Cytotoxicity and antiproliferative activity of essential oils from lemon, wild orange and petitgrain against MCF-7, HepG2 and HeLa cancer cells


  • Worachot SAENGHA Mahasarakham University, Faculty of Technology, Department of Biotechnology, Natural Antioxidant Innovation Research Unit, Mahasarakham 44150 (TH)
  • Thipphiya KARIRAT Mahasarakham University, Faculty of Technology, Department of Biotechnology, Natural Antioxidant Innovation Research Unit, Mahasarakham 44150 (TH)
  • Benjaporn BURANRAT Mahasarakham University, Faculty of Medicine, Mahasarakham 44000 (TH)
  • Teeraporn KATISART Mahasarakham University, Faculty of Science, Department of Biology, Mahasarakham 44150 (TH)
  • Nyuk Ling MA Universiti Malaysia Terengganu, Faculty of Science and Marine Environment, 21030, Kuala Nerus, Terengganu (MY)
  • Vijitra LUANG-IN Mahasarakham University, Faculty of Technology, Department of Biotechnology, Natural Antioxidant Innovation Research Unit, Mahasarakham 44150 (TH)



antimigration, apoptosis, Citrus aurantium, Citrus limon, Citrus sinensis, MTT assay


The purpose of this study was to determine the chemical composition and biological properties of the citrus essential oils (EOs) derived from orange rinds (peels) of lemon (Citrus limon), wild orange (Citrus sinensis) from Brazil extracted by the cold pressed/expressed method, and leaves and twigs of petitgrain (Citrus aurantium) from Paraguay extracted by steam distillation. These food grade EOs were evaluated for their cytotoxic activity in breast, liver, and cervical cancer cells (MCF-7, HepG2 and HeLa) via MTT assay, antiproliferative activity via colony formation assay, and antimigratory activity via wound healing assay, and apoptosis via DNA fragmentation and morphology assessment. The major compounds found in lemon EO were D-limonene (66.75%), beta-pinene (12.82%), and gamma-terpinene (11.57%), totaling over 90% of the identified compounds. For wild orange, the only predominant compound was limonene (96.60%), and the rest, found in minor amounts, included alpha-pinene, bicyclohexane, beta-pinene, beta-myrcene, 3-carene, and o-cymene. For petitgrain EO, linalyl isobutyrate (51.76%) and linalool (26.86%) were mainly detected. Based on the MTT assay, petitgrain EO was the most effective against MCF-7, HepG2 and HeLa. However, wild orange EO was the most antiproliferative and antimigratory against all three cells using the anticolony formation assay and wound healing assay, respectively. The results showed that cell death is associated with the apoptotic process, with morphological hallmarks of apoptosis including membrane blebbing and DNA fragmentation. These findings imply that the three citrus EOs might be used as active components in functional food products for chemopreventive benefits.


Asjad H, Akhtar M, Bashir S, Gulzar B, Khalid R, Asad M (2013). Phenol, flavonoid contents and antioxidant activity of six common citrus plants in Pakistan. Journal of Pharmaceutical and Cosmetic Sciences 1:1-5.

Bai X, Tang J (2020). Myrcene exhibits antitumor activity against lung cancer cells by inducing oxidative stress and apoptosis mechanisms. Natural Product Communications 15(9):1-7.

Ben Hsouna A, Ben Halima N, Smaoui S, Hamdi N (2017). Citrus lemon essential oil: chemical composition, antioxidant and antimicrobial activities with its preservative effect against Listeria monocytogenes inoculated in minced beef meat. Lipids in Health and Disease 16(1):146.

Chebet JJ, Ehiri JE, McClelland DJ, Taren D, Hakim IA (2021). Effect of d-limonene and its derivatives on breast cancer in human trials: a scoping review and narrative synthesis. BMC Cancer 21(1):902.

Chidambara Murthy KN, Jayaprakasha GK, Patil BS (2012). D-limonene rich volatile oil from blood oranges inhibits angiogenesis, metastasis and cell death in human colon cancer cells. Life Science 91:429-439.

Cho M, So I, Chun JN, Jeon JH (2016). The antitumor effects of geraniol: Modulation of cancer hallmark pathways (Review). International Journal of Oncology 48(5):1772-1782.

Cholke PB, Bhor AK, Shete AM, Sonawane RK (2017). Extraction and GC-MS analysis of orange (Citrus sinensis) peel oil. Research Journal of Life Sciences, Bioinformatics, Pharmaceutical and Chemical Sciences 2(5):41-51.

