INTRODUCTION:

Cancer incidence continues to increase despite significant progress in recent years in the development of appropriate therapies and prevention strategies. The International Agency for Research on Cancer (IARC) has projected a nearly twofold increase in new cancer cases and deaths influenced by aging and population growth by 2030¹.

Research shows that the burden of cancer is not only on patients but also on caregivers^2,3 which significantly affects socio-economic development. Cancer is the second leading cause of death in the world after cardiovascular disease. Deaths due to cancer account for 2-3% of annual deaths recorded worldwide^4,5. According to the Global Cancer Observatory data from 2020, Indonesia's cancer incidence rate of 144.1 per 100,000 population ranks 8th in Southeast Asia and 23rd in Asia. Breast cancer has the highest incidence, with 24.1 cases per 100,000 population and an average mortality rate of 8.2 per 100,000 population, followed by cervical cancer with an incidence rate of 13.4 per 100,000 population and an average mortality rate of 7.7 per 100,000 population⁶. Current chemotherapy approaches to cancer have many challenges, including the emergence of resistance, side effects, and even the emergence of relapse after therapy⁷. The discovery of new anticancer compounds can be done by looking at the mechanisms of the cell cycle and apoptosis. Dysregulation of the cell cycle is regarded as the initial step in carcinogenesis, as seen in renal cell carcinoma, where it plays a critical role in tumour invasion and metastasis. Identification of changes in progression through the cell cycle is also important during drug testing. It provides important data that help explain the mechanism of action of these drugs. Therefore, the techniques used must allow detection of various aspects of the life cycle of the cells under study, such as the total length of the cell cycle, the length and variability of the different cell cycle phases, the presence of quiescent or senescent cells, or the presence of dead cells⁸. Apoptosis (programmed cell death) plays an important role in embryonic development, homeostasis, immune system function and wound repair⁹. The ability to avoid apoptosis induction has been used by cancer cells to defend against the body's defense mechanisms¹⁰. The molecular mechanisms involved in cancer cell apoptosis have been well documented and involve specific biochemical events such as DNA fragmentation, chromatin condensation, degradation of cell organelles and protein cleavage¹¹.

New chemotherapy candidates in the active compounds of Opuntia elatior Mill (OpE) extract have the potential as cytotoxic agents through cell cycle and apoptosis approaches¹². Previous studies have shown that Opuntia ficus indicia extract contains various phenolic metabolite compounds, flavonoids, tannins, alkaloids, and anthocyanins that have anticancer activity against various types of cancer cells¹³. Phenolic active compounds and kaempferol from Opuntis ficus-indica L. Mill extract have strong cytotoxic activity through apoptosis induction in PC3 prostate cancer cells¹⁴. Opuntia spp. is traditionally used in conditions of obesity, cardiovascular disease, inflammation, diabetes and cancer¹⁵. The antioxidant activity of Opuntia cochenillifera (L.) Miller can inhibit free radicals and is used in cancer treatment. The phytochemical content found in the plant is phenolic acid, flavonoids, betalains and betacyanins¹⁶. Opuntia ficus-indica has a phytochemical content similar to OpE. Phenolic and flavonoid components that have aromatic rings can bind free radicals so that they are used as antioxidants. Anticancer activity was tested using trypan blue with the Ehrlich Ascites Carcinoma Cells (EACC) technique. The results showed that extracts with chloroform and ethanol solvents had strong antioxidant and anticancer activities. This is due to the 17-hydroxy betanin component, total phenolics such as tannins, and flavonoids contained in the extract¹⁷.

Molecular docking is a highly effective and widely used method for analysing drug interactions with proteins¹⁸. This method is valuable for uncovering the binding conformations and interaction profiles of drugs with critical breast cancer targets, highlighting their therapeutic potential¹⁹. As a comprehensive approach, this computational drug design method is considered more cost-effective and time-efficient compared to traditional methods in cancer treatment. Additionally, it offers the advantage of enhancing productivity and quality in pharmaceutical research²⁰.

This study aims to isolate secondary metabolites with potential anticancer activity and investigate their mechanisms of action on the cell cycle and apoptosis in cancer cells. Furthermore, in silico analysis was conducted on the isolates to evaluate their molecular interactions with specific receptors.

