Indonesian Journal of Cancer Chemoprevention.
October 2023 ISSN: 2088Ae0197 e-ISSN: 2355-8989 Cytotoxic Activity of Cambodian Leaves Extract (Plumeria acuminat.
on Breast Cancer Cells and COX-2 Targeted Prediction of Its Chemical Contents Inggita Hasi Rahmah1.
Hayfa Salsabila Harsan1.
Faaza Aulia Rahman1.
Edy Meiyanto1,2.
Ratna Asmah Susidarti1,2* Cancer Chemoprevention Research Center (CCRC).
Fakultas Farmasi.
Universitas Gadjah Mada.
Sekip Utara.
Yogyakarta 55281.
Indonesia Department of Pharmaceutical Chemistry.
Faculty of Pharmacy.
Universitas Gadjah Mada.
Sekip Utara Yogyakarta 55281 Indonesia Abstract Cambodian leaves are suspected to contain stigma sterol which may target CycloOxygenase-2 (COX-.
or Estrogen Receptor (ER) to contribute to its cytotoxic activity on breast cancer cells.
This study aimed to determine the potential of Cambodian leaf compounds and extracts as chemopreventive agents for luminal breast cancer with a molecular target of COX-2.
Ethanol was used to extract the active compound of Cambodian leaves.
The study on chemical activity against COX-2 employed molecular docking with Molecular Operating Environment (MOE) and the cytotoxic property of Cambodian leaf extract (CLE) on T47D was determined using the trypan blue exclusion method.
The extraction yielded as 4.
87% w/w CLE.
Thin layer chromatography showed that Cambodian leaves contain sterol.
Molecular docking confirmed that several sterol compounds have greater affinity to COX-2 than native ligands indicating that they are potent as COX-2 inhibitors.
They are Stigmast-7-en-3-ol.
Lupeol Acetate, and Lupeol carboxylic acid with docking scores of -14.
3874, -13.
8098, and 1045 kcal/mol respectively.
The CLE exhibited cytotoxic activity on T47D cells with an IC50 value of 18 AAg/mL.
Therefore.
CLE has a potential effect as a chemopreventive agent for breast cancer and potentially as a COX-2 inhibitor.
Keywords: Cambodian leaf extract, breast cancer.
COX-2 inhibitor, chemopreventive.
INTRODUCTION
Breast cancer therapy, including luminal breast cancer, often causes numerous side effects, as its mechanism of action lacks selectivity, consequently posing a risk of harming normal cells (Patel, 2.
Consequently, there is a requirement for a chemopreventive compound that can complement chemotherapy medications, leading to a more effective treatment outcome and a reduction in undesirable side effects.
Chemopreventive agents not only obstruct the initiation of cancer but also aid in the recovery of cancer patients, promoting Submitted: October 25, 2021 Revised: March 04, 2024 Accepted: March 07, 2024 Published online: March 26, 2024 Corresponding author: ratna_asmah@ugm.
Rahmah, et al.
, 2023
Indones.
Cancer Chemoprevent.
, 14.
, 199-206
their return to a healthy state.
Chemopreventive substances can be prepared from medicinal plants and their secondary metabolite derivatives, including compounds like flavonoids, flavonols, saponins, terpenoids, alkaloids, steroids, and various others with the potential to inhibit cancer (Yu, et al .
, 2.
Breast cancer has various potential targets in prevention, one of which is COX-2 (Harris, et , 2.
The relationship between COX-2 and chemopreventive agents is an area of interest in cancer research and prevention.
COX-2 is an enzyme that plays a role in the development and progression of certain types of cancer.
Specifically, overexpression of COX-2 has been observed in various cancer types including breast cancer (Harris.
In breast cancer.
COX-2 is overexpressed at every stage of its development (Hugo, et al.
, 2.
Plumeria sp.
, a plant native to Cambodia, had many activities including anti-inflammatory, pro-apoptotic, accuminata.
drastic, and P.
phagidenica, are known for their medicinal activities.
Cambodian contains various compounds, including steroids, flavonoids, tannins, alkaloids, and glycosides triterpenoids, which may function as anti-inflammatory agents (Gupta, et al.
, 2.
There are also indications of additional potentials within Cambodian leaf extract that warrant further exhibits pharmacological activities, including the ability to induce cell apoptosis (Gai, et al.
, 2.
, and the presence of metabolites with anti-tumor properties has been documented (Riaz, et al.
, 2.
Moreover, the methanolic extract of Plumeria acuminata is reported to have significant anti-inflammatory effects in animal models with carrageenaninduced edema, both in acute and chronic settings.
Additionally.
rubra has exhibited cytotoxic activities against breast cancer cells.
Consequently, experimental research is needed to assess the potential of compounds found in Cambodian leaf extract as inhibitors of COX-2 and to investigate the potential molecular mechanisms through which the active compound in Cambodian leaves (P.
may interact with COX-2 enzyme.
MATERIALS AND METHODS
Material Collection.
