J Gandhara Med Dent Sci

October - December 2025

ORIGINAL ARTICLE

ABSTRACT

OBJECTIVES

To investigate the in vitro cytotoxic potential of Papaya Leaves on 3 distinct

cell lines: BJ, cervical, and prostate.

METHODOLOGY

Papaya (Red Lady) samples were obtained. Acetone and n-hexane extracts

were used in a Soxhlet extractor at the University of Karachi. The cytotoxic

activity was assessed using the MTT (3- (4,5-dimethylthiazol-2-yl)- 2,5-

diphenyltetrazolium bromide) assay. Cells were cultured in 75cm² ﬂasks with

5% CO2 at 37°C, 5% fetal-bovine serum, 100IU/ml penicillin, and 100µg/ml

streptomycin. Cells were counted using a hemocytometer, diluted, and then

added to 96-well plates at a concentration of 6 × 10^4 cells/mL. After 48

hours, MTT reduction to formazan was measured using a microplate reader.

RESULTS

The acetone extract exhibited potent cytotoxic activity against the BJ cell,

with a % inhibition (mean ± SD) of 60.4 ± 0.85 at a concentration of 30 µM,

and an IC50 of 18.19 ± 0.19 µM (p < 0.001). In contrast, the n-hexane

extract revealed minimal activity. In cervical, both extracts fell below the

50% inhibition threshold, unlike the eﬀective Doxorubicin. The acetone

extract showed moderate activity for prostate, while the n-hexane extract was

inactive.

CONCLUSION

The acetone extract displays signiﬁcant activity against BJ cells, while the n-

hexane extract is inactive. Both extracts have minimal eﬀects on HeLa and

none on PC3 cells.

KEYWORDS: Fibroblasts, Cervical Cell Lines, Prostate Cell Lines

How to cite this article

Asadullah, Saeed, Asif, Unar MA. In-

Vitro Cytotoxic Potential of Carica

Papaya Leaves On Bj-Human Fibroblast,

Cervical-Hela and Prostate Cell Lines. J

Gandhara Med Dent Sci.2025;12(4):18.

-22. http://doi.org/10.37762/jgmds.12-4.

736

Date of Su bmission: 29-05-2025

Date Revised: 30-08-2025

Date Acceptance: 30-08-2025

PhD Scholar, Baqai Medical University,

Karachi

PhD Scholar, Baqai Medical University,

Karachi

Professor, Baqai Medical University,

Karachi

Correspondence

PhD Scholar, Baqai Medical University,

Karachi

+92-334-7092779

fastmasood7@gmail.com

IN-VITRO CYTOTOXIC POTENTIAL OF CARICA PAPAYA LEAVES ON BJ -HUMAN

FIBROBLAST, CERVICAL-HELA AND PROSTATE CELL LINES

Asadullah

, Saeed

, Asif

, Masood Ahmed Unar

INTRODUCTION

Cancer is a leading global cause of mortality, marked

by uncontrolled cell proliferation. Lung cancer is

prevalent in males, while breast cancer aﬀects females.

Treatment options vary by cancer type, stage, and

location, including surgery, chemotherapy,

radiotherapy, immunotherapy, and combination

therapies.

Chemotherapy, using drugs like Irinotecan

and Doxorubicin, is particularly eﬀective for highly

metastatic cancers.

Despite their eﬀectiveness,

chemotherapeutic drugs have drawbacks like limited

bioavailability, toxicity, lack of speciﬁcity, rapid

clearance, and adverse eﬀects such as cytotoxicity,

neuropathy, cardiovascular issues, and gastrointestinal

disturbances.

Consequently, to address these

challenges, researchers are exploring alternative

treatments with minimal or no side eﬀects.

Extensive

studies suggest phytochemicals are a promising anti-

cancer option due to their potential eﬃcacy and fewer

side eﬀects compared to traditional medicine.

The

tropical fruit papaya is a family of caricaceae and

several varieties have been utilized as ethnomedicine

against a diﬀerent of disorders.

Papaya is cultivated for

its roots, leaves, seeds, and fruits, which are extensively

used in conventional medicine to treat a wide range of

disorders.

