INTRODUCTION:

Averrhoa bilimbi (Oxalidaceae) has significant medicinal potential ^1,2, with fruit decoctions treating hepatitis, fever, and diarrhea, and leaves used for coughs, skin eruptions, and swelling ^3-5. In India, it combats obesity, while Mascarene Island uses it for infections and hypertension^6,7. This study explores A. bilimbi leaf extracts' cytotoxic and apoptosis-inducing effects on cancer cell lines (AsPC-1, A549, KYSE 150, RD) using MTT and Caspase 3/7 assays.

MTT assesses cell viability via formazan formation, while Caspase 3/7 measures apoptosis. This research, employing established methods, investigates A. bilimbi’s unexplored anticancer potential on these specific cell lines.

Experimental Methods :

Averrhoa bilimbi (Oxalidaceae) from Sultan-ul-Uloom College, Hyderabad (voucher specimen GACE-219), was tested on cancer cell lines (AsPC-1, A549, KYSE 150, RD, Vero) from Sigma Aldrich, cultured in DMEM with FBS and PSA. Extracts used N-hexane, ethyl acetate, methanol. Key reagents included MTT, DMSO, SDS, PBS, and Caspase-Glo® 3/7.

Preparation of Leaf Extracts:

Averrhoa bilimbi leaves (200 g) were washed, dried, and extracted using Soxhlet with n-hexane, ethyl acetate, and methanol (1:2 solvent-to-plant ratio). Filtration, solvent concentration, and storage at <4°C followed. Extract yields were calculated.

Sample Preparation for Screening:

Dried extracts were reconstituted in DMSO (30 mg/mL stock), filtered (0.2 μm), and stored at 4°C. A 500 µg/mL sub-stock was prepared by diluting stock in 10% DMEM. Two-fold dilutions were used for MTT and Caspase 3/7 assays to evaluate apoptosis-inducing potential. Cell viability and luminescence indicated enzymatic activity.

Cell Lines and Experimental Setup:

Cells were cultured in T75 flasks at 37°C with 5% CO₂ in DMEM containing 10% FBS and penicillin/ streptomycin (100 units/mL). At 80% confluence, cells were washed with PBS, trypsinized (1 mL, 7 minutes), neutralized, centrifuged, and re-suspended in 5 mL fresh media. Cell viability was determined using a hemocytometer and trypan blue exclusion.

Cells (1x10⁴/well) were seeded in 96-well plates (100 µL) and incubated overnight for adhesion. Upon confluency, cells were treated with Averrhoa bilimbi extracts (62.5–500 µg/mL; n-hexane, ethyl acetate, methanol) and compared to untreated controls.

MTT and Caspase Assay:^8,9

After a 24-hour incubation, 20 µL of MTT (5 mg/mL) was added to each well and incubated for 4 hours at 37°C in 5% CO2. Then, 100 µL of sodium dodecyl sulfate (10%)/hydrochloric acids (0.01 M) was added to solubilize formazan. Absorbance was measured at 570 nm.

(Treated Optical Density)

% Inhibition = 100 - -------------------------------- x 100

(Non-treated Optical Density)

Graphs showed the link between restricting growth and extract concentrations, revealing each cancer cell line's IC50. The Selectivity Index (SI) was the ratio of Vero cell IC50 to cancer cell IC50. In the Caspase 3/7 Assay, cells were seeded at 1.0×10^4 per well in 96-well plates and incubated for 24 hours. After 2 hours of extract treatment, 100 μL of Caspase-Glo® 3/7 reagent was added and incubated for an hour. Measured luminescent signals showed caspase-3 and caspase-7 activity.

(Ic50 non cancer cell

SI = ----------------------------

(IC50 cancer cell)

A statistical analysis of duplicate experiments was conducted. Results are presented as mean ± SD and evaluated using two-way ANOVA and Bonferroni post hoc test in Graph Pad Prism 8.0. Statistical significance was set at p < 0.05.

