TABLE 2.

Detailed description of the studies that used thymol, included in the systematic review.

Model	Concentration/Incubation time	Experimental methods for testing IC50 values	Results/targets			Conclusion	Authors (Year), Country
			Increase	Decrease	IC50
Monoterpene thymol
In vitro studies
HepG2	150–900 μmol	–	Cytotoxic effects	DNA damage level	–	HepG2 cells were slightly more sensitive to the effects	Horváthová et al. (2006), Slovakia
Caco-2	24 of incubation
K-562	200, 400, 600, 800, 1,000 μM	Trypan blue exclusion	Cytotoxic effects	DNA damage level	500 μM–24 h	Thymol has cytotoxic, antioxidant effects and has a protective action against DNA damage	Horvathova et al. (2007), Slovakia
	24 or 48 h of incubation
P-815	0.004–0.5% v/v	MTT assay	Cytotoxic effects	–	0.015% v/v–48 h	Thymol is cytotoxic	Jaafari et al. (2007), Morocco
	48 h of incubation
HepG2	150–1,000 μM	Trypan blue exclusion	Cytotoxic effects	Cell proliferation	HepG2 - 400 μM–24 h	Thymol has antiproliferative and protective effects	Slamenová et al. (2007), Slovakia
			Resistance to harmful DNA effects (antioxidant properties)
Caco-2	24 of incubation				Caco-2 - 700 μM–24 h
HeLa	15, 30.5, 61, 122, 244 ng/mL	–	Cytotoxic effects	Cell survival	–	Thymol has strong antitumor activity against the HeLa cell line	Abed, (2011), Iraq
Hep	72 h of incubation
MG63	100, 200, 400, 600 μmol/L	–	Cytotoxic effects	Cell viability	–	Thymol showed antitumor activity in MG63 cells, moreover, its apoptotic effect is related to the pronounced antioxidant activity	Chang et al. (2011), China
	24 h of incubation		Apoptosis induction
			Generation of ROS
HL-60	5, 25, 50, 75, 100 μM	–	Cytotoxic effects	Cell viability	–	Apoptosis induced by thymol in HL-60 cells involves the dependent and independent pathways of caspase	Deb et al. (2011), India
	24 h of incubation		Apoptosis induction	Cells in phases G0/G1, S and G2/M
			Cells in sub phase G0/G1 generation of ROS	Cell cycle stop in phase G0/G1 Bcl-2
			Caspase-9, -8 and -3
DBTRG-05MG	200, 300, 400, 500, 600, 800 μM	–	Cytotoxic effects	Cell viability	–	Thymol induces cell death in human glioblastoma cells	Hsu et al. (2011), China
			Apoptosis induction and necrosis
	24 h of incubation
HepG2	20–200 μg/mL	CellTiter-Blue® cell viability assay	Cytotoxic effects	Membrane damage	60.01 μg/mL–24 h	Thymol exhibits antioxidant activities and anti-cancer effects on cells	Özkan and Erdogan, (2011), Turkey
			Antiproliferative effects
	24 h of incubation
P-815	0.05–1.25 μM	MTT assay	Cytotoxic effects	Cell cycle stop in phase G0/G1	P-815–0.15 μM–48 h	Thymol showed relevant cytotoxic effects in all tested strains	Jaafari et al. (2012), Morocco
CEM	48 h of incubation				CEM - 0.31 μM–48 h
K-562					K-562–0.44 μM–48 h
MCF-7					MCF-7 - 0.48 μM–48 h
MCF-7gem					MCF-7gem -
H1299	10–2,000 μM	CellTiter-Blue® cell viability assay	Cytotoxic effects	Membrane and DNA damage	497 μM–24 h	Thymol exhibited a cytotoxic and antioxidant effect	Ozkan and Erdogan, (2012), Turkey
	24 and 48 of incubation		MDA		266 μM–48 h
			8-OHdG
B16-F10	75, 150, 300, 600, 1,200 μM	Trypan blue and MTT assay	Cytotoxic effects	Cell viability	400 μM	Thymol showed antitumor effect with moderate cytotoxicity	Satooka and Kubo, (2012), United States
	24 h of incubation		Generation of ROS
				Density of melanoma cells
HepG2	1.56–50 μg/mL	Trypan blue assay	Cytotoxicity only for B16-F10 cells	–	HepG2 - > 25 μg/mL	Thymol showed cytotoxicity to B16-F10 cells	Ferraz et al. (2013), Brazil
