Phytochemical profiling and anticancer potential of Cymbopogon citratus extract

Abstract

Objective: To evaluate the anticancer potential of Cymbopogon citratus extract. Methods: GC-MS analysis was used to identify phytocomponents in the methanolic extract of Cymbopogon citratus. A fractionation method was employed to isolate and assess the bioactivity of different fractions and their cytotoxic activities against cancer cell lines HCT116, LoVo, Caco-2, and HT-29 were investigated. A dual staining method with acridine orange and ethidium bromide was used to assess the effect of the extract on cell apoptosis. Additionally, the expression levels of Bax and TP53 were quantified using real-time PCR in Caco-2 cells treated with the ethyl acetate fraction of Cymbopogon citratus extract. A protein array was employed to profile key pro- and anti-apoptotic proteins in Caco-2 cells. Moreover, molecular docking studies were conducted to investigate the interactions between key compounds of Cymbopogon citratus extract and specific apoptosis-related protein domains (PDB IDs: 7wql and 4bkx). Results: A significant growth inhibition was observed in Caco-2 cells treated with Cymbopogon citratus extract. Among the seven fractions of the plant extract, the ethyl acetate fraction showed the highest cytotoxicity against Caco-2 cells with an IC₅₀ value of (6.16 ± 0.01) µg/mL. The immunofluorescence assay showed that the ethyl acetate fraction could induce apoptosis of Caco-2 cells. Moreover, the fraction upregulated the gene expressions of Bax and TP53 in a dose-dependent manner. The docking analysis demonstrated the interaction of five compounds isolated from the ethyl acetate fraction with key proteins in Caco-2 cells, indicating their anticancer properties. Conclusions: Cymbopogon citratus extract shows anticancer activity against Caco-2 cells by inducing apoptosis. It may be a promising candidate for the treatment of colon cancer, which needs further investigation.

Keywords

Cymbopogon citratus / Anticancer / Caco-2 cell / Apoptosis / Colon cancer

Acknowledgments

The authors extend their appreciation to Researchers Supporting Project number (RSP2025R414) King Saud University, Riyadh, Saudi Arabia.

Conflict of interest statement

The authors declare no conflict of interest.

Funding

This work was supported by Researchers Supporting Project number (RSP2025R414) King Saud University, Riyadh, Saudi Arabia.

Data availability statement

The data supporting the findings of this study are available from the corresponding authors upon request.

Authors’ contributions

All authors contributed to the study’s conception and design. Material preparation, data collection, and analysis were performed by BA, MA, BAD, and AR. The first draft of the manuscript was written by BA and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

References

[1] Soerjomataram I, Bray F. Planning for tomorrow: Global cancer incidence and the role of prevention 2020-2070. Nat Rev Clin Oncol 2021; 18(10): 663-672.

[2] World Health Organization. World Health Statistics 2016: Monitoring health for the sustainable development goals (SDGs). [Online] Available from: https://www.who.int/publications/i/item/9789241565264 [Accessed on December 12, 2023].

[3] Wong MC, Huang J, Lok V, Wang J, Fung F, Ding H, et al. Differences in incidence and mortality trends of colorectal cancer worldwide based on sex, age, and anatomic location. Clin Gastroenterol Hepatol 2021; 19(5): 955-966.

[4] Sawicki T, Ruszkowska M, Danielewicz A, Niedźwiedzka E, Arłukowicz T, Przybyłowicz KE. A review of colorectal cancer in terms of epidemiology, risk factors, development, symptoms and diagnosis. Cancers (Basel) 2021; 13(9). doi: 10.3390/cancers13092025.

[5] Greenwell M, Rahman P. Medicinal plants: Their use in anticancer treatment. Int J Pharm Sci Res 2015; 6(10). doi: 10.13040/IJPSR.0975-8232.6(10).4103-12.

[6] Khan T, Ali M, Khan A, Nisar P, Jan SA, Afridi S, et al. Anticancer plants: A review of the active phytochemicals, applications in animal models, and regulatory aspects. Biomolecules 2019; 10(1). doi: 10.3390/biom10010047.

[7] Cooper GM, Hausman R. The cell: A molecular approach. USA: Oxford University Press: Sinauer Associates; 2000, p. 719-728.

