The rhizomes of Boesenbergia pandurata were harvested from the Southwest region of Viet Nam and subsequently identified at the Southern Institute of Ecology, Viet Nam. Fresh rhizomes were thoroughly cleaned and ground before undergoing solvent extraction. Specifically, 100 grams of the ground rhizomes were subjected to extraction with 500 mL of absolute ethanol for 72 hours using the maceration technique. The resultant extract was then filtered and subjected to freeze-drying at -40°C. The resulting dry extract was reconstituted in absolute ethanol to achieve a final concentration of 200 mg/mL. This stock solution was stored at 4°C for subsequent experimental procedures.

The foreskin fibroblast cell line was provided by the Stem Cell Institute, University of Science - Viet Nam National University, Ho Chi Minh City, Viet Nam. These cells were cultured in D'MEM/F12 (Lonza, UK), supplemented with 10% FBS (Gibco, UK) and 1% antibiotic (Gibco, UK) under 5% CO₂ and 37°C.

Human fibroblasts (2.5 x 10⁴ cells/mL) were seeded in a 96-well plate in 100 μL of culture medium. After 24 hours, hFs were treated with etoposide (Abcam, UK) at concentrations of 240 µM, 120 µM, 60 µM, 30 µM, 15 µM, 7.5 µM, 3.75 µM, 1.875 µM, 0.9375 µM and control for 48 hours. Cells were evaluated for their viability by staining with Alamar Blue for 1 hour and reading the measurements using a DTX 880 multimode reader (Beckman Coulter, CA).

For testing the Boesenbergia pandurata extract (BPE) potential, human fibroblasts (hFs) (2.5 x 10⁴ cells/mL) were seeded in a 96-well plate in 100 μL of culture medium. After 24 hours, hFs were treated with BPE at concentrations of 2000 µg/ml, 1000 µg/ml, 500 µg/ml, 250 µg/ml, 125 µg/ml, 60 µg/ml, 30 µg/ml, 15 µg/ml, 7.5 µg/ml and control for 48 hours. Cells were evaluated for their viability by staining with Alamar Blue for 1 hour and reading the measurements using a DTX 880 multimode reader (Beckman Coulter, CA).

Human fibroblasts (5 x 10³ cells/well) were cultured in a 48-well plate in 200 µL of culture medium. After 24 hours, the cells were treated with etoposide at concentrations of 10 µM, 20 µM, 30 µM and control for 48 hours. The suspension cells were observed under 40X magnification. Three ROIs (Regions of Interest) per well were captured under a light microscope to measure their diameter using AxioVision 4.8 software (Zeiss, Germany).

For the BPE testing, hFs (5 x 10³ cells/well) were cultured in a 48-well plate in 200 µL of culture medium. After 24 hours, the cells were treated with 20 µM etoposide to make a senescent fibroblast model for 48 hours. After that, the cells were washed with 200 µL PBS-. The experimental groups were treated with the BP extract at concentrations of 60 µg/ml, 30 µg/ml, 15 µg/ml. The senescent group and the control group were cultured with culture medium. After 48 hours, the suspension cells were observed under 40X magnification. Three ROIs per well were captured under a light microscope to measure their diameter using AxioVision 4.8 software (Zeiss, Germany).

Human fibroblasts (5 x 10³ cells/well) were seeded in a 48-well plate in 200 µL of culture medium. After 24 hours, the cells were treated with etoposide at 10 µM, 20 µM, 30 µM and control for 48 hours. Afterward, the cells were stained following the protocol of the beta Galactosidase Staining Kit (ab102534) (Abcam, UK). Three ROIs per well were captured under a light microscope. The β-galactosidase-positive cell ratio was then calculated by dividing the total number of β-galactosidase-positive cells by the total number of cells.

For the BPE testing, human fibroblasts (5 x 10³ cells/well) were cultured in a 48-well plate in 200 µL of culture medium. After 24 hours, the cells were treated with 20 µM etoposide to make a senescent fibroblast model for 48 hours. After that, the cells were washed with 200 µL PBS^-. The experimental groups were treated with the BPE at concentrations of 60 µg/ml, 30 µg/ml, 15 µg/ml. The senescent group and the control group were cultured with culture medium. After 48 hours, cells were stained according to the beta Galactosidase Staining Kit protocol (ab102534) (Abcam, UK). Three ROIs per well were captured under a light microscope. The β-galactosidase-positive cell ratio was then calculated by dividing the total number of β-galactosidase-positive cells by the total number of cells.