RESULTS:

Air-dried Averrhoa bilimbi leaves (200 g) were extracted with n-hexane, ethyl acetate, and methanol. Methanol yielded the highest extract (7.25%), followed by ethyl acetate (3.85%) and n-hexane (1.88%). After 24 hours, cytotoxicity was tested on KYSE 150, A549, RD, AsPC-1, and Vero cells¹⁰. Hexane extract showed the highest cytotoxicity with IC50 values of 13.8±0 µg/mL for KYSE 150 and 14.13±0.46 µg/mL for Vero cells. Ethyl acetate had IC50 values of 19.09±12.86 µg/mL (RD) and 20.22±1.65 µg/mL (KYSE 150). No activity was observed for A549 and AsPC-1 cells.

Vero Cells:¹¹

Viability of Vero cells was assessed using hexane, ethyl acetate, and methanolic extracts of Averrhoa bilimbi at concentrations of 62.5, 125, 250, and 500 µg/mL. The hexane extract showed 91.51% ± 7.5 inhibition at 500 µg/mL. Ethyl acetate achieved 90.5% ± 10.9 inhibition, while methanol showed 53.11% ± 2.63 inhibition at 500 µg/mL. Significant differences (p < 0.05) were observed compared to the control and among extracts (Fig-1).

RD Cell:¹²

Averrhoa bilimbi extracts (hexane, ethyl acetate, methanol) were tested at 62.5, 125, 250, and 500 µg/mL. The hexane extract inhibited viability by 50%, peaking at 86.23% ± 0.6 at 500 µg/mL. Ethyl acetate showed 85.51% ± 6.11 inhibition at 125 µg/mL, and methanol reached 91.47% ± 2.01 at 500 µg/mL. Significant differences (p < 0.05) were noted (Figure-2).

KYSE 150 Cells:¹³

Averrhoa bilimbi extracts (hexane, ethyl acetate, methanol) were tested at 62.5, 125, 250, and 500 µg/mL. The hexane extract showed a peak inhibition of 93.87% ± 4.16 at 500 µg/mL. Ethyl acetate inhibited 77.42% ± 6.37 at 125 µg/mL, while methanol reached 85.21% ± 1.4 at 500 µg/mL. Significant differences (p < 0.05) were observed, particularly between hexane and ethyl acetate at 500 µg/mL (Figure-3).

AsPC-1 Cells:¹⁴

Ethyl acetate extracts at 125, 250, and 500 µg/mL inhibited AsPC-1 cell viability, with a peak of 72.6% ± 10.63 at 250 µg/mL. Methanolic extracts showed a maximum inhibition of 79.73% ± 0.15 at 500 µg/mL. The hexane extract could not provide an IC50 value as it exceeded the measured range. Significant differences (p < 0.05) were found between hexane and ethyl acetate, as well as between hexane and methanolic extracts. No significant differences were observed between ethyl acetate and methanolic extracts (Figure-4).

A549 Cells:¹⁵ A549 cell viability decreased concentration-dependently, with a peak inhibition of 76.48% ± 8.21 at 500 µg/mL. Ethyl acetate and methanolic extracts exhibited >50% inhibition at concentrations of 125 µg/mL and higher, with ethyl acetate reaching 83.58% ± 4.87 at 250 µg/mL and methanolic extract 63.17% ± 8.98 at 500 µg/mL. Significant differences (p < 0.05) were observed, especially between hexane and ethyl acetate at 125 µg/mL, and ethyl acetate and methanolic extracts at 250 µg/mL (Figure-5).