Dahham SS, Tabana YM, Iqbal MA, Ahamed, MB, Ezzat MO, Majid AS, Majid AM (2015). The anticancer, antioxidant and antimicrobial properties of the sesquiterpene β-Caryophyllene from the essential oil of Aquilaria crassna. Molecules (Basel, Switzerland)20(7):11808-11829.

De Pasquale F, Siragusa M, Abbate L, Tusa N, De Pasquale C, Alonzo G (2006). Characterization of five sour orange clones through molecular markers and leaf essential oils analysis. Scientia Horticulturae 109:54-59.

Di Sotto A, Di Giacomo S, Rubini E, Macone A, Gulli M, Mammola CL, …, Mazzanti G (2020). Modulation of STAT3 signaling, cell redox defenses and cell cycle checkpoints by β-Caryophyllene in cholangiocarcinoma cells: possible mechanisms accounting for doxorubicin chemosensitization and chemoprevention. Cells 9(4):858.

Do TKT, Hadji-Minaglou F, Antoniotti S, Fernandez X (2015). Authenticity of essential oils. Trends in Analytical Chemistry 66:146-157.

Dosoky NS, Setzer WN (2018). Biological activities and safety of Citrus spp. Essential oils. International Journal of Molecular Sciences 19(7):1966.

Ellouze I, Abderrabba M (2014). Kinetics of extraction of Citrus aurantium essential oil by hydrodistillation: influence on the yield and the chemical composition. Journal of Materials and Environmental Science 5:841-848.

Fernández-Bedmar Z, Anter J, de La Cruz-Ares S, Muñoz-Serrano A, Alonso-Moraga A, Pérez-Guisado J (2011). Role of citrus juices and distinctive components in the modulation of degenerative processes: genotoxicity, antigenotoxicity, cytotoxicity, and longevity in Drosophila. Journal of Toxicology and Environmental Health. Part A74(15-16):1052-1066.

Fitsiou E, Anestopoulos I, Chlichlia K, Galanis A, Kourkoutas I, Panayiotidis MI, Pappa A (2016). Antioxidant and antiproliferative properties of the essential oils of Satureja thymbra and Satureja parnassica and their major constituents. Anticancer Research 36(11):5757-5763. anticanres.11159

Gezici S (2018). Promising anticancer activity of lavender (Lavandula angustifolia Mill.) Essential oil through induction of both apoptosis and necrosis. Annals of Phytomedicine 7:38-45.

Girola N, Figueiredo CR, Farias CF, Azevedo RA, Ferreira AK, Teixeira SF, … Lago JH (2015). Camphene isolated from essential oil of Piper cernuum (Piperaceae) induces intrinsic apoptosis in melanoma cells and displays antitumor activity in vivo. Biochemical and Biophysical Research Communications 467(4):928-934.

Gniewosz M, Kraśniewska K, Kosakowska O, Pobiega K, Wolska I (2017). Chemical compounds and antimicrobial activity of petitgrain (Citrus aurantium L. var. amara) essential oil. Herba Polonica 63:18-25.

Grandview Research Essential Oils Market Size (2022). Share & Trends Analysis Report by Application (Cleaning & Home, Medical, Food & Beverages, Spa & Relaxation), By Product, By Sales Channel, And Segment Forecasts, 2019–2025. Retrieved 2022 March 5 from

Hata T, Sakaguchi I, Mori M, Ikeda N, Kato Y, Minamino M, Watabe K (2003). Induction of apoptosis by Citrus paradisi essential oil in human leukemic (HL-60) cells. In Vivo 17:553-559.

Iwasaki K, Zheng YW, Murata S, Ito H, Nakayama K, Kurokawa T, … Ohkohchi N (2016). Anticancer effect of linalool via cancer-specific hydroxyl radical generation in human colon cancer. World Journal of Gastroenterology 22(44):9765-9774.

Jo H, Cha B, Kim H, Brito S, Kwak BM, Kim ST, … Lee MG (2021). α-Pinene enhances the anticancer activity of natural killer cells via ERK/AKT pathway. International Journal of Molecular Sciences 22(2):656.

Kulthanachairojana N, Chansriwong P, Thokanit NS, Sirilerttrakul S, Wannakansophon N, Taychakhoonavudh S (2021). Home-based chemotherapy for stage III colon cancer patients in Thailand: Cost-utility and budget impact analyses. Cancer Medicine10(3):1027-1033.

Lee JH, Lee K, Lee DH, Shin SY, Yong Y, Lee YH (2015). Anti-invasive effect of β-myrcene, a component of the essential oil from Pinus koraiensis cones, in metastatic MDA-MB-231 human breast cancer cells. Korean Society for Applied Biological Chemistry 58:563-569.