MATERIALS AND METHODS:

Extraction and Fractionation:

Opuntia elatior Mill (OpE) was obtained from Balongmulyo village, Krajan sub-district, Rembang regency, Central Java province. Plant determination was carried out by the Plant Taxonomy Laboratory, Faculty of Mathematics and Natural Sciences, Universitas Negeri Semarang with voucher number 530. OpE was dried indirect sunlight to produce simplicia powder. Extraction of 100 grams of OpE powder was carried out by maceration using 1000 mL of 96% ethanol. Maceration was carried out for 1x24 hours and repeated 3 times with the replacement of new solvents. The extracted filtrate was evaporated with a rotary evaporator at a temperature of 50°C to obtain a thick ethanolic extract (EE). Fractionation was carried out on 1 gram of EE dissolved in 10 mL of n-hexane. The mixture was stirred using a vortex mixer for 5 minutes and then separated using a centrifuge at a speed of 3000 rpm for 5 minutes. The supernatant was separated from the insoluble part and then dried in a porcelain dish to obtain the n-hexane fraction (HF). The insoluble part was then fractionated successively with ethyl acetate and methanol using a similar procedure to produce the ethyl acetate fraction (EAF) and methanol fraction (MF)^21,22.

Isolation of Secondary Metabolites from Ethyl Acetate Fraction (EAF):

Ethyl Acetate Fraction (EAF) was further separated using vacuum liquid chromatography (VLC). A sintered glass column was prepared to be filled with silica gel 60 PF₂₅₄, then vacuumed and the top was precipitated. The weighed EAF (1.207) was ground together with silica until homogeneous to produce sample powder. The top layer of the column was filled with sample powder to be further vacuumed and precipitated again. The VLC fraction process was carried out using the following mobile phase compositions: n-hexane 100%, n-hexane: ethyl acetate (3:1), n-hexane: ethyl acetate (1:1), n-hexane: ethyl acetate (1:3), ethyl acetate 100%, and chloroform: methanol (1:1). Sub fraction G (0.4471) was further separated using preparative thin layer chromatography (P-TLC) on a 20 x 20 cm glass plate with a mobile phase of n-hexane: ethyl acetate (1:3). The purity test of the isolate used three different types of mobile phases, namely n-hexane: ethyl acetate (1:3), chloroform: ether (9:1), and chloroform: acetone (9:1).

Isolate Characterization:

Determination of the functional group of the isolate was carried out using Agilent FTIR through IR spectra analysis at the wave numbers that appeared (cm^-1). Determination of the type and number of hydrocarbons was carried out using NMR, both ¹H-NMR and ¹³C-NMR by looking at the chemical shift (ppm) of the NMR spectra that appeared²³.

Anticancer Assays:

Cancer cell lines (HeLa, T47D, 4TI, MCF-7, and WiDr) in 80-90% confluent conditions for harvesting were taken from the CO₂ incubator. The number of cells was counted, and cell dilutions were made with complete medium. Cells with a density of 10,000 cells/well were transferred into a 96-well plate, 100 µl each. The condition of the cells was observed with an inverted microscope to see the distribution of cells. The cells were incubated in the CO₂ incubator overnight^24–26. After the cells returned to normal for 1x24 hours, a sample concentration of 500 µg/mL was made (including cell control and media control). The microplate containing the cells was taken from the CO₂ incubator, then the cell media was discarded. A sample of 100 µL was put into the well (triplicate) and incubated in the CO₂ incubator for 24 hours. The cell media was discarded and 100 µl of MTT reagent was added to each well. The cells were incubated for 4 hours in a CO₂ incubator. 100 µl of DMSO was added and the microplate was wrapped in aluminium foil, shaken and incubated in the dark at room temperature for 15 minutes. The absorbance of each well was read with a microplate reader at a wavelength of 595 nm^27–29. The data is statistically tested using t-test to see if there is significant difference between groups.

Effect of Isolates on Cell Cycle and Apoptosis of T47D Cells:

A total of 3 ml of T47D cell suspension with a density of 2x10 cells/well was distributed into the wells of a 6-well plate, then incubated in a CO₂ incubator for 1x24 hours. The medium was discarded and then the isolates were added at concentrations of 700, 350, and 175 µg/mL and incubated again overnight. Cell morphology was observed under a microscope and then the cells were harvested. The supernatant was collected in a sterile conical tube, the wells were washed 3 times with PBS and also collected. Cells were released from the plate by adding trypsin-EDTA (0.05%) and then collected in a tube. Furthermore, the plate was washed again with the medium 2 times and then collected. The tube was centrifuged at 3000 rpm for 3 minutes, then the supernatant was discarded, and the sediment was resuspended with 1 ml of PBS. The cell suspension was centrifuged again at 3000 rpm for 3 minutes. The supernatant was discarded, the precipitate was resuspended with 250 µl of staining solution (propidium iodide 50 µg/ml in PBS), incubated at room temperature and ready for distribution analysis using a flow cytometer²⁹. Apoptotic cell population was determined using PI-Annexin V assay (Annexin V-FITC Apoptosis Detection Kit). A total of 2 x 10 cells/well were transferred into a 6-well plate. Cells were treated with the isolate for 24 hours. Cells were harvested, added with 120 µL of binding buffer, plus reagent with PI-Annexin V each 2 µL, and incubated at room temperature in the dark for 5 minutes. Cell suspensions were analysed using flow cytometry^30,31.