K-562	72 h of incubation		Apoptosis induction in HepG2 cells		K-562–72 h
B16-F10			Induction of caspase-3-dependent apoptotic cell death in HepG cells		B16-F10–18.23 μg/mL–72 h
PC-3	10, 30.50, 70, 100 μg/mL	MTT assay	Cytotoxic effects	Cell viability	PC-3 - 18 μg/mL–48 h	Thymol exhibited cytotoxicity and induced apoptosis	Pathania et al. (2013), India
MDA-MB 231			Apoptosis induction	Cell proliferation	MDA-MB 231–15 μg/mL–48 h
A549	48 h of incubation		DNA fraction sub G0	PI3K/AKT/mTOR	A549–52 μg/mL–48 h
MCF-7			TNF-R1		MCF-7 - 10 μg/mL–48 h
HL-60			Bax	Bcl-2	HL-60–45 μg/mL–48 h
			Caspase-8 and 9
Caco-2	100–2,500 μM	–	–	–	–	The cells exposed to thymol remained unchanged and did not produce any cytotoxic, apoptotic or necrotic effects at any of the tested concentrations	Llana-Ruiz-Cabello et al. (2014), Spain
	24 and 48 h of incubation
A549	1–1.000 μM	SRB assay	Cytotoxic effects	–	A549–0.187 ± 0.061 mΜ–72 h	Thymol exhibited more effective cytotoxicity against cells (Hep3B), while cells (A549) were less sensitive to treatment and cells (HepG2) were more resistant	Fitsiou et al. (2016), Greece
HepG2	72 h of incubation		Antiproliferative effects		HepG2 - 0.390 ± 0.01 mΜ–72 h
Hep3B					Hep3B- 0.181 ± 0.016 mΜ–72 h
AGS	100, 200, 400 μM	–	Cytotoxic effects	Cell viability	–	Thymol has potent anticancer effects on gastric cancer cells	Kang et al. (2016), Republic of Korea
			Apoptosis induction
			Sub-G1 phase	Cell growth
	6, 12, 24 h of incubation		Generation of ROS
			Bax	MMP
			Caspase-8, -7 and -9
C6	0.1, 0.3, 1, 3, 10,	–	–	Cell viability	–	Thymol is a potential candidate for the treatment of malignant gliomas	Lee et al. (2016), Republic of Korea
	30, 100, 200 µM			Cell migration
	24 h of incubation			p-ERK1/2
				MMP-2 and -9
A549	0–250 μM	–	Cytotoxic effects	Cell viability	–	Thymol has cytotoxic and antioxidant activity and its cytotoxic effect was greater than that of carvacrol	Coccimiglio et al. (2016), Canada
	24 h of incubation
HCT-116	100, 150, 200 μg/mL	–	Cytotoxic effects	Cell proliferation	–	Thymol can be used as a potent drug against colon cancer due to its lower toxicity	Chauhan et al. (2018), Republic of Korea
	24 h of incubation		Apoptosis induction	Clonogenic potential
			Generation of ROS
			Caspase-3
			p-JNK
			Cyt C
HepG2	0.06, 0.11, 0.22, 0.45, 0.90 μg/μL	MTT assay	–	Cell viability	289 mg/L–24 h	Thymol has therapeutic potential in tumor cells without adverse effects on healthy cells	Elshafie et al. (2017), Italy
	24 h of incubation			Hepatocarcinoma cells
T24	25, 50, 100, 150 μM	MTT assay	Cytotoxic effects	Cell viability	T24–90.1 ± 7.6 μM–24 h	Thymol can be used as a promising anticancer agent against bladder cancer	Li et al. (2017), China
SW780	24 h of incubation or 100 μM –		Apoptosis induction	Cell cycle stop in phase G2/M	SW780–108.6 ± 11.3 μM–24 h
			p21	Cyclin A and B1
J82	6, 12, 24, 36 h of incubation		Caspase-3 and -9	CDK2	J82–130.5 ± 10.8 μM–24 h
			p-JNK
			p-p38
			MAPK	PI3K/Akt
			Generation of ROS
PC-3	100, 300, 500, 700, 900 μM	–	Cytotoxic effects	Cell viability	–	Thymol was cytotoxic to PC-3 cells	Yeh et al. (2017), China
	24 h of incubation		Induction of cell death
Cal7	200–800 µM	Cell Titer 96 ® Aqueous non-Radioactive cell Proliferation assay	Cytotoxic effects	Cell viability	350 μM–500 μM	Thymol had cytotoxic, antiproliferative and antitumor effects	De La Chapa et al. (2018), United States