Papaya is revered as a nutritionally plentiful

source of several vitamins, such as A, B, and C,

providing a reasonable quantity of Ca and Fe.

It has

papain, an enzyme that helps with the digestion and is

utilized to treat ulcers and some microbial illnesses.

Benzyl isothiocyanate, found in papaya fruit seed

extract, has bacteriostatic, fungicides, and bactericide

effects at a safe dose of 4-5g of seeds.

Papaya has

strong antioxidant properties that help scavenge free

radicals and stop the onset of disease.

Papain, glycyl-

endopeptidase, chymopapain, and caricain are among

the many proteinases found in latex, which is one of the

most signiﬁcant components of papaya.

New

attention has been drawn to the rich phytochemicals

found in tropical plants like papaya, prompting global

research eﬀorts towards developing phytochemical-

based treatments with minimal side eﬀects for

combating cancer.

Papaya leaves have been used to

treat several illnesses, such as asthma, beriberi, colic,

fever, and jaundice. In addition, Papaya leaves have

been conventionally utilized for anti-cancerous

activities in the native Gold Coast of Australia, based

J Gandhara Med Dent Sci

October - December 2025

on a few sketchy pieces of information of signiﬁcant

cases acknowledged in various publications.

Diﬀerent

varieties of papaya are cultivated worldwide, varying in

color and size. The Red Lady variety is commonly

grown globally due to its excellent yield.

Consequently, this study aimed to explore the potential

of Red Lady papaya leaves as a therapeutic option for

cancer by evaluating the cytotoxic eﬀects of acetone

and n-hexane extracts against various cell lines.

METHODOLOGY

An in vitro experimental study was conducted at the

Department of Pharmacology and Therapeutics, Baqai

Medical College and University of Karachi, spanning a

duration of 6 months. Selected plant was collected

from University of Karachi, and authenticated by an

ethno-botanist (G.#H97627). Extraction was used at the

International Centre for Chemical and Biological

Sciences (ICCBS) University of Karachi, Industrial

Analytical Centre. The technique involved inserting the

ground sample into the Soxhlet apparatus thimble

chamber. Heating the acetone and n-hex extraction

solution in the bottom ﬂask caused it to evaporate into

the sample thimble, condense in the condenser, and drip

back. The procedure was repeated as the ﬂuid ﬁlled th e

bottom ﬂask and cleared into it as it approached the

siphon arm. The extraction process takes place in a

Soxhlet apparatus for 72 hours, after weighing between

10g and 82g of the semi-solid sample. By evaporating

the extract at 60°C, the extract was concentrated.

Acetone and n-hexane were used for the extraction of

distinctly diﬀerent classes of phytochemicals. Acetone,

with a moderate polarity, extracts ﬂavonoids, phenolics,

and other moderately polar compounds that potentially

contribute to bioactivity. N-hexane, being a nonpolar

solvent, facilitates the separation of lipophilic

constituents, such as fatty acids, terpenoids, and

chlorophyll derivatives. Together, this might oﬀer a

complementary option for polar-non-polar portfolios.

This choice of solvents is selected because most

ethanol, methanol, and water, as well as other solvents

used in previous studies on papaya leaves, have already

been utilized, which prompted consideration of less-

explored fractions for novel activities. The cell lines

were obtained from the Bio-bank facility of ICCBS,

which were purchased from the American Type Culture

Collection: i.e., HeLa (cervix), PC3 (prostate), and BJ

(normal human skin ﬁbroblast). HeLa and BJ cells were

cultured in Dulbecco's Modiﬁed Eagle‘s Medium

(DMEM), supplemented with 10% heat-inactivated

fetal bovine serum (FBS), 2 mM L-glutamine,

penicillin (100 U/mL), and streptomycin (100 μg/mL).

The PC3 cell line was maintained in RPMI-1640,

supplemented with 10% (v/v) FBS, penicillin

(100U/mL), streptomycin (100μg/mL), and 1% of non-

essential amino acids. All cell lines were incubated in a

T-75 cell culture-treated ﬂask with 10mL of complete

medium at 37°C in a humidiﬁed incubator with 5%

CO2 until the medium was replaced. A cell culture was

prepared and added to 96-well plates (100 µL/well) at a

concentration of 6×10^4 cells/mL. Following an

overnight incubation period, the medium was removed

and 200µL of new medium containing a variable

dosage of compounds (1-30µM) was added. Each well

received 200 µL of MTT (0.5mg/mL), and after 2 days

and 4 more hours of incubation, the cells were required.