In Silico Analysis of Opuntiol:

The molecular docking validation method was performed as described by Mani et al³². The Estrogenic Receptor (ER) and Progesterone Receptor (PR) structures were retrieved from the Protein Data Bank (PDB), with PDB IDs 1A52³³ and 1A28³⁴, respectively. For docking studies, chain A of each receptor structure was selected using AutoDockTools-1.5.7. To validate the docking procedure, the co-crystallized ligands of each receptor were removed, and the receptors were re-docked. Tamoxifen and Opuntiol structures were obtained from PubChem. Tamoxifen served as a reference compound, while Opuntiol was the experimental ligand under investigation.

The active sites of estrogen receptor (ER) and progesterone receptor (PR) were identified using the Computed Atlas of Surface Topography of the Universe of Protein Folds (CASTpFold)³⁵. The amino acid residues constituting the predicted active sites were subsequently validated by comparison with the active site residues observed in the experimentally determined crystal structures of ER and PR in complex with their respective biological ligands, estradiol and progesterone.

Molecular docking of the selected ligands with their respective receptors was conducted using AutoGrid4 and AutoDock4³⁶. The ligand-receptor combinations examined in this study included estradiol, tamoxifen, and opuntiol docked with the estrogen receptor (ER), and progesterone, tamoxifen, and opuntiol docked with the progesterone receptor (PR). Docking simulations were performed within a defined grid box size of 30 × 30 × 30 Å, centered on the co-crystal structure to encompass all residues forming the receptor's active site pocket. The docking process generated a list of ligand poses and their corresponding binding affinities as output data. The results were further analyzed using Discovery Studio 2024, which enabled visualization of key interactions, such as hydrogen bonds and hydrophobic interactions, between the ligands and target proteins.

RESULT AND DISCUSSION:

Separation and purification of isolates:

Vacuum liquid chromatography (VLC) is carried out with the aim of obtaining a simpler separation of compounds. Before the VLC process is carried out, the chromatography column is prepared first. The solvents used in the EAF liquid vacuum chromatography process are solvents that have different levels of polarity, starting from solvents with low polarity to solvents that are more polar. Each solvent comparison is made as much as 100 mL which is used for the elution process four times. Every twenty-five milliliters of solvent that is put into the column is eluted with the help of a vacuum pump and is stopped when there is no more solvent dripping into the collection flask³⁷. The results of the separation using VLC obtained 23 fractions. Based on the chromatogram profile of the TLC results of 23 fractions with the mobile phase n-hexane: ethyl acetate (1:3), there are similarities in the chromatogram profile so that the fractions of 23 fractions were combined into 8 fractions. Based on Figure 1, it shows the TLC results of the combined fractions of the VLC EAF results (subfractions A-H) after being sprayed with anisaldehyde-sulfuric acid spot detector. The combined fractions were weighed and the percentage yield obtained was calculated as listed in Table 1.

Figure 1. TLC Profile of subfraction A-H. stationary phase: silica gel F₂₅₄. mobile phase: n-hexane:ethyl acetate (1:3). anisaldehyde-sulfuric acid spot appearance.

Subfraction G is the selected subfraction used for P-TLC. Subfraction G was chosen because it is easy to separate and has the highest yield among other subfractions. Subfraction G used for P-TLC was 447.1 mg. Separation of subfraction G with P-TLC was divided into three parts, namely G1, G2, and G3. From the results of P-TLC, the yield of isolate G1 was 61.015%, isolate G2 was 6.218%, and isolate G3 was 5.457%.

Table 1. A-H subfraction profile of EAF

Subfraction	Merging Fraction	Subfraction Weight (gram)	Yield (%)
A	1, 2, 3, 4, 5, 6	0.1364	11.30
B	7, 8, 9, 10	0.0558	4.42
C	11	0.0221	1.83
D	12	0.0213	1.76
E	13, 14	0.0928	7.69
F	15, 16, 17, 18	0.1555	12.88
G	19, 20	0.4471	37.04
H	21, 22, 23	0.1444	11.96

The TLC profile of the G subfraction separation results obtained spots with Rf 0.6 which were easily separated in isolates G2 and G3. These spots were only visible under UV light at 254 nm and in the anisaldehyde-sulfuric acid spot-detecting reagent which was identified as red (Figure 2). These spots were not visible under UV light at 366 nm. Isolates G2 and G3 were separated again to obtain purer isolates by referring to the results of observations using UV light at 254 nm³⁸.