Apoptotic-Assay:^16,17

The study evaluated the apoptotic efficacy of Averrhoa bilimbi leaf extracts on KYSE 150 and RD cells utilizing the MTT assay and Selectivity Index (SI). The ethyl acetate extract demonstrated considerable cytotoxicity and enhanced selectivity towards both cancer cell lines, resulting in its selection for subsequent apoptosis studies. The cells were exposed to two doses (31.25 and 15.625 µg/mL) of ethyl acetate extract for 2 hours, after which apoptosis was evaluated using the Caspase 3/7 Glo® test kit. The results indicated that the ethyl acetate extract elicited the most significant apoptotic activity in KYSE 150 cells at a concentration of 15.625 µg/mL, a pattern similarly noted in RD cells. Marked disparities were seen in treated cells relative to controls (Figure-6).

Selectivity Index Evaluation:

Table 3 presents the Selectivity Index (SI) of Averrhoa bilimbi extracts for cancer cells (KYSE 150, A549, RD, AsPC-1) versus Vero cells. SI >1.0 signifies higher cancer selectivity. Ethyl acetate extracts showed SI values of 1.86 ± 0.48 (RD) and 1.69 ± 0.05 (KYSE 150), while methanolic extracts had 1.74 ± 0.01 (RD) and 2.26 ± 0.03 (KYSE 150). RD and KYSE 150 cells were chosen for apoptosis evaluation.

Selection of Cells for Further Experimentation:

RD cells and KYSE 150 cells were chosen for further experimentation because to their advantageous Selectivity Index (SI) values. Caspase 3/7 levels are measured in this follow-up study to determine whether apoptosis is present. RD cells and KYSE 150 cells were selected for subsequent experimentation due to their favorable Selectivity Index (SI) values. This follow-up study measures Caspase 3/7 levels to ascertain the presence of apoptosis.

Notable cytotoxic effects were observed, with hexane and methanolic extracts showing promising IC50 values against KYSE 150 cells (13.85 ± 0.01 µg/mL and 14.24 ± 0.19 µg/mL, respectively). Ethyl acetate extract displayed enhanced cytotoxicity against RD and KYSE 150 cells (IC50: 18.97 ± 11.16 µg/mL, 19.92 ± 1.15 µg/mL). SI values raised concerns for hexane extracts (below 1 for A549, AsPC-1 cells), suggesting toxicity to Vero cells. Ethyl acetate and methanolic extracts showed favorable selectivity for RD and KYSE 150, warranting further optimization for non-specific toxicity.

Polyphenols, flavonoids, and saponins contribute to the cytotoxicity of ethyl acetate and methanolic extracts. Ethyl acetate extract, selected for apoptotic studies, induced apoptosis in KYSE 150 and RD cells at 15.625 and 31.25 µg/mL, with maximum activity at 15.625 µg/mL. Morphological changes, including cell rounding, membrane blebbing, and apoptotic bodies, were observed, unlike control cells. These findings suggest that the ethyl acetate extract of Averrhoa bilimbi promotes apoptosis in RD and KYSE 150 cells, highlighting its potential for cancer therapy and necessitating further mechanistic studies.

Morphological Changes Induced by Ethyl Acetate Extract of Averrhoa bilimbi Leaf in KYSE 150 and RD Cells:

The study observed significant morphological changes in KYSE 150 and RD cells after treatment with 31.25 µg/mL & 125 µg/mL ethyl acetate extract of Averrhoa bilimbi for 2 hours. These changes included reduced cell count, membrane blebbing, cellular shrinkage, nuclear condensation, and apoptotic bodies with organelles and nuclear fragments, indicating apoptosis and growth inhibition (Fig-8 and 9).

DISCUSSION:

Averrhoa bilimbi leaf extracts (hexane, ethyl acetate, and methanolic) contain anticancer properties and limitations across cell lines due to their cytotoxic activities. The hexane extract demonstrated notable activity against KYSE 150 cancer cells, exhibiting an IC50 value of 13.85 µg/mL, which indicates its efficacy against esophageal squamous cell carcinoma. Nonetheless, the moderate efficacy against RD and A549 cells, indicated by IC50 values of 188.38 µg/mL and 110.92 µg/mL, raises concerns regarding selectivity, particularly in light of the low IC50 of 14.12 µg/mL noted in Vero normal cells. The available data regarding the AsPC-1 cell line is scarce. The ethyl acetate extract displayed significant cytotoxic effects on RD and KYSE 150 cells, with IC50 values of 18.97 and 19.92 µg/mL, respectively, and showed moderate activity against A549 and AsPC-1 cells. The IC50 value of 150.70 µg/mL for the Vero cell line indicates enhanced selectivity for cancer cells, positioning it as a potential anticancer agent.