Determination, and Extraction The Cambodian leaves utilized in this study were sourced from the Gunungkidul regency and determined at the Laboratory of the Department of Pharmaceutical Biology.
Faculty of Pharmacy.
Universitas Gadjah Mada.
Yogyakarta.
Indonesia.
The collected leaves were sliced into small pieces and dehydrated for 10 h at a temperature range of 2847AC using a dehydrator.
Subsequently, the leaves were subjected to pollination with a pollinator and filtered through a 40 mesh sieve.
The extraction procedure in this study used maceration technique.
The extraction process utilized digital scales, a dryer, a rotary evaporator, a grinding machine, a measuring cup, an erlenmeyer, a dehydrator, a grinder, and a maceration flask.
total of 200 grams of Cambodian leaf powder were weighed and placed into an erlenmeyer.
The sample and compound had a ratio of 1:4, and were then dissolved in PA ethanol.
The Cambodian leaves were macerated for 20 h and then filtered using a vacuum press separation technique (Shofi, et al.
Identification of Phytochemical Profile Identification of phytochemical compounds in Cambodian leaves was carried out by thin-layer 5 mg Cambodian leaf extract was dissolved in 500 l so the solution contained 000 g/mL CLE.
The standard solution for TLC was ursolic acid (Sigma.
Missouri St.
Louis.
USA) which was spotted 1 cm from the extractAos spot.
The stationary phase that was used for TLC is the F254 silica gel plate (Merck.
Darmstadt.
German.
The Indonesian Journal of Cancer Chemoprevention.
October 2023 ISSN: 2088Ae0197 e-ISSN: 2355-8989 mobile phase was toluene and ethyl acetate with a ratio of 8:2.
Then the spot result was visualized in visible light which was documented with a camera.
Active Compound Activity Test The active compound test evaluates the inhibitory effect of the active compound through the KNIME (Konstanz Information Mine.
software using the TeachOpenCADD workflow template.
The active compounds tested were stigmast-7-enol, lupeol acetate, lupeol carboxylic acid, and ursolic Data on the structure of the compound to be tested for its inhibitory effect can be obtained by entering the SMILES code for the Cambodian leaf extract from PubChem .
ttps://pubchem.
Molecular Docking Molecular docking was conducted to assess the binding affinity of the four identification compounds present in the Cambodian leaf extract.
We obtained data from the molecular docking simulation and visualized the interactions using the MOE 2010 software, licensed by the Faculty of Pharmacy at UGM.
We acquired the COX-2 model with the code 3LN1 from PDB.
The MOE provides a direct description of the compound structure to be docked by entering the SMILES code for the CLE compound from PubChem .
ttps://pubchem.
In the molecular docking procedure, the validation of the molecular docking technique takes This validation phase involves reattaching the original ligand to the receptor, which was initially The docking method is deemed successful if it produces Root Mean Square Deviation (RMSD) value of 2 (Puratchikody, et al.
, 2.
Cytotoxic Assay with Direct Counting The cytotoxic assay utilized a 24-well plate, micropipette, microtube, and hemocytometer.
Each well of the 24-well plate was seeded with 2x104 T47D cells and incubated for 24 h in DulbeccoAos Modified Eagle Medium (DMEM) at 37AC, with CO2 levels of 0.
A cambodian leaf extract test solution was added to each well.
After 24 h of incubation, the cells were washed 2-3 times with PBS in each well.
Then, the Trypsin-EDTA solution was evenly added and incubated in the incubator for 3 minutes.
Next, media was added to inactivate The cells were resuspended using a micropipette until they were released.
Finally, the cells were transferred into a new sterile microtube, and trypan blue was added to the cell suspension Cell suspension treated with trypan blue was pipetted into a hemocytometer and counted under a light or inverted microscope using a counter.
Viable cells .
were then counted under a light microscope.
The number of viable cells in each sample treatment was normalized to the number of untreated .
cells, and the data was plotted on a concentration vs.
% cell viability graph.
The IC50 value was calculated using this data.
Analysis Method Yield Calculation The Cambodian leaf extract that has been obtained is weighed to determine the total yield using the following formula:
%Extract yield=(Cambodian Leaf Extract gained weight .
/sample weight .
)x100% Compound Interaction Strength Four compounds were collected as possible binding interactions on COX-2.
The compound that had a lower docking score than the native ligand COX-2 was concluded as the compound with the greatest interaction strength with the COX-2.
Cytotoxic assay The data was obtained in the form of variations in the concentration of Cambodian leaf extract and the viability cells.
The percentage of cell viability was calculated using the data of cell viability results for each concentration compared Rahmah, et al.
, 2023
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Cancer Chemoprevent.
, 14.
, 199-206
with the results of cell viability in the control group.
Then calculated the concentration of IC50 using the linear regression method between the concentration of the extract and the percentage of living cells.
ll viability=[(Absorbance by treatmentAbsorbance control medi.
/(Absorbance control cells-Absorbance control medi.
]x100% RESULTS Determination.
Extraction, and Chemical Detection The cambodian leaves were obtained from the Gunungkidul area for the extraction process and based on the determination test showed that the sample was indeed Plumeria acuminate.