Figure 2. TLC profile of isolates G1, G2, and G3. stationary phase: silica gel F₂₅₄. mobile phase: n-hexane:ethyl acetate (1:3). detection under UV₂₅₄ (A), under UV₃₆₆ (B) and anisaldehyde-sulfuric acid spot detection (C).

Characterization of isolate structure:

Based on the results of ¹H and ¹³C-NMR analysis in CDCl₃ which can be seen in Table 2, it is known that this sample is identical to the Opuntiol compound where the 3 most downfield carbons (C-1, C-3 and C-5) refer to Cq-sp² olefinic, two carbons (C-2 and C-4) refer to CH-sp² olefinic and two carbons (C-6 and C-7) refer to -C-O-C- sp³-aliphatic. In terms of correlation or relationship based on the coupling constant, it is also seen that H-2 and H-4 have values of 2.3 Hz and 2.25 Hz respectively, which means that these two protons are neighbors in a -meta manner in the aromatic system. The hydroxyl group in Opuntiol is also confirmed by IR spectra analysis, which shows an absorption band with moderate intensity at a wave number of 3373 cm^-1. In addition, the appearance of an absorption band at wave number 1058 cm^-1 with very strong intensity strengthens the presence of a C-O-C bond in the ether group³⁹.

Table 2. Correlation of ¹H-NMR and ¹³C-NMR of Opuntiol Isolates

No	¹³C (ppm)	¹H (mult., J in Hz, ∑H)
1	164.12	-
2	88.39	5.45 (d, 4J = 2.3 Hz, 1H)
3	171.15	-
4	99.05	6.09 (d, 4J = 2.25 Hz, 1H)
5	163.19	-
6	61.10	4.42 (s, 2H)
7	55.99	3.82 (s, 3H)

Anticancer activity of ethanolic extract (EE) and Opuntiol isolate:

Opuntiol isolate has the highest anticancer activity against T4D cancer cells compared to other types of cancer cells with a percentage inhibition value of 20.61 ± 3.23% at a sample concentration of 500 µg/mL presented in Table 3. This value is not significantly different from the results of the percentage inhibition of the EE. Based on this phenomenon, it can be seen that the EE has the active compound Opuntiol which acts as a marker compound as well as an active compound if further developed into a standardized pharmaceutical preparation⁴⁰.

Table 3. Inhibition percentage (%) of anticancer activity of ethanolic extract (EE) and Opuntiol Isolate at a concentration of 500 µg/mL

Cancer Cell Lines	Ethanolic Extract (EE)	Opuntiol Isolate
HeLa	10.49 ± 2.97^a	16.98 ± 5.67^a
T47D	21.81 ± 4.80^a	20.61 ± 3.23^a
4TI	21.61 ± 5.93^a	19.87 ± 2.02^a
MCF-7	24.03 ± 4.97^a	3.34 ± 2.23^b
WiDr	40.93 ± 1.64^a	13.07 ± 0.53^b

Note: different letters indicate no significant difference between EE and the oputiol isolate (p value < 0.05, independent sample t-test)

Mechanism of action of Opuntiol isolate on T47D cell cycle:

Based on cell cycle analysis using flow cytometry method, it shows that administration of Opuntiol isolate in various concentrations can inhibit the T47D cell cycle in the G0/G1 phase. The concentration of Opuntiol isolate of 700 µg/mL causes 67.8% of cells to accumulate in the G0/G1 phase, indicating the highest percentage of the G0/G1 phase compared to other concentrations and controls, which can be shown in Figure 3. The higher percentage of T47D cell accumulation compared to the control indicates that the cells are unable to move to the next phase in the cell cycle⁴¹. This study is in line with previous studies that found that there was inhibition of the cell cycle and the occurrence of cycle arrest in the G0/G1 phase of U87MG cancer triggered by Opuntia humifusa extract. The study revealed that induction of Opuntia humifusa extract triggered the occurrence of cell cycle inhibition and cycle arrest caused by an increase in reactive oxygen species (ROS) which led to damage to cancer cell DNA⁴².

Figure 3. Histogram (A) and cell distribution number (B). the concentration of opuntiol isolates was 175, 350, 700 µg/mL. cell cycle analysis was performed once.