The methanolic extract exhibited the greatest cytotoxicity towards KYSE 150 cells, with an IC50 of 14.24 µg/mL, and demonstrated significant cytotoxicity against RD, A549, and AsPC-1 cells, presenting IC50 values of 33.5, 96.95, and 52.63 µg/mL, respectively. The IC50 value of 464.71 µg/mL in Vero cells demonstrates significant selectivity. The methanolic extract demonstrates the greatest potential for targeting KYSE 150 cells among the three extracts; however, additional optimization is required. The observed cytotoxic effects of the ethyl acetate and methanolic extracts are attributed to the presence of specific secondary metabolites, including polyphenols, flavonoids, and particularly saponins^18,19. These studies highlight A. bilimbi's potential as a natural anticancer agent, demonstrating varying efficacy and selectivity among different extracts and cell lines.

CONCLUSION:

Averrhoa bilimbi extracts were tested for cytotoxicity and apoptosis on cancer cell lines. The ethyl acetate extract showed excellent anticancer activity against RD (18.97 ± 11.16 µg/mL) and KYSE 150 (19.92 ± 1.15 µg/mL) cells with the lowest IC50 values. Additionally, the methanolic extract was effective against KYSE 150 cells (14.24 ± 0.19 µg/mL). IC50 values of 150.70 ± 19.18 µg/mL for ethyl acetate and 464.71 ± 57.11 µg/mL for methanol indicate selectivity for cancer cells, since both extracts showed lesser cytotoxicity towards normal Vero cells These extracts contain higher amounts of phytochemicals such flavonoids, neoflavonoids, alkaloids, and tannins, which may fight cancer. To investigate Averrhoa bilimbi's anticancer potential, molecular docking, apoptotic pathways, and phytochemical identification are needed.

Figure-1: Vero cell viability inhibition percentage using MTT test using hexane, ethyl acetate, and methanolic Averrhoa bilimbi extracts at doses from 62.5 to 500 µg/mL.

(Note: *P<0.05 versus concentration at 0 µg/mL (Control group)

# indicates significant difference between hexane and ethyl acetate extracts

δ indicates significant difference between hexane and methanolic extracts

ϕ indicates significant difference between ethyl acetate and methanolic extracts)

Figure-2: RD viability inhibition percentage using MTT test using hexane, ethyl acetate, and methanolic Averrhoa bilimbi extracts at doses from 62.5 to 500 µg/mL.

(Note: *P<0.05 versus concentration at 0 µg/mL (Control group)

# indicates significant difference between hexane and ethyl acetate extracts

δ indicates significant difference between hexane and methanolic extracts

ϕ indicates significant difference between ethyl acetate and methanolic extracts)

Figure-3: KYSE 150 viability inhibition percentage using MTT test using hexane, ethyl acetate, and methanolic Averrhoa bilimbi extracts at doses from 62.5 to 500 µg/mL.

Note: P<0.05 versus concentration at 0 µg/mL (Control group)

# indicates significant difference between hexane and ethyl acetate extracts

δ indicates significant difference between hexane and methanolic extracts

ϕ indicates significant difference between ethyl acetate and methanolic extracts

Figure 4: Pancreatic-AsPC-1 viability inhibition percentage using MTT test using hexane, ethyl acetate, and methanolic Averrhoa bilimbi extracts at doses from 62.5 to 500 µg/mL.