[8] Kumar S, Pathania AS, Saxena A, Vishwakarma R, Ali A, Bhushan S. The anticancer potential of flavonoids isolated from the stem bark of Erythrina suberosa through induction of apoptosis and inhibition of STAT signaling pathway in human leukemia HL-60 cells. Chem Biol Interact 2013; 205(2): 128-137.

[9] Mosmann T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J Immunol Methods 1983; 65(1-2): 55-63.

[10] Atanasov AG, Zotchev SB, Dirsch VM, Supuran CT. Natural products in drug discovery: Advances and opportunities. Nat Rev Drug Discov 2021; 20(3): 200-216.

[11] Talib WH, Alsalahat I, Daoud S, Abutayeh RF, Mahmod AI. Plant-derived natural products in cancer research: Extraction, mechanism of action, and drug formulation. Molecules 2020; 25(22). doi: 10.3390/molecules25225319.

[12] Aftab K, Ali M, Aijaz P, Beena N, Gulzar H, Sheikh K, et al. Determination of different trace and essential element in lemon grass samples by X-ray fluorescence spectroscopy technique. Int Food Res J 2011; 18(1): 265-270.

[13] Ajayi E, Sadimenko A, Afolayan A. GC-MS evaluation of Cymbopogon citratus (DC) Stapf oil obtained using modified hydrodistillation and microwave extraction methods. Food Chem 2016; 209: 262-266.

[14] Chen H, Zhu B, Zhao L, Liu Y, Zhao F, Feng J, et al. Allicin inhibits proliferation and invasion in vitro and in vivo via SHP-1-mediated STAT3 signaling in cholangiocarcinoma. Cell Physiol Biochem 2018; 47(2): 641-653.

[15] Oladeji OS, Adelowo FE, Ayodele DT, Odelade KA. Phytochemistry and pharmacological activities of Cymbopogon citratus: A review. Sci Afr 2019; 6. doi: 10.1016/j.sciaf.2019.e00137.

[16] Karkala Manvitha KM, Bhushan Bidya BB. Review on pharmacological activity of Cymbopogon citratus. Int J Herb Med 2014; 1(6): 5-7.

[17] Toropov O. Electron ionization library component of the NIST/EPA/NIH Mass Spectral Library and NIST GC Retention Index Database. [Online] Available from: https://www.nist.gov/programs-projects/electron-ionization-library-component-nistepanih-mass-spectral-library-and-nist-gc [Accessed on December 18, 2023].

[18] Li T, Jiao R, Ma J, Zang J, Zhao G, Zhang T. Zinc binding strength of proteins dominants zinc uptake in Caco-2 cells. RSC adv 2022; 12(33): 21122-21128.

[19] Liu K, Liu PC, Liu R, Wu X. Dual AO/EB staining to detect apoptosis in osteosarcoma cells compared with flow cytometry. Med Sci Monit Basic Res 2015; 21: 15-20.

[20] Fan X, Guo H, Teng C, Zhang B, Blecker C, Ren G. Anti-colon cancer activity of novel peptides isolated from in vitro digestion of quinoa protein in Caco-2 cells. Foods 2022; 11(2). doi: 10.3390/foods11020194.

[21] Umar HI, Saliu TP, Josiah SS, Ajayi A, Danjuma JB. In silico studies of bioactive compounds from selected African plants with inhibitory activity against nitric oxide synthase and arginase implicated in asthma. Egypt J Med Hum Genet 2021; 22: 1-16.

[22] Avoseh O, Oyedeji O, Rungqu P, Nkeh-Chungag B, Oyedeji A. Cymbopogon species; ethnopharmacology, phytochemistry and the pharmacological importance. Molecules 2015; 20(5): 7438-7453.

[23] Almutairi BO, Almutairi MH, Alrefaei AF, Alkahtani S, Alarifi S. HSPB6 is depleted in colon cancer patients and its expression is induced by 5-aza-2′-deoxycytidine in vitro. Medicina 2023; 59(5). doi: 10.3390/medicina59050996.

[24] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72(1-2): 248-254.

[25] Almutairi BO, Almutairi SM, Alarifi S. Synthesis of 1-(4-chlorobenzoyl)-4-(dimethylamino) pyridin-1-ium chloride and determination of cytotoxicity and apoptosis in human colon cancer cells. J King Saud Univ Sci 2024; 36(3). doi: 10.1016/j.jksus.2024.103101.