Then, 100 µL of DMSO was added to every well, and

the absorbance at 570nm was measured using a

microplate reader (Spectra-Max plus, Molecular

Devices, CA, USA) to determine the degree of MTT

reduction to formazan within the cells. The

concentration that caused 50% growth inhibition (IC

)

for BJ, HeLa, and PC-3 was used to measure the

cytotoxicity. The subsequent equation was used for

calculating the % Inhibition: %Inhibition=100-(mean of

test compounds O.D.-mean of negative-control O.D.)/

(mean of positive-control O.D.-mean of negative-

control O.D.)*100).

18,19

For Statistical Analysis, all

cytotoxicity values were compared using one-way

ANOV A to measure diﬀerences between treatment

groups. In instances where a diﬀerence was detected (p

< 0.05), pairwise signiﬁcance was evaluated

by Tukey‘s HSD post hoc test. The cytotoxic activity

was deﬁned with reference to the 50% inhibition cut-

oﬀ. This threshold is widely applied in cytotoxicity and

natural product screening studies for separating active

extracts from inactive ones. Although the choice of

50% inhibition seems somewhat arbitrary, it is

consistently applied in other works, allowing some

comparisons of the results obtained. In this work, such

a comparison was made possible by adopting the same

threshold as used in prior literature.

RESULTS

The results for BJ Human ﬁbroblast cell lines in Table

1 suggest that the acetone extract of Papaya leaves (RL)

demonstrates remarkable cytotoxic eﬀects, with a

%Inhibition (mean±SD) of 60.4 ± 0.85, at a

concentration of 30µM. The IC

±SD value for this

extract was calculated to be 18.19±0.19 µM, with p <,

highlighting its eﬃcacy in impeding cell growth. In

contrast, the n-hexane extract from the same Papaya

variety showed insigniﬁcant activity, with a 40.5 ± 0.70

inhibition (mean ± SD) at a 30 µM concentration,

falling below the 50% threshold and thus deemed

inactive. Instead, the positive control, Doxorubicin,

showed vigorous activity, with a 93.7 ± 0.45%

Inhibition (mean ± SD) at a 30 µM concentration and

In-Vitro Cytotoxic Potential of Carica Papaya Leaves

J Gandhara Med Dent Sci

October - December 2025

an exceptionally low IC50 ± SD value, emphasizing its

potent ability to suppress cell growth. For the cervical-

HeLa cell line, the results shown in Table 2 exhibited

that the acetone extract displayed a 26.9 ± 0.72%

Inhibition (mean ± SD). In comparison, the n-hexane

extract illustrated a 38.9 ± 0.64% Inhibition (mean ±

SD). Despite these ﬁndings, both extracts fell short of

the predeﬁned cut-oﬀ value of 50%, rendering them

inactive at the tested concentrations. In contrast, the

standard anti-cancer drug Doxorubicin showed notable

efﬁcacy against cervical cancer cells, with a remarkable

% inhibition (mean ± SD) of 98.7 ± 0.47 at a

concentration of 30 µM. The low IC

value of

1.13±0.16µM for Doxorubicin emphasizes its potent

ability to suppress cancer cell growth. For the prostate

PC3 cell line, the results, as demonstrated in Table 3,

illustrated that the acetone extract, when administered

at a concentration of 30 µM, showed a moderate %

inhibition (mean ± SD) of 30.5 ± 0.69. Although it

showed signiﬁcant activity, it did not meet the required

threshold of 50% inhibition for classiﬁcation as active.

Similarly, the n-hexane extract, administered at a

concentration of 30 µM, showed a relatively low

percentage of inhibition (mean ± SD), 8.0 ± 0.44,

classifying it as inactive. In contrast, Doxorubicin, at a

concentration of 30 µM, revealed a signiﬁcant %

inhibition (mean ± SD) of 80.8 ± 0.62, indicating a

positive effect against prostate cancer cells.