Mechanism of action of Opuntiol isolate on T47D cell apoptosis:

Opuntiol isolate was able to induce apoptosis at a concentration of 175 µg/mL by 12.9%. The increase in the percentage of cells undergoing apoptosis increased along with the increase in sample concentration. Figure 4 shows that T47D breast cancer cells undergo apoptosis by emitting fluorescent light. Immuno/DNA observations show that T47D cancer cells experience morphological changes in cellular. Wyllie (2010) revealed that changes in cellular morphology due to this apoptosis mechanism can occur in several stages, namely shrinkage of cell density, condensation, and fragmentation of cell chromatin and fragmentation of cell nuclei⁴³. Previous studies have reported that trans-taxifolin and dihydrokaempferol are flavonoid compounds in Opuntia humifusa that can reduce the survival of human bladder and colon cancer cells through apoptosis induction. Both compounds inhibit cell proliferation by stopping the G1 phase which leads to decreased expression of cyclin D1, Cdk4, and phospho-Rb proteins, as well as increased expression of p21^WAF1/Cip1 and p53 in mRNA which will cause apoptosis in cancer cells. Treatment of Opuntia ficus-indica bark extract to K562 cancer cells also induces the release of cytochrome C into the cytosol which results in decreased Bcl-2 regulation and reduced membrane potential which leads to apoptosis^44,45.

Figure 4. Histogram (A) and number of apoptotic cell distribution (B). The concentration of opuntiol isolates was 175, 350, 700 µg/mL. cell cycle analysis was performed once.

In Silico Analysis:

Molecular docking is a key technique in in-silico drug discovery, enabling the prediction of optimal binding conformations between small molecule ligands and their target binding sites. This study focuses on estrogen receptor (ER) and progesterone receptor (PR), two critical proteins associated with breast cancer and found T47D cell line⁴⁶. Using molecular docking, we investigated the interactions of these targets with Opuntiol.

As an initial investigation, we examined Estrogenic Receptor (ER) and Progesterone Receptor (PR) interactions using three distinct compounds. This included a control set comprising the natural biological ligands estradiol and progesterone for ER and PR, respectively, alongside tamoxifen, a widely used anti-breast cancer drug specifically designed to target these receptors. The active sites of both receptors were identified using CASTpFold. Contributing residues forming the active site pocket illustrated in Figure 5 for PR and Figure 6 for ER. The grey regions represent the amino acid residues, while the red regions highlight the binding pockets.

Figure 5. Active site prediction of PR

Figure 6. Active site prediction of ER

Molecular docking of the selected ligands was conducted against the predicted active site pockets of their respective receptors using AutoGrid4 and AutoDock4. The docking outcomes were expressed as free binding energy values, along with their corresponding conformations, as presented in Table 4.

The control ligands were initially docked with their respective receptors, yielding binding affinities of -11.63 kcal/mol (PR with progesterone), -9.56 kcal/mol (PR with tamoxifen), -10.17 kcal/mol (ER with estradiol), and -9.44 kcal/mol (ER with tamoxifen). In contrast, docking of opuntiol resulted in binding affinities of -4.59 kcal/mol with ER and -4.51 kcal/mol with PR. Although these values exhibit a noticeable difference compared to the controls, further analysis was conducted to evaluate the bond-forming tendencies of the ligands.

Table 4. Docking results of ligands to Progesterone and Estrogen Receptors

Receptor	Ligands	Binding Energy (kcal/mol)
PR	Progesterone	-11.63
	Tamoxifen	-9.56
	Opuntiol	-4.59
ER	Estradiol	-10.17
	Tamoxifen	-9.44
	Opuntiol	-4.51

CONCLUSION:

In summary, opuntiol isolated from Opuntia elatior Mill (OpE) demonstrates significant potential as a candidate for chemotherapy. The compound has been identified and characterized using Infrared Spectrometry, H-NMR, and C-NMR, confirming its identity as opuntiol. The isolate exhibits activity against the T47D breast cancer cell line, with an inhibition rate of 20.61% ± 3.23 at a concentration of 500 µg/mL and can inhibit the T47D cell cycle in the G0/G1 phase. In addition, opuntiol isolates can induce apoptosis with a minimum sample concentration of 175 µg/ml. Molecular docking analysis revealed binding affinities of -4.59 kcal/mol with estrogen receptor (ER) and -4.51 kcal/mol with progesterone receptor (PR). Opuntiol interacts with the PR via hydrogen bonds with Gln725 and Arg766 and hydrophobic interactions with Met756 and Met759, mimicking the binding mechanism of progesterone. Similarly, it binds to ER through hydrogen bonds with Glu353 and Arg394 and hydrophobic interactions with Leu387, Leu391, and Phe404, resembling the binding pattern of estradiol.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

ACKNOWLEDGMENTS:

This study was supported by a grant under the Fundamental Research - Reguler scheme (grant no. 070/E5/PG.02.00.PL/2024 and 114.12.6/UN37/PPK.10/2024) by the Indonesian Ministry of Education, Culture, Research and Technology.