Human fibroblasts (2 x 10⁵ cells/mL) were seeded in a 6-well plate in 1 mL of culture medium. After 24 hours, the cells were treated with etoposide at 10 µM, 20 µM, 30 µM and control for 48 hours. After that, the total RNA from the fibroblasts was extracted using the Easy-Blue Total RNA Extraction Kit (iNtRON Biotechnology).

For the BPE testing, human fibroblasts (5 x 10³ cells/well) were cultured in a 48-well plate in 200 µL of culture medium. After 24 hours, the cells were treated with 20 µM etoposide to make a senescent fibroblast model for 48 hours. After that, the cells were washed with 200 µL PBS^-. The experimental groups were treated with the BPE at concentrations of 60 µg/ml, 30 µg/ml, 15 µg/ml. The senescent group and the control group were cultured with culture medium. After 48 hours, the total RNA from the fibroblasts was extracted using the Easy-Blue Total RNA Extraction Kit (iNtRON Biotechnology).

Real-Time Reverse Transcription Quantitative PCR (Real-Time RT-qPCR)^Ct method (Ct is the threshold cycle value).

Human fibroblasts were seeded in a 6-well plate. The cell density was 2 x 10⁵ cells per well (in 1 mL volume). These cells were treated with 10 μM, 20 μM, 30 μM etoposide and incubated for 48 hours. Afterwards, the cells were labeled with 5 μL CD90 (BD Biosciences, Franklin Lakes, NJ) in 100 μL of staining buffer for 15 minutes. The cells were detected using a FACSCalibur flow cytometer. Data were analyzed using CellQuest Pro software (BD Biosciences, Franklin Lakes, NJ).

For the BPE testing, human fibroblasts (2 x 10⁵ cells/well) were cultured in a 6-well plate in 1 mL of culture medium. After 24 hours, the cells were treated with 20 µM etoposide to make a senescent fibroblast model for 48 hours. After that, the cells were washed with 200 µL PBS^-. The experimental groups were treated with the BPE at concentrations of 60 µg/ml, 30 µg/ml, 15 µg/ml. The senescent group and the control group were cultured with culture medium. After 48 hours, the cells were labeled with 5 μL CD90 (BD Biosciences, Franklin Lakes, NJ) in 100 μL of staining buffer for 15 minutes. The cells were detected using a FACSCalibur flow cytometer. Data were analyzed using CellQuest Pro software (BD Biosciences, Franklin Lakes, NJ).

Data were collected and processed using GraphPad Prism 9 software. The results are presented as mean ± SD. Differences between treatments were assessed using one-way ANOVA, with statistical significance set at p < 0.05.

Table 1

Gene	Primers
p53	F: 5’-GGTTTCCGTCTGGGCTTCTT-3’
	R: 5’-GGGCCAGACCATCGCTATC-3’
p21	F: 5’-CTTCGACCTTTGTCACCGAGA-3’
	R: 5’-AGGTCCACATGGTCTTCCTC-3’
p16	F: 5’-CATAGATGCCGCGGAAGGT -3’
	R:5’-CTAAGTTTCCCGAGGTTTCTCAGA-3’
GADPH	F: 5’-GAGTCCACTGGCGTCTTC-3’
	R: 5’-GGGGTGCTAAGCAGTTGGT-3’

To evaluate the impact of etoposide on human fibroblast cells, we determined the IC₅₀ value through the Alamar Blue assay, measuring the level of cell metabolism after a 48-hour treatment with etoposide. The results show that the IC₅₀ value was found to be 32.08 ± 2.85 µM. Based on these data, the etoposide concentrations used for further evaluation were 10 µM, 20 µM, and 30 µM (Figure 1).

Figure 1

Abbreviations: conc: Concentration; hFs: human fibroblasts

The presence of the SA-β-Galactosidase enzyme through an X-gal staining kit at pH 6.0 is used to confirm senescent cells. In this experiment, senescent fibroblasts were treated with BPE at concentrations of 60 µg/ml, 30 µg/ml, and 15 µg/ml for 48 hours to examine the effect of this extract on senescent cells. Then, the cells were stained with a Galactosidase Staining Kit (Abcam, UK), and senescent cells exhibited a blue color.