SCC4	48 h of incubation
SCC9
HeLa
H460
MDA-231
PC-3
AGS	10, 20, 30, 50, 100, 200, 400, 600 µM	CellTiter-Glo Luminescent cell viability assay	Apoptotic effects	Cell viability	75.63 ± 4.01 µM–24 h	Thymol has cytotoxic, apoptotic, genotoxic and dose-dependent ROS-generating effects	Günes-Bayir et al. (2018), Turkey
	24 h of incubation		Necrosis	Bcl-2
			Bax
			Caspase-3 and -9
			Generation of ROS	GSH levels
			Genotoxic effect
MCF-7	10, 15, 30, 50, 80, 100, 200 μg/mL	MTT assay	Cytotoxic effects	Bcl-2	MDA-MB 231–56 μg/mL–24 h	Thymol has antiproliferative effects	Jamali et al. (2018), Iran
MDA-MB 231	24 h of incubation		Antiproliferative effect	Interruption of cell cycle progression in the S phase	MCF-7 - 47 μg/mL–24 h
			Apoptosis induction
			Caspase-3
			Bax
			Generation of ROS
			Sub-G1 phase
MCF-7	5, 10, 20, 30, 40, 50, 75, 100 g/mL	MTT assay	Cytotoxic effects	Number of cancer cells	54 μg/mL - 48 h	Thymol can induce the process of apoptosis in MCF-7 and, therefore, can be considered an anticancer agent	Seresht et al. (2019), Iran
	48 and 72 h of incubation		p53	Cell cycle arrest induction	62 μg/mL - 72 h
			p21
HT-29	62.5, 125, 250, 500, 750, 1,000 ppm	Trypan Blue exclusion assay	Cytotoxic effects	–	152.1 ± 18.0 ppm–24 h	Thymol induces cytotoxicity and provides genoprotective effects	Thapa et al. (2019), United Kingdom
	24 h of incubation		Genoprotective effects
MDA-MB 231	100, 200, 400, 600, 800 µM	MTT assay	Cytotoxic effects	–	MDA-MB 231–208.36 μM–72 h;	Thymol has apoptotic and antiproliferative properties and can serve as a potential therapeutic agent	Elbe et al. (2020), Turkey
PC-3	24, 48 and 72 h of incubation		Antiproliferative effect		PC-3 - 711 μM–24 h, 601 μM–48 h and 552 μM–72 h;
DU 145			Apoptosis induction		DU 145–799 μM–24 h, 721 μM–48 h and 448 μM–72 h
KLN 205					KLN 205–421 μM–48 h and 229.68 μM–72 h
SKOV-3	100, 200, 400, 600 μM	MTT assay	Apoptosis induction	Cell viability	316.08 μM–24 h	Thymol was cytotoxic to the ovarian cancer cell line and it was more potent than carvacrol	Elbe et al. (2020), Turkey
	24 and 48 h of incubation				258.38 μM–48 h
HCT116	10, 20, 40, 80, 120 μg/mL	CCK-8 Kit	Apoptosis induction	Proliferative capacity	LoVo - 41.46 μg/mL - 48 h	Thymol treatment reduced the proliferative capacity of cells and suppressed cell migration and invasion	Zeng et al. (2020), China
					HCT116–46.74 μg/mL - 48 h
LoVo	24, 48 and 72 h of incubation		Bax	Cell migration and invasion
			Caspase-3 and PARP	Cell cycle stop
			Cells in phase G0/G1	Bcl-2
				Cells in S and G2/M phases
AGS	0–600 μM	CellTiter-Glo Luminescent cell viability assay	Cytotoxic effects	Cell viability	75.63 ± 4.01 μM–24 h	Thymol has cytotoxic and antioxidant effects in gastric adenocarcinoma	Günes-Bayir et al. (2020), Turkey
	24 h of incubation		Generation of ROS	GSH levels
			Apoptosis induction
			Bax	Bcl-2
			Caspase-3 and -9
			DNA damage
A549	25–200 μg/mL	MTT assay	Antiproliferative effect	Cell viability	745 μM–24 h	Thymol can act as a safe and potent therapeutic agent to treat non-small cell lung cancer	Balan et al. (2021), India
	12 and 24 h of incubation		Apoptosis induction	MMP
			DNA damage	Bcl-2
			Generation of ROS
			Caspase-3 and -9
			Bax	SOD
			Cells in phase G0/G1
			TBARBS
			CARBONIL