REFERENCES:

1. Stewart BW, Bray F, Forman D, et al. Cancer prevention as part of precision medicine: “Plenty to be done.” Carcinogenesis. 2016; 37(1). doi:10.1093/carcin/bgv166

2. Ge L, Mordiffi SZ. Factors Associated With Higher Caregiver Burden Among Family Caregivers of Elderly Cancer Patients: A Systematic Review. Cancer Nurs. 2017; 40(6): 471-478. doi:10.1097/NCC.0000000000000445

3. Johansen S, Cvancarova M, Ruland C. The Effect of Cancer Patients’ and Their Family Caregivers’ Physical and Emotional Symptoms on Caregiver Burden. Cancer Nurs. 2018; 41(2): 91-99. doi:10.1097/NCC.0000000000000493

4. American Cancer Society. Breast Cancer Facts and Figures 2007-2008. American Cancer Society, Inc.; 2007.

5. Antoniou A, Pharoah PDP, Narod S, et al. Average risks of breast and ovarian cancer associated with BRCA1 or BRCA2 mutations detected in case Series unselected for family history: a combined analysis of 22 studies. Am J Hum Genet. 2003; 72(5): 1117-1130. doi:10.1086/375033

6. WHO. Cancer Insiden in Indonesia. 2020; 858: 1-2.

7. Mooradian MJ, Sullivan RJ. Immunomodulatory effects of current cancer treatment and the consequences for follow-up immunotherapeutics. Future Oncol. 2017; 13(18): 1649-1663. doi:10.2217/fon-2017-0117

8. Ligasová A, Frydrych I, Koberna K. Basic Methods of Cell Cycle Analysis. Int J Mol Sci. 2023;24(4). doi:10.3390/ijms24043674

9. Strasser A, O’Connor L, Dixit VM. Apoptosis Signaling. Annu Rev Biochem. 2000; 69(1): 217-245. doi:10.1146/annurev.biochem.69.1.217

10. Lowe SW, Lin AW. Apoptosis in cancer . Carcinogenesis. 2000; 21(3): 485-495. doi:10.1093/carcin/21.3.485

11. Elmore S. Apoptosis: a review of programmed cell death. Toxicol Pathol. 2007; 35(4): 495-516. doi:10.1080/01926230701320337

12. Abdul Ahad H, Abdalla Abdelaziz MA, Bakrey H, Abdu A, Elshiekh Mohamed YB, A. Noureldeen Amal. Profession for the Magnitude of Temperature and Exposure time on Opuntia elatior cladode extraction on percent yield using design expert software. Res J Pharm Technol. 2023; 16(12): 5760-5764. doi:10.52711/0974-360X.2023.00932

13. El-Beltagi H, Mohamed H, Elmelegy A, Eldesoky S, Safwat G. Phytochemical screening, antimicrobial, antiaxidant, anticancer activities and nutritional values of cactus (opuntia ficus indicia) pulp and peel. Fresenius Environ Bull. 2019; 28: 1534-1551.

14. Heikal A, Abd El-Sadek ME, Salama A, Taha HS. Comparative study between in vivo- and in vitro-derived extracts of cactus (Opuntis ficus-indica L. Mill) against prostate and mammary cancer cell lines. Heliyon. 2021; 7(9): e08016. doi:10.1016/j.heliyon.2021.e08016

15. Del Socorro Santos Díaz M, Barba de la Rosa AP, Héliès-Toussaint C, Guéraud F, Nègre-Salvayre A. Opuntia spp.: Characterization and Benefits in Chronic Diseases. Oxid Med Cell Longev. 2017; 2017: 8634249. doi:10.1155/2017/8634249

16. Monrroy M, García E, Ríos K, García JR. Extraction and Physicochemical Characterization of Mucilage from Opuntia cochenillifera (L.) Miller. J Chem. 2017; 2017(1): 4301901. doi:https://doi.org/10.1155/2017/4301901

17. M. Abou Elella F, Farouk Mohammed Ali R. Antioxidant and Anticancer Activities of Different Constituents Extracted from Egyptian Prickly Pear Cactus (Opuntia ficus-indica) Peel. Biochemistry and Analytical Biochemistry. 2014; 03(02). doi:10.4172/2161-1009.1000158

18. Akash S, Kumer A, Rahman MdM, et al. Development of new bioactive molecules to treat breast and lung cancer with natural myricetin and its derivatives: A computational and SAR approach. Front Cell Infect Microbiol. 2022; 12. doi:10.3389/fcimb.2022.952297

19. Ayub MA, Tyagi AR, Srivastava SK, Singh P. Quantum DFT analysis and molecular docking investigation of various potential breast cancer drugs. J Mater Chem B. 2025; 13(1): 218-238. doi:10.1039/D4TB01803F

20. Arjmand B, Hamidpour SK, Alavi-Moghadam S, et al. Molecular Docking as a Therapeutic Approach for Targeting Cancer Stem Cell Metabolic Processes. Front Pharmacol. 2022; 13. doi:10.3389/fphar.2022.768556