Note: P<0.05 versus concentration at 0 µg/mL (Control group)

# indicates significant difference between hexane and ethyl acetate extracts

δ indicates significant difference between hexane and methanolic extracts

ϕ indicates significant difference between ethyl acetate and methanolic extracts

Figure 7: Assessment of Apoptotic (Caspase 3/7) Activity in Rhabdomyosarcoma (RD) cancer cell lines Treated with Ethyl Acetate Extract at Conc. of 15.625 µg/mL & 31.25 µg/mL for 2hrs.

Note: The data presented are the average values with standard deviations (±SD) obtained from two separate experimental trials. Statistical significance is denoted by*, indicating p-values less than 0.05 compared to control levels."

Table-1: IC₅₀values (µg/mL) of Averrhoa bilimbi leaf extracts against cancer and normal cell lines as determined by MTT assay. Data were obtained from two independent experiments and presented as mean± standard deviation (SD)

Type of Extracts	Cancer cell lines													Normal cell line
	RD			KYSE 150			A549			AsPC-1				Vero
	1	2	Mean ± SD (µg/mL)	1	2	Mean ± SD (µg/mL)	1	2	Mean± SD (µg/mL)	1	2	Mean± SD (µg/mL)	1		2	Mean± SD (µg/mL)
Hexane	188.2	188.55	188.38± 4.31	14.1	13.6	13.85± 0.01	90.9	130.93	110.92± 22.78	-	-	-	13.8		14.45	14.12± 1.93
Ethyl acetate	9.96	27.98	18.97± 11.16	18.95	19.88	19.92± 1.15	70.08	94.13	82.11± 12.76	82.98	110.92	96.96± 11.9	145.2		156.19	150.70± 19.18
Methanolic	31.56	33.51	33.5± 0.86	15.54	14.95	14.24± 0.19	90.98	101.89	96.95± 2.25	53.08	52.18	52.63± 0.91	438.22		491.18	464.71± 57.11

Table-2: Selectivity index (SI) of Averrhoa bilimbi leaf extracts calculated by the IC₅₀ against Vero cells and cancer cell lines. Data were obtained from two independent experiments and presented as mean± standard deviation (SD).

Type of Extracts	Cancer cell lines
	RD			KYSE 150			A549			AsPC-1
	Exp-1	Exp-2	Mean ± SD	Exp-1	Exp-2	Mean ± SD	Exp-1	Exp-2	Mean ± SD	Exp-1	Exp-2	Mean ± SD
Hexane	1.16	1.18	1.17± 1.06	0.96	0.98	0.97± 0.02	0.60	0.57	0.585± 1.13	-	-	-
Ethyl acetate	1.79	1.98	1.88± 0.63	1.92	1.35	1.635± 1.08	1.28	1.08	1.18± 1.16	1.19	1.02	1.10± 0.93
Methanolic	1.82	1.69	1.76± 1.09	2.64	2.08	2.36± 1.91	1.49	1.26	1.38± 0.09	1.73	1.29	1.51± 0.05

(a)

(b)

(c)

Figure 8: Morphological Changes in KYSE 150 Cells Treated with Ethyl Acetate Extract of Averrhoa bilimbi at Different Concentrations for 2 Hours, Compared to (a) Untreated control (b) 31.25 µg/mL and (c) 125 µg/mL (Magnification: 10x)

(a)

(b)

(c)

Figure 9: Morphological Changes in RD Cells Treated with Ethyl Acetate Extract of Averrhoa bilimbi at Different Concentrations for 2 Hours, Compared to (a) Untreated control (b) 31.25 µg/mL and (c) 125 µg/mL (Magnification: 10x)

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Received on 29.10.2024 Revised on 20.02.2025

Accepted on 29.04.2025 Published on 13.01.2026

Available online from January 17, 2026

Research J. Pharmacy and Technology. 2026;19(1):301-307.

DOI: 10.52711/0974-360X.2026.00043

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