KG1	25, 50, 100 μM	–	Cell death	Cell viability	–	KG1 cells treated with 50 µM thymol were more sensitive compared to the other two lines. At 100 μM, thymol induced complete cell death of KG1 and HL60 cells, while about 50% of K562 cells resisted cell death after 48 h of treatmentl	Bouhtit et al. (2021), Belgium
K-562	24 and 48 h of incubation
HL-60

Model	Concentration	Results/Targets		Conclusion	Authors (Year), country
		Increase	Decrease
In vivo studies
Female athymic nude rats were injected subcutaneously in the right flank with 3 × 106 Cal27 or HeLa cells in 0.1 mL of sterile PBS	4.3 mM thymol (32 μg diluted in 50 μl sterile saline with a final concentration of 0.25% DMSO)	Apoptotic cells	Tumor volume reduction	Thymol had cytotoxic, antiproliferative and antitumor effects	De La Chapa et al. (2018), United States
			Proliferative cells
Xenograft model: BALB/c male nude mice were injected subcutaneously with HCT116 cells (1 × 107 cells in 0.2 mL of PBS) on the back	Xenograft model and lung metastasis model: Intraperitoneal injection for 30 days with thymol at 75 mg/kg 1x on alternate days or thymol at 150 mg/kg 1x on alternate days	Necrotic lesions	Tumor growth and metastasis	Thymol inhibits the growth and metastasis of colorectal cancer in vivo by suppressing Wnt/β-catenin signaling and the EMT program	Zeng et al. (2020), China
			Average number of tumor nodules on the surface of the lungs
		Bax	Ki-67 expression level
Lung metastasis model: HCT116 cells (1 × 106) were intravenously injected into the tail vein of each mouse			Cell proliferation
			Bcl-2
			Wnt/β-catenin signaling pathway
		Caderina-E	Vimentina
			Cyclin D1
			C-myc
			Survivin
Male Wistar rats injected with DMH (40 mg/kg intraperitoneally, twice a week) for 16 consecutive weeks	20 mg/kg/day, orally, for 16 weeks	Final body weight	Mortality	Thymol administration had promising preclinical protective efficacy by promoting inhibition of oxidative stress, inflammation and induction of apoptosis	Hassan et al. (2021), Egypt
		Weight gain	Incidence of ACF
		Growth rate	Serum CEA levels
		NRF2	Serum levels of CA19-9
			Caspase-3
			TNF-α
		GST, GSH, SOD, CAT	NF-κB
			IL-6
			Tissue content of MDA (colon lipid peroxidation)

Abbreviations: 5RP7, Mouse embryonic fibroblast with transformation of H-ras oncogenes; 8-OHdG, 8-hydroxy-2′-deoxyguanosine; A375, Melanoma (skin) cancer cell line; A549, Lung Carcinoma Cell Line; ACF, Aberrant crypt foci; AFP, Alpha-fetoprotein serum; AFU, Alpha l-fucosidase; AgNORs, Proteins Associated with the Argyrophilic Nucleolar Organizing Region; AGS, Human gastric carcinoma cell line; ALP, Alkaline Phosphatase; ALT, Alanine transaminase; AST, Aspartate transaminase; AXL, Tyrosine Kinase Receptor; B[a]P, 3.4 benzopurene; B16-F10, Mouse melanoma cells; BT-474, Breast ductal carcinoma; BT-483, Breast ductal carcinoma; C6, Glioma cell line; Caco-2, Cell line derived from human colon carcinoma; CA 19–9, Tumor markers carbohydrate antigen 19–9; Cal27, Cell line of the squamous cell carcinoma of the tongue; CAT, Catalase; CEA, Carcinoembryonic antigen; CCK-8, Cell Counting Kit-8; CCND1, Gene encoding the cyclin D1 protein; CDK4 or 6, Cyclin-dependent kinases; cGT, Glutamyl transpeptidase Range; CyT C, Cytochrome C; c-Myc, Proto-oncogene; CO25, Mouse muscle cell line; COX-2, Cyclooxygenase; DAPK1, Protein kinase 1 associated with death; DBTRG-05MG, Human Glioblastoma Cells; DEN, Diethylnitrosamine; DMH, 1,2-dimethylhydrazine; DMBA, 7,12-dimethylbenz[a]anthracene; DMSO, Dimethylsulfoxide; DNA, Deoxyribonucleic