21. Fernandese Mendonca LM, Bhimrao Joshi A, Bhandarkar A, Joshi H. Antioxidant, Antiproliferative, Pro-apoptotic and cell cycle arrest properties of crude extract and biofractions of Hybanthus enneaspermus Linn. to combat breast cancer. Res J Pharm Technol. 2023; 16(9): 4127-4134. doi:10.52711/0974-360X.2023.00675

22. Eden WT, Rakainsa SK, Widhihastuti E. Antioxidant and Anticancer Activity of Opuntia elatio Mill. Ethanol Extract and the Fractions: http://www.doi.org/10.26538/tjnpr/v7i12.27. Tropical Journal of Natural Product Research (TJNPR). 2023; 7(12 SE-Articles): 5558-5565. https://tjnpr.org/index.php/home/article/view/3193

23. Asha S, Thirunavukkarasu P, Mohamed Sadiq A. Preparative HPLC Method for the Isolation of Compounds from Euphorbia hirta Linn. Ethanol Extract. Res J Pharm Technol. 2018; 11(6): 2541-2545. doi:10.5958/0974-360X.2018.00469.9

24. Priya K, Setty MM, Pai KSR. In vitro and In vivo Evaluation of Anticancer Properties of Clerodendrum indicum (L.) Kuntze in Colon Cancer. Res J Pharm Technol. 2020; 13(5): 2321. doi:10.5958/0974-360X.2020.00418.7

25. Kuswanti N, Widyarti S, Widodo W, Rifa’i M. Cytotoxicity of Ethanolic Extract of Plumeria rubra L. Stem bark to Cancer Cells and Lymphocytes. Res J Pharm Technol. 2018; 11(12): 5545. doi:10.5958/0974-360X.2018.01009.0

26. Al-Kubaisi ZA, Al-Shmgani HS, Salman MJ. Evaluation of In vivo and In vitro protective effects of quercetin on lipopolysaccharide-induced inflammation and cytotoxicology. Res J Pharm Technol. 2020; 13(8): 3897. doi:10.5958/0974-360X.2020.00690.3

27. Fadholly A, Ansori ANM, Utomo B. Anticancer Effect of Naringin on Human Colon Cancer (WiDr Cells): In Vitro Study. Res J Pharm Technol. 2022; 15(2): 885-888. doi:10.52711/0974-360X.2022.00148

28. Kurnijasanti R, Fadholly A. Cytotoxic Effect of Capsicum annum L. extract on T47D Cells: In vitro Study. Res J Pharm Technol. 2021; 14(6): 3389-3392. doi:10.52711/0974-360X.2021.00589

29. Eden WT, Wahyuono S, Cahyono E, Astuti P. Phytochemical, Antioxidant, and Cytotoxic Activity of Water Hyacinth (Eichhornia crassipes) Ethanol Extract. Tropical Journal of Natural Product Research. 2023; 7(8): 3606-3612. doi:10.26538/tjnpr/v7i8.5

30. Shabana P, Varalakshmi KN. A bioactive fraction from Turbinaria ornata inducing apoptosis via cell cycle arrest. Res J Pharm Technol. 2020; 13(9): 4263. doi:10.5958/0974-360X.2020.00752.0

31. Sushma B, Raveesha H. Effect of Elicitors treated callus of Baliospermum montanum (Willd.) Muell. Arg. on cell cycle arrest and apoptosis in HEP-2 and PC-3 cells. Res J Pharm Technol. 2020; 13(11): 5443-5450. doi:10.5958/0974-360X.2020.00950.6

32. Mani S, Swargiary G, Gulati S, Gupta S, Jindal D. Molecular docking and ADMET studies to predict the anti-breast cancer effect of aloin by targeting estrogen and progesterone receptors. Mater Today Proc. 2023; 80: 2378-2384. doi:10.1016/j.matpr.2021.06.362

33. Tanenbaum DM, Wang Y, Williams SP, Sigler PB. Crystallographic comparison of the estrogen and progesterone receptor’s ligand binding domains. Proceedings of the National Academy of Sciences. 1998; 95(11): 5998-6003. doi:10.1073/pnas.95.11.5998

34. Williams SP, Sigler PB. Atomic structure of progesterone complexed with its receptor. Nature. 1998; 393(6683): 392-396. doi:10.1038/30775

35. Ye B, Tian W, Wang B, Liang J. CASTpFold: Computed Atlas of Surface Topography of the universe of protein Folds. Nucleic Acids Res. 2024; 52(W1):W194-W199. doi:10.1093/nar/gkae415