Table 1: Cytotoxic Eﬀect of Papaya Leaves Variety of Red Lady

Extract on BJ-Human Fibroblast Cell Lines (Conc.30 µM)

Sample %Inhibi

tion

(mean±SD)

IC50±

(µM)

One-

way

ANOVA

Tukey’s HSD

Post Hoc Test

(Pairwise

comparisons)

Acetone

ext. of

CPL. Var.RL

60.4 ± 0.85

18.19±

0.19

P<0.001

Acet vs n-

Hex∼20(P<0.001)

Acet vs Std∼33.0

(P<0.01)

N -hexane

ext. of

CPL. Var.RL

40.5 ± 0.70

Inactive

n -Hex vs

Std ∼53.0(P<0.001)

Doxorubicin

(Std)

93.7 ± 0.45

0.16±0.

Table 2: Cytotoxic Eﬀect of Papaya Leaves Variety of Red Lady

Extract on Cervical-Hela Cell Line (Conc. 30 µM)

Sample %Inhibi

tion

(Mean±

SD)

IC50±

(µM)

One-

way

ANO

Tukey’s HSD

Post Hoc Test

(Pairwise

Comparisons)

Acetone

ext. of

CPL. Var.RL

26.9 ±

0.72

Inactive

P<0.001

Acet vs n-

Hex∼12(P<0.001)

Acet vs

Std∼71.7(P<0.01)

N-hexane

ext. of

CPL. Var.RL

38.9 ±

0.64

Inactive

n-Hex vs

Std∼59.8(P<0.001)

Doxorubicin

(Std)

98.7

±0.47

1.13±0.

Table 3: Cytotoxic Eﬀect of Papaya Leaves Variety of Red Lady

Extract on Prostate-Pc3 Cell Line (Conc. 30µM)

Sample %Inhibi

tion

(mean±

SD)

IC50±

(µM)

One -

way

ANOVA

Tukey’s HSD

Post Hoc Test

(Pairwise

Comparisons)

Acetone

ext. of

CPL. Var.

30.5 ±

0.69

Inactive

P<0.0

Acet vs

nHex∼22.8(P<0.0

01)Acet vs

Std∼50.0(P<0.01)

N-hexane

ext. of

CPL. Var.

8.0 ±

0.44

Inactive

n-Hex vs

Std∼72.8(P<0.001)

Doxorubicin

(Std)

80.8 ±

0.62

1.18±0.

Figure 1: MTT method in PC3, HeLa, and BJ cells exposed to

diﬀerent concentrations of Carica Papaya Leaves variety of Red

Lady extracts, and assays were performed in triplicate.

DISCUSSION

Many anti-cancer drugs derived from botanical sources

are currently recognized as successful treatments in

medical settings. Vinca alkaloids,

Taxanes, Podophyllotoxin and its analogues, and

Camptothecin and the derivatives are the four main

categories into which these agents fall.

In the leaves

of papaya, numerous compounds have been reported to

possess potential anti-tumor activity, including α-

tocopherol, lycopene, ﬂavonoids, and benzyl-

isothiocyanate.

21,22

The results for BJ-Human ﬁbroblast

cell lines suggest that the acetone extract of these leaves

showed important activity, as indicated by a substantial

% Inhibition (mean±SD) of 60.4 ± 0.85, at a

concentration of 30 30µM, suggesting that Papaya

leaves could be a valuable source of agents. The

relatively low IC

value indicates a strong inhibitory

effect on cell growth, underscoring the therapeutic

potential of the acetone extract from Papaya leaves.

Whereas the n-hexane extract from the Papaya (RL)

Leaf demonstrated moderate activities, with a 40.5 ±

0.70 %Inhibition (mean±SD) at (30μM conc.), falling

below the 50% threshold and thus deemed inactive. In

In-Vitro Cytotoxic Potential of Carica Papaya Leaves

J Gandhara Med Dent Sci

October - December 2025

the research examination of Pawlicka, MA et al.