acid; DU 145, Human Prostate Cancer Cell Line; EC50, Half of the maximum effective concentration; EMF, Acute T Lymphoblastoid Leukemia; EMT, Epithelial-mesenchymal transition; ERK 1/2, Kinase 1/2 regulated by extracellular signal; ERO, Reactive Oxygen Species; GGT, Gamma-Glutamyltransferase; GPx, Glutathione Peroxidase; GR, Glutathione reductase; GSH, Reduced Glutathione; H1299, Parental and Drug Resistant Human Lung Cancer Cell Line; H460, Non-small cell lung cancer cell line; HCT116, Colorectal adenocarcinoma cell line; HeLa, Human Cervical Cancer Cell Line; Hep, Human Laryngeal Squamous Cell Carcinoma; Hep3Β, Human Hepatocellular Carcinoma Cell Line; HepG2, Human Hepatocellular Carcinoma Cell Line; HL-60, Human Acute Promyelocytic Leukemia Cell Line; HT-29, Colorectal adenocarcinoma cell line; IC50, Half of the maximum inhibitory concentration; IL-6, Interleukin-6; J82, Bladder Cancer Cell Line; Jagged-1, Jagged Canonical Notch Ligand 1; JAR, Human Choriocarcinoma Cell Line; JEG3, Human Choriocarcinoma Cell Line; Jurkat, Lymphocytes derived from T-cell lymphoma; KG1 and K-562, Human Myelogenous Leukemia Cell Line; Kelly, Neuroblastoma cell line; Ki-67, Antigen, biomarker; KLN 205, Non-small cell lung cancer; LDH, Lactate dehydrogenase; LoVo, Colorectal Adenocarcinoma Cell Line; MAPK, Protein kinase activated by mitogen; MTT, Methyl Tetrazolium Test; MTS, Tetrazolium salt reduction; MCF-7, Human breast cancer cell line; MCF-7gem, Gemcitabine-resistant human breast adenocarcinoma; MDA, Malondialdehyde; MDA-MB 231, Human metastatic breast adenocarcinoma cell line; MDA-MB 453, Human metastatic breast adenocarcinoma cell line; MDPK, Myotonic dystrophy protein kinase; MG63, Human Osteosarcoma Cell Line; MMP, Potential of the mitochondrial membrane; MMP-2 or 9, Metalloproteinase-2 or 9 of the matrix; N2a, Rat neuroblastoma cell line; NDEA, N-nitrosodiethylamine; Notch-1, Signaling path; NSCLC, Non-small cell lung cancer; OC2, Human oral cancer cells; OSCC, Human oral squamous cell carcinoma; p21, WAF1 encoding gene; p38, Mitogen-activated protein kinases; p53, tumor protein; P-815, Murine Mastocytoma Cell Line; p-AKT, Phospho-protein kinase B; PBS, Sterile phosphate buffered saline; PC-3, Human Prostate Cancer Cell Line; PCNA, Proliferating Cell Nuclear Antigen; PI3K/AKT/mTOR, Phosphoinositide-3-kinase/Akt/mammalian target; PI3K/Akt, Phosphoinositide-3-kinase-Akt; p-JNK, Fosto-c-Jun N-terminal kinase; p-p38, Phospho-p38; PPP2R2A, Serine/threonine-protein phosphatase 2A; p-STAT3, Phospho-signal transducer and transcription activator; SRB, Sulforhodamine B; SCC-25, Human squamous cell carcinoma cell line; SCC4 and SCC9, Human oral squamous cell carcinoma cell line; SH-SY5Y, Neuroblastoma cell line; SiHa, Human Cervical Cancer Cell Line; SKOV-3, Ovarian cancer cell line; SOD, Superoxide dismutase; SW780, Bladder cancer cell line; T24, Bladder Cancer Cell Line; TAC, Total antioxidant capacity; TBARS, Thiobarbituric Acid Reactive Substances; TCA-8113, Human tongue squamous cell carcinoma cell line; TNF-α, Tumor Necrosis Factor-Alpha; TNFR1, Tumor necrosis factor 1 receptor; TOS, Total oxidant status; TRPM7, Subfamily M of the cation channel of the potential transient receptor Member 7; U87, Human glioblastoma cell line; VEGF, Vascular endothelial growth factor; XXT, 2.3‐bis(2‐methoxy‐4‐nitro‐5‐sulfophenyl)‐2H‐tetrazolium-5-carboxanilide inner salt.