36. Morris GM, Huey R, Lindstrom W, et al. AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility. J Comput Chem. 2009; 30(16): 2785-2791. doi:10.1002/jcc.21256

37. Astuti P, Erden W, Wahyuono S, Hertiani T. Pyrophen produced by endophytic fungi Aspergillus sp isolated from Piper crocatum Ruiz and Pav exhibits cytotoxic activity and induces S phase arrest in T47D breast cancer cells. Asian Pacific Journal of Cancer Prevention. 2016; 17(2): 615-618. doi:10.7314/APJCP.2016.17.2.615

38. Veeramani Kandan P, Dhineshkumar E, Karthikeyan R, et al. Isolation and characterization of opuntiol from Opuntia Ficus indica (L. Mill) and its antiproliferative effect in KB oral carcinoma cells. Nat Prod Res. 2021; 35(18): 3146-3150. doi:10.1080/14786419.2019.1690484

39. Abbasi MA, Shahzadi T, Akkurt M, Aziz-Ur-Rehman, Riaz T. 6-Hydroxy-methyl-4-meth-oxy-2H-pyran-2-one (Opuntiol). Acta Crystallogr Sect E Struct Rep Online. 2009; 66(Pt 1): o199. doi:10.1107/S1600536809053860

40. Öncül Ş, Becer E, Mega Tiber P, Teralı K, Aykac A. In vitro and in silico investigations of the pro-apoptotic activity of Opuntia ficus-indica cladode extracts against K562 cells. 2024; 49(4): 533-541. doi:doi:10.1515/tjb-2023-0229

41. Yuniarti A, Sundhani E, Nurulita N. The potentiation effect of Bawang Dayak (Sisyrinchium palmifolium L.) extract on T47D cell growth inhibition after 5-fluorouracil treatment. Pharmaciana. 2018; 8: 204. doi:10.12928/pharmaciana.v8i2.9480

42. Hahm SW, Park J, Oh SY, et al. Anticancer properties of extracts from Opuntia humifusa against human cervical carcinoma cells. J Med Food. 2015; 18(1): 31-44. doi:10.1089/jmf.2013.3096

43. Wyllie AH, Kerr JF, Currie AR. Cell death: the significance of apoptosis. Int Rev Cytol. 1980; 68: 251-306. doi:10.1016/s0074-7696(08)62312-8

44. Fatma F, Kumar A. The Cell Cycle, Cyclins, Checkpoints and Cancer. Asian Journal of Research in Pharmaceutical Sciences. 2021; 11(2): 175-183. doi:10.52711/2231-5659.2021-11-2-14

45. Sreekanth D, Arunasree MK, Roy KR, Chandramohan Reddy T, Reddy G V, Reddanna P. Betanin a betacyanin pigment purified from fruits of Opuntia ficus-indica induces apoptosis in human chronic myeloid leukemia Cell line-K562. Phytomedicine. 2007; 14(11): 739-746. doi:10.1016/j.phymed.2007.03.017

46. Yu S, Kim T, Yoo KH, Kang K. The T47D cell line is an ideal experimental model to elucidate the progesterone-specific effects of a luminal A subtype of breast cancer. Biochem Biophys Res Commun. 2017; 486(3): 752-758. doi:10.1016/j.bbrc.2017.03.114

47. Agu PC, Afiukwa CA, Orji OU, et al. Molecular docking as a tool for the discovery of molecular targets of nutraceuticals in diseases management. Sci Rep. 2023; 13(1): 1-18. doi:10.1038/s41598-023-40160-2

Received on 31.12.2024 Revised on 21.04.2025

Accepted on 02.06.2025 Published on 01.12.2025

Available online from December 06, 2025

Research J. Pharmacy and Technology. 2025;18(12):5971-5980.

DOI: 10.52711/0974-360X.2025.00863

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

Progesterone Receptor
	Van der Waals Interaction	Hydrogen Bonds	Hydrophobic Interactions
Progesterone		Gln725, Arg766, Cys891	Leu718, Trp755, Met756, Met759, Leu797, Leu887, Tyr890
Tamoxifen	Phe905	Asn719	Leu719, Leu721, Met756, Met759, Val760, Leu797, Leu887, Met801, Cys891
Opuntiol		Gln725, Arg766, Met756	Met756, Met759, Phe 778
Estrogen Receptor
	Van der Waals Interaction	Hydrogen Bonds	Hydrophobic Interactions
Estradiol		Glu353, Arg394, Gly521, His524	Ala350, Leu387, Met388 Leu384, Leu391, Phe404, Ile424, Leu525
Tamoxifen		Phe404	Met343, Ala350, Leu387, Met388, Met421, Ile424, Leu525
Opuntiol		Glu353, Arg394	Leu387, Leu391, Phe404