[26] Ferrer RJ, Ong MJ, Jacinto SD. Extract of Codiaeum luzonicum Merr. overcomes multidrug resistance in human colon cancer cells by modulating P-glycoprotein. Asian Pac J Trop Biomed 2022; 12(9): 400-410.

[27] Labib MM, Amin M, Alzohairy A, Elashtokhy M, Samir O, Hassanein S. Inhibition analysis of aflatoxin by in silico targeting the thioesterase domain of polyketide synthase enzyme in Aspergillus ssp. J Biomol Struct Dyn 2022; 40(10): 4328-4340.

[28] Shoeb M. Anti-cancer agents from medicinal plants. Bangladesh J Pharmacol 2006; 1(2): 35-41.

[29] Kumar S, Korra T, Thakur R, Arutselvan R, Kashyap AS, Nehela Y, et al. Role of plant secondary metabolites in defence and transcriptional regulation in response to biotic stress. Plant Stress 2023; 8. doi: 10.1016/j.stress.2023.100154.

[30] Jin Z, Chai YD, Hu S. Fatty acid metabolism and cancer. Adv Exp Med Biol 2021; 1280: 231-241.

[31] Ukwubile C, Ahmed A, Katsayal U, Ya’u J, Mejida S. GC-MS analysis of bioactive compounds from Melastomastrum capitatum (Vahl) Fern. leaf methanol extract: An anticancer plant. Sci Afr 2019; 3. doi: 10.1016/j.sciaf.2019.e00059.

[32] Liu XQ, Zou QP, Huang JJ, Yook CS, Whang WK, Lee HK, et al. Inhibitory effects of 3α-hydroxy-lup-20 (29)-en-23, 28-dioic acid on lipopolysaccharide-induced TNF-α, IL-1β, and the high mobility group box 1 release in macrophages. Biosci Biotechnol Biochem 2017; 81(7): 1305-1313.

[33] Kaur P, Arora S, Singh R. Isolation, characterization and biological activities of betulin from Acacia nilotica bark. Sci Rep 2022; 12(1). doi: 10.1038/s41598-022-13338-3.

[34] Bharath B, Perinbam K, Devanesan S, AlSalhi MS, Saravanan M. Evaluation of the anticancer potential of hexadecanoic acid from brown algae Turbinaria ornata on HT-29 colon cancer cells. J Mol Struct 2021; 1235. doi: 10.1016/j.molstruc.2021.130229.

[35] Kumari R, Sharma N, Karwasra R, Khanna K. Colon cancer and their targeting approaches through nanocarriers: A review. Asian Pac J Trop Biomed 2023; 13(3): 104-111.

[36] Harada H, Yamashita U, Kurihara H, Fukushi E, Kawabata J, Kamei Y. Antitumor activity of palmitic acid found as a selective cytotoxic substance in a marine red alga. Anticancer Res 2002; 22(5): 2587-2590.

[37] Kasibhatla S, Amarante-Mendes GP, Finucane D, Brunner T, Bossy-Wetzel E, Green DR. Acridine orange/ethidium bromide (AO/EB) staining to detect apoptosis. Cold Spring Harbor Protocols 2006; 2006(3). doi: 10.1101/pdb.prot4493.

[38] Im K, Mareninov S, Diaz MFP, Yong WH. An introduction to performing immunofluorescence staining. Methods Mol Biol 2019; 1897: 299-311.

[39] Shinde V, Desai K. In vitro cytotoxicity, macromolecular interaction and antioxidant potential of dual coated selenium nanoparticles. J Biomed Mater Res B 2022; 110(6): 1400-1411.

[40] Peña-Blanco A, García-Sáez AJ. Bax, Bak and beyond-mitochondrial performance in apoptosis. FEBS J 2018; 285(3): 416-431.

[41] Carneiro BA, El-Deiry WS. Targeting apoptosis in cancer therapy. Nat Rev Clin Oncol 2020; 17(7): 395-417.

[42] Imbabi TA, Habashy WS, Abol-Fetouh GM, Labib MM, Osman A, Elkelish A, et al. Enhancing semen quality, brain neurotransmitters, and antioxidant status of rabbits under heat stress by acacia gum, vitamin C, and lycopene as dietary supplements: An in vitro and in silico study. Ital J Anim Sci 2023; 22(1): 321-336.