The extraction method was maceration and obtained weight of 9.
75 grams and an extract yield 87% w/w.
The Cambodian leaf extract (CLE) Figure Cambodian phytochemical profile.
The cambodian leaf extract (A) was obtained by maceration using ethanol PA .
87% yield The extract was identified by thin layer chromatography (B.
and showed the same stain (Rf value : 4.
as Ursolic Acid Standard (B.
and also gave the positive reaction with Liebermann burchard reagent.
Both stigmast-7-en-3-ol and ursolic acid showed the similar chemical structure (C).
is a blackish green thick extract (Figure 1A).
This extract was analyzed by thin layer chromatography (TLC).
Reagan standard for TLC used ursolic acid that sterolAos derivatives.
The result showed that cambodian leaves extract had the same Rf value with ursolic acid and also gave a positive reaction with Liebermann burchard reagent (Figure 1B-C).
The Rf value is 4.
It means that Cambodian leaves contain sterol.
This result confirmed the previous research that the Cambodian leaves contain four phytochemical compounds, including Stigmast7-en-3-ol.
Lupeol acetate, ursolic acid, and lupeol carboxylic acid (Farooque, et al .
, 2.
Active Compound Activity Test We then analyzed the main compounds, stigmast-7-en-3-ol, lupeol acetate, ursolic acid, and lupeol carboxylic acid (Farooque, et al.
, 2.
, in Cambodian leaves using machine learning KNIME to obtain compounds that were predicted to have inhibitory activity against COX-2.
The results of screening potential compounds of cambodian leaf extract which have activity as COX-2 inhibitors with KNIME resulted in an overall accuracy >70% which is 0.
978 and the ROC curve with p-value >0.
7 which is 0.
907 which means that the method used is valid.
The results of the analysis showed that all compounds have inhibitory activity against COX-2 (Table .
Table 1.
Potential compounds of cambodian leaf extract which have activity as COX-2 Compound Inhibition Prediction Lupeol acetate Stigmast-7-en-3-ol Lupeol carboxylic acid Ursolic acid Indonesian Journal of Cancer Chemoprevention.
October 2023 ISSN: 2088Ae0197 e-ISSN: 2355-8989 Molecular Docking Prediction of compound activity is also done using molecular docking where compounds with low bond energies will be easier to bind to the proteins.
The test of the interaction strength of compounds in Cambodian leaf extract with COX2 was carried out by molecular docking through the MOE application.
Based on the results of the molecular docking test, the compounds that have the lowest docking scores are stigmast-7-en-3-ol, lupeol acetate, lupeol carboxylic acid, and ursolic acid (Table .
Celecoxib (CEL) was used as a native ligand of COX-2 in performing molecular docking and showed a docking score of -13.
3398 kcal/ mol (Table .
CEL, celecoxib ligand is a COX-2 inhibitor that is used as a reference for comparison with the test compound.
If the docking score of the Table 2.
Docking scores of compounds against COX-2.
Ligand Score .
cal/mo.
CEL .
ative ligan.
Stigmast-7-en-3-ol Lupeol acetate Lupeol carboxylic acid Ursolic acid compound being tested has a more negative value than the ligand, it can be said that the compound being tested has a strong potential to become a COX-2 inhibitor.
In addition to the docking value, the estimated interaction between the test compound and native ligand with COX-2 was also obtained.
Figure 2.
The 2D and 3D visualization of the interaction for several compounds in Cambodian leaf extract with COX-2.
Molecular docking was performed using MOE as described in the methods.
The interaction visualization of native ligand CEL (A) of COX2 compared to the interaction of Lupeol carboxylic acid (B).
Lupeol Acetate (C).
Stigmast-7-en-3-ol (D) to COX-2.
The results showed that three compounds were potent as COX-2 inhibitors, namely Stigmast7-en-3-ol.
Lupeol Acetate, and Lupeol Carboxylic Acid.
The three compounds illustrated the interactions between COX-2 and related compounds showing the tight interaction at the same site with the native ligand with the lower docking score (Figure .
Rahmah, et al.
, 2023
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Cancer Chemoprevent.
, 14.
, 199-206
Figure 3.
The cytotoxic effect of Cambodian leaf extract on T47D.
The cytotoxic activity of Cambodian leaf extract on T47D cells was assessed using the direct counting for 24 h.
Cambodian leaf extract at the respective concentrations was tested for its cytotoxicity on T47D cells using the direct counting method.
Cytotoxic Assay with Direct Counting A cytotoxic assay was conducted to determine the cytotoxic ability of Cambodian leaf extract against T47D breast cancer cells which were assessed with the IC50 parameter.
The IC50 value is the concentration of the test material that gives 50% growth inhibition to living cells.
The result showed that CLE suppressed the growth of T47D cells progressively with the IC50 value of 18 AAg/mL (Figure .
This means that Cambodian leaf extract has a strong inhibitory activity on the growth of luminal breast cancer cells.
DISCUSSION