The results show that senescent fibroblasts treated with BPE displayed a noticeable decrease in SA-β-Galactosidase expression, which is a common marker of cellular aging. Senescent cells treated with BPE at a concentration of 15 µg/ml exhibited a 1.2-fold reduction in SA-β-Galactosidase positivity compared to untreated cells (26.311% ± 1.177%) (p < 0.0001). At a higher concentration of 30 µg/ml, the proportion of SA-β-Galactosidase positive cells was 25.905% ± 0.431%, representing a 1.22-fold decrease compared to senescent cells (p < 0.0001). At the highest concentration tested, 60 µg/ml, the SA-β-Galactosidase positivity rate was 27.735% ± 1.093%, which is a 1.14-fold reduction compared to senescent cells (p < 0.001).

To evaluate the potential of the BP extract on senescent cells, the decrease in aging-related genes illustrates these effects9. The results show that treating senescent cells with BPE decreases the expression of genes p16, p21, and p53, in contrast to untreated senescent cells (Figure 9). Senescent cells treated with BPE demonstrate a reduction in p16 gene expression by 5.89 times (p < 0.0001), 21.3 times (p < 0.0001), and 18.85 times (p < 0.0001) at concentrations of 15 µg/ml, 30 µg/ml, and 60 µg/ml, respectively, compared with the senescent group. Similarly, BP-treated senescent fibroblasts at concentrations of 15 µg/ml, 30 µg/ml, and 60 µg/ml also reduced p21 and p53 gene expressions by 5.68 times (p < 0.001), 6.74 times (p < 0.001), and 3.79 times (p < 0.001) for the former gene, and by 3.26 times (p < 0.001), 5 times (p < 0.0001), and 2.73 times (p < 0.001) for the latter gene, respectively, compared to the senescent group.

The results indicate that cells expressing the CD90 marker were positive in both the extract-treated and control groups (Figure 10). In the control group, the highest proportion of cells positive for the CD90 marker was 98.76%. This was followed by senescent cells with a CD90 positivity rate of 98.71%. Senescent cells treated with BPE also exhibited high CD90 expression (above 98%).

Figure 10

Abbreviations: BPE: Boesenbergia Pandurata extract; “+”: aging cells, “-”: normal cells.

This study successfully established a senescent fibroblast model using the etoposide agent. The optimal etoposide concentration for inducing fibroblast senescence was determined to be 20 μM, with a treatment duration of 48 hours. The IC₅₀ of etoposide for fibroblasts is 32.08 μM ± 2.854 μM. Furthermore, this study demonstrated the potential of the ethanol extract of Boesenbergia pandurata root in modulating specific senescence characteristics induced by etoposide. The extract showed an IC₅₀ for fibroblast inhibition of 212.1 μg/ml ± 5.27 μg/ml. When applied at 15 μg/ml, B. pandurata extract improved several markers of cellular aging, including reduced cell size, decreased SA-β-Galactosidase activity, and downregulated expression of p16, p53, and p21.

BPE - Boesenbergia Pandurata Extract, CD90 - Cluster of Differentiation 90, FBS - Fetal Bovine Serum, GAPDH - Glyceraldehyde 3-phosphate dehydrogenase, hFs - human fibroblasts, IC₅₀ - Half-maximal inhibitory concentration, µM - Micromolar, µg/ml - Micrograms per milliliter, mRNA - Messenger RNA, PBS - Phosphate Buffered Saline, pH - Potential Hydrogen, PCR - Polymerase Chain Reaction, pRB - Retinoblastoma Protein, p16^INK4A - p16 Inhibitor of Cyclin-dependent Kinase 4A, p21^CIP1 - p21 Cyclin-dependent kinase Inhibitor 1, p53 - Tumor protein p53, qPCR - Quantitative Polymerase Chain Reaction, SA-β-Gal - Senescence-associated beta-Galactosidase, SASP - Senescence-associated secretory phenotype, UVB - Ultraviolet B

I would like to express my sincere gratitude to Assoc. Prof. Dr. Phuc Van Pham, thank you for your technical support throughout this project. His expertise and guidance have been crucial in overcoming challenges and achieving the research goals.

Phuc Hong Vo performed the experiments, analyzed the results, and wrote the manuscript. Nghia Minh Do supports the identification and verification of Boesenbergia pandurata. Sinh Truong Nguyen supervised the research and reviewed the manuscript. All authors read and approved the final manuscript.

This study was granted by Stem Cell Institute, University of Science, Viet Nam National University Ho Chi Minh City, Viet Nam.

Data and materials used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Not applicable.

Not applicable.

The authors declare that they have no competing interests.