Reported in 2022 that the experienced isoﬂavonoid is

intimately connected to its strength. Increasing the

concentrations of Genistein (50μM and 100μM)

reduces the number of ﬁbroblast cells with longer

contact times (48 hours). However, the decrease in

strength of Genistein (10 & 20μM) exhibited a higher

number of dermal ﬁbroblasts. 23 Our research study

results further support the thought that the cytotoxic

activities of Papaya leaves (RL) might vary depending

on the extraction solvent utilized. Overall, our ﬁndings

highlight the potential of Papaya leaves, particularly the

acetone extract from the RL variety, as a source of

novel anti-proliferative agents. The research ﬁndings

reveal the cytotoxic potential of Papaya Leaves against

the cervical HeLa cell line. Both acetone and n-hexane

extracts exhibited cancer cell growth inhibition; their

potency was insigniﬁcant at the tested concentrations.

The result for the prostate-PC3 cell line showed that the

acetone extract of Papaya RL Leaf yielded a negligible

effect when dispensed at a concentration of 30μM,

illustrating a reasonable % inhibition (mean ± SD) of

30.5 ± 0.69. Even though it demonstrated considerable

activity. The cytotoxicity of the acetone extract is

recorded against BJ ﬁbroblasts, but not in HeLa or PC3

cells, possibly due to the presence of mid-polarity

phytoconstituents that exert nonspeciﬁc toxic properties

on normal cells. In contrast, cancer cells evade death by

modulating apoptosis or eﬄux mechanisms. Further

phytochemical studies are necessary to understand the

selectivity better. Likewise, the n-hexane extract of

Papaya Leaf, at a concentration of 30 μM, showed a

comparatively low inhibition rate of 8.01%, indicating

it to be inactive. In 2020, Abankwa JK et al.

investigated the antioxidant and anti-cancer activities of

Moringa oleifera, Phyllanthus amarus, and Carica

papaya. Aqueous extract of Carica papaya in particular

exhibited signiﬁcant anti-prostate cancer activity,

selective enough toward PC3 prostate cancer cells to

record an IC₅₀ value of 45.68 ± 1.16 µg/mL and a very

high selectivity index (SI = 18). This provides evidence

for the therapeutic promise of C. papaya as a rich

source of bioactive compounds capable of selectively

targeting cancer cells with minimal eﬀects on normal

cells, thereby supporting its traditional application in

complementary medicine. There remains a further need

to deepen mechanistic and in vivo studies to validate its

application in the management of prostate cancer.

Further optimization of Papaya leaf extracts is

recommended to enhance their cytotoxic eﬃcacy across

diﬀerent cell lines. Future studies should focus on

identifying and isolating speciﬁc bioactive components

responsible for the observed moderate activity against

the BJ-cell line, with the goal of developing targeted

therapies.

LIMITATIONS

This study is limited by its reliance on in-vitro assays,

which may not fully replicate the complex interactions

in living organisms. Only two extraction solvents were

tested, which may not capture the complete

phytochemical potential of papaya leaves. Furthermore,

the study focused on a limited number of cell lines, and

the sample size for assays was relatively small,

restricting the generalizability of the ﬁndings.

Additional in-vivo studies, broader solvent systems, and

mechanistic analyses are required to validate and

expand upon these results.

CONCLUSIONS

The acetone extract displays signiﬁcant activity against

BJ-cells (59.5, 60.48, and 61.2%), while the n-hexane

extract is inactive, based on a cut-oﬀ value of 50%.

Both extracts have minimal eﬀects on HeLa cells and

none on PC3 Cells, falling short of the predeﬁned cut-

oﬀ value of 50%.

CONFLICT OF INTEREST: None

FUNDING SOURCES: None

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Asadullah - Concept & Design; Data Acquisition; Drafting

Manuscript; Final Approval

Saeed - Concept & Design; Data Acquisition; Data

Analysis/Interpretation; Drafting Manuscript; Final Approval

Asif - Concept & Design; Data Acquisition; Drafting

Manuscript; Critical Revision; Final Approval

Masood Ahmed Unar- Concept & Design; Data Acquisition;

Drafting Manuscript; Final Approval

AUTHORS CONTRIBUTION

The authors accept responsibility for all aspects of the work

and will ensure that any concerns regarding the accuracy or

integrity of any part are properly investigated and resolved.

In-Vitro Cytotoxic Potential of Carica Papaya Leaves