Mitropoulou G, Fitsiou E, Stavropoulou E, Papavassilopoulou E, Vamvakias M, Pappa A, … Kourkoutas Y (2015). Composition, antimicrobial, antioxidant, and antiproliferative activity of Origanum dictamnus (dittany) essential oil. Microbial Ecology in Health and Disease 26:26543.

Monajemi R, Oryan S, Haeri-Roohani A, Ghannadi A, Jafarian A. (2010). Cytotoxic effects of essential oils of some Iranian Citrus peels. Iranian Journal of Pharmaceutical Research 4(3):183-187.

Negreiros HA, de Moura KG, Barreto do Nascimento M, do Nascimento Rodrigues DC, Ferreir P, Braz DC, … de Castro E Sousa JM (2021). Alpha-Terpineol as antitumor candidate in pre-clinical studies. Anti-cancer Agents in Medicinal Chemistry 21(15):2023-2031.

Ngân TTK, Nguyen OB, Mười NV, Truc TT, My VT (2020). Chemical composition and antibacterial activity of orange (Citrus sinensis) essential oils obtained by hydrodistillation and solvent free microwave extraction. IOP Conference Series: Materials Science and Engineering 991:012023.

Nipin SP, Kang DY, Joung YH, Park JH, Kim WS, Lee HK, …, Yang YM (2017). Nobiletin inhibits angiogenesis by regulating Src/FAK/STAT3-mediated signaling through PXN in ER+ breast cancer cells. International Journal of Molecular Sciences 18:935.

Njoroge SM, Lan Phi NT, Sawamura M (2009). Chemical composition of peel essential oils of sweet oranges (Citrus sinensis) from Uganda and Rwanda. Journal of Essential Oil Bearing Plants 12(1):26-33.

Salehi B, Upadhyay S, Erdogan Orhan I, Kumar Jugran A, LD Jayaweera S, A Dias D, … Sharifi-Rad J (2019). Therapeutic potential of α- and β-Pinene: a miracle gift of nature. Biomolecules 9(11):738.

Sherif AE, Marzouk AM, Zaghloul MG, Halim AF (2015). Chemical composition and cytotoxic activity of petitgrain essential oil of Citrus aurantium L. "Russian colon". Journal of American Science 11(8):64-68

Su YC, Hsu KP, Wang EI, Ho CL (2013). Composition and in vitro anticancer activities of the leaf essential oil of Neolitsea variabillima from Taiwan. Natural Product Communications 8(4):531-532.

Virani S, Bilheem S, Chansaard W, Chitapanarux I, Daoprasert K, Khuanchana S, ... Sriplung H (2017). National and subnational population-based incidence of cancer in Thailand: assessing cancers with the highest burdens. Cancers 9(8):108.

Wang Y, Wang L, Tan J, Li R, Jiang ZT, Tang SH (2021). Comparative analysis of intracellular and in vitro antioxidant activities of essential oil from white and black pepper (Piper nigrum L.). Frontiers in Pharmacology 12:680754.

Yang C, Chen H, Chen H, Zhong B, Luo X, Chun J (2017). Antioxidant and anticancer activities of essential oil from Gannan navel orange peel. Molecules (Basel, Switzerland)22(8):1391.

Yu L, Yan J, Sun Z (2017). D-limonene exhibits anti-inflammatory and antioxidant properties in an ulcerative colitis rat model via regulation of iNOS, COX-2, PGE2 and ERK signaling pathways. Molecular Medicine Reports 15:2339-2346.

Yu X, Lin H, Wang Y, Lv W, Zhang S, Qian Y, … Qian B (2018). D-limonene exhibits antitumor activity by inducing autophagy and apoptosis in lung cancer. OncoTargets and Therapy11:1833-1847.

Zu Y, Yu H, Liang L, Fu Y, Efferth T, Liu X, Wu N (2010). Activities of ten essential oils towards Propionibacterium acnes and PC-3, A-549 and MCF-7 cancer cells. Molecules (Basel, Switzerland)15(5):3200-3210.



How to Cite

SAENGHA, W., KARIRAT, T., BURANRAT, B., KATISART, T., MA, N. L., & LUANG-IN, V. (2022). Cytotoxicity and antiproliferative activity of essential oils from lemon, wild orange and petitgrain against MCF-7, HepG2 and HeLa cancer cells. Notulae Botanicae Horti Agrobotanici Cluj-Napoca, 50(3), 12713.



Research Articles
DOI: 10.15835/nbha50312713

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