogen bonding with Arg394 and Glu353 and interact by forming hydrophobic bonding with the estrogen alpha Ligand Binding Domain (LBD)³⁴.

Palmarosa essential oil contains two main components, geraniol and geranyl acetate, in the leaves, stems, or herbs. This essential oil is included in GRAS, recognized as an essential oil claimed to be safe by the US FDA³⁵ Each part of the C. martinii plant could produce essential oils with different characteristics and components. The leaves and stems of Chilean herbal medicine plants show differences in the type of the main compound and the amount of its content³⁶. Essential oil produced from C. martini leaf had the greatest amount of essential oil in the herbs, and the lowest production was in the stems. The results of other studies showed that the amount of essential oil in the leaf of C. martini is also greater than its flowers³⁷. Therefore, the cytotoxic activity of leaves PEO was higher than the stem and herbs PEO. These different amounts of the compound in all parts contribute to inhibiting the T-47D cancer cell line. The leaves showed stronger cytotoxicity than other plant parts³⁸. The herbs PEO contained the highest geraniol compared to the other parts, but it showed the weakest inhibition. It is caused by compounds other than geraniol that contribute to detaining the growth of cancer cells. Leaves PEO were proven to have strong cytotoxic activity on MCF-7, DU-145, and WI-38 cells¹².

All this time, the discovery of cancer drugs from plants has focused on observing the target mechanism of action, one of which is the process of apoptosis (programmed cell death)¹⁵. Essential oil from Cymbopogon flexuosus has been proven to induce apoptosis in HL-60 leukemia cells³⁹. Apoptosis assays could well characterize and predict a range of gene-controlled morphologies and biochemicals. It allows the discovery of biomarkers that could influence the clinical progression of cancer⁴⁰. The initial steps include chromatin condensation, swelling of the nuclear membrane, and shrinking of the cytoplasm. These processes are characterized by using protein cleavage, breakdown of DNA, and recognition of apoptotic cells. These processes, triggered by either internal factors like mitochondria or external signals like death receptors, culminate in the final stage known as the execution phase of apoptosis⁴¹. The simplest apoptotic changes could be observed using the microscope.

The intact cell membranes would give an even bright green fluorescence color. In Fig. 3, DNA staining of control cells shows an even bright green fluorescence in control cells, whereas treatment with Palmarosa essential oil shows an uneven color of green mixed with orange. The orange color was caused by ethidium bromide entering the cells due to membrane blebbing on the cells which indicates an apoptosis. In addition, some cells experience nuclear condensation, indicated by the yellowish color in the nucleus. It indicates an early apoptosis event⁴². PEO from the leaves has an IC₅₀ value of 83.41+3.24µg/mL with moderate-level potential against T47D cells (Table 2). However, the observations of cell morphology after treatment showed the phenomenon of cell death (Fig. 2). This was confirmed by the results of DNA staining, which indicated cell death was caused by apoptosis.

The potential of PEO in stimulating cell apoptosis may be due to the geraniol content. Geraniol has been proven to have antitumor potential against colon cancer⁴³, prostate⁴⁴, and great radical-scavenging activity similar to butylated hydroxytoluene (BHT), α- tocopherol, and ascorbic acid⁴⁵. Geraniol could induce programmed cell death in Ishikawa cell lines by affecting various pathways. This resulted in a notable upregulation of genes involved in cell death, such as caspase-3, caspase-8, Bax, cytochrome C, and Fas, while the gene Bcl-2 was downregulated significantly⁴⁶. Bax, Bak, Bcl2, and Bcl-xl are Bcl-2 family proteins. Bax and Bak are proapoptotic proteins, while Bcl-2 and Bcl-xl are antiapoptotic proteins. These proteins mediate the apoptosis mechanism in the intrinsic pathway. During the cell cycle, Bcl2 attaches to the outer mitochondrial membrane, blocking the release of cytochrome C, while Bcl-xl binds to Apaf-1. Cytochrome C and Apaf-1 have a crucial role in the intrinsic pathway of apoptosis by triggering the activation of caspase 9. The cell's ability to survive is counteracted by the cell's death function, which is controlled by Bax and Bak. Bax can attach to the external surface of the mitochondria, it’s cause the release of cytochrome C from the mitochondria. Meanwhile, Bak can bind to Bcl-xl, releasing Apaf-1. If the expression of Bax or Bak is increased and Bcl-2 or Bcl-xl is decreased, cell arrangement will occur toward death through apoptosis⁴⁷.

Besides the intrinsic pathway, geraniol is also able to increase the expression of the Fas gene, which is part of the TNF receptor in the extrinsic apoptosis pathway⁴⁶. In the binding of certain ligands such as Fas ligand, TNF-a and lymphotoxin could change conformation when interacting with special intracellular adapter proteins. After ligand binding, there is a change in the conformation of the intracellular domain and the presence of a death domain such as TRADD (TNFR-Associated death domain), resulting in a change in the conformation of the intracellular domain that can trigger a cell death signal. The final step in this extrinsic mechanism is the activation of Caspase 8, which will initiate apoptosis⁴⁷. Additionally, TRADD could associate with FADD (FAS-associated death domain), which will induce apoptosis through recruitment and cleavage of procaspase 8⁴⁸.

The process of molecular docking involves attaching drug molecules to the receptor's active site where the drug exerts its pharmacological effects. This process involves aligning two molecules in 3D space and can be used to study activities like anti-cancer effects⁴⁹. This study results demonstrated that 4,11-Selinadiene has the best affinity for the progesterone receptor compared to other compounds. This compound was only found in the leaves. In addition, β-Caryophyllene has the highest affinity for ER-α and was highest in leaves PEO, followed by stems and herbs PEO. Our study showed that 4,11-selinadiene and β-Caryophyllene increased the cytotoxic activity in the PEO of C. martinii. β-Caryophyllene is proven to have moderate cytotoxic activity against several breast cancer cell lines, including T-47D (IC₅₀ 160µM), MCF-7 (IC₅₀ 285µM), MDA-MB-231-LM2 (IC₅₀ 230µM), MCF-10A (IC₅₀ 680µM), UFH-001 (IC₅₀ 580µM)^50,51. the β-Caryophyllene content was highest in the leaves PEO, followed by stems and herbs PEO. This sesquiterpene compound may contribute to the cytotoxic activity of PEO.

CONCLUSION:

In conclusion, the major volatile constituents in each part of C. martinii were Geraniol and Geranyl acetate, however, 4,11-Selinadiene and β-Caryophyllene showed the best affinity at progesterone receptors. PEO has a cytotoxic effect on breast cancer cells T-47D and the possibility of inducing apoptosis through various mechanisms. The mechanisms behind these effects still need to be identified. Nevertheless, the current data indicates that PEO may be a pledging option for breast cancer medication.

CONFLICT OF INTEREST:

The authors do not have any personal interests that could influence this research.

ACKNOWLEDGMENTS:

The authors would like to thank Rumah Atsiri Indonesia and Sekolah Tinggi Ilmu Kesehatan Nasional for the funding of this research.

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Received on 20.06.2024 Revised on 06.02.2025

Accepted on 14.06.2025 Published on 08.11.2025

Available online from November 13, 2025

Research J. Pharmacy and Technology. 2025;18(11):5191-5200.

DOI: 10.52711/0974-360X.2025.00749

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