Comparative Effects of Ethanol Concentration on Phytochemical Profiles and In Vitro Bioactivities of Lime (Citrus aurantifolia) and Lemon (Citrus limon) Peel Extracts

Amornrat  Srisukong; Orapin  Komutiban; Khwunjit  Itsarasook; Lueacha  Tabtimmai

doi:10.65763/jfhb.2026.e270559

Authors

Amornrat Srisukong Faculty of Science and Technology, Suan Dusit University, Bangkok, 10700 Thailand
Orapin Komutiban Faculty of Science and Technology, Suan Dusit University, Bangkok, 10700 Thailand
Khwunjit Itsarasook Faculty of Science, Ramkhamhaeng University, Bangkok, 10240 Thailand
Lueacha Tabtimmai Faculty of Applied Science, King Mongkut's University of Technology North Bangkok, Bang Sue, Bangkok, 10800 Thailand

DOI:

https://doi.org/10.65763/jfhb.2026.e270559

Keywords:

Lime and lemon peel extracts, Antioxidant activity, Nitric oxide production, UHPLC–ESI–QTOF–MS

Abstract

This study comparatively evaluated the effects of ethanol concentration on the phytochemical characteristics and in vitro biological responses of lime (Citrus aurantifolia) and lemon (Citrus limon) peel extracts. Extractions were performed using 50, 70, and 95% (v/v) ethanol. Extraction yield, total phenolic content (TPC), total flavonoid content (TFC), antioxidant activity using DPPH and ABTS assays, nitric oxide (NO) production and cell viability in LPS-stimulated RAW 264.7 macrophages, and cellular metabolic activity in HeLa and A549 cancer cells were evaluated. Targeted phenolic and flavonoid profiles were also characterized using UHPLC–ESI–QTOF–MS. Lemon peel extracts consistently showed higher extraction yields than lime peel extracts across the tested ethanol concentrations, with the highest yield obtained using 50% ethanol (16.94% for lemon and 6.66% for lime). In contrast, lime peel extracts showed higher TPC values across all ethanol concentrations, with the highest TPC observed in the 95% ethanol lime peel extract (18.08 mg GAE/g extract), whereas the 95% ethanol lemon peel extract showed the highest TFC among all samples (27.82 mg QE/g extract). Among the tested extracts, the 95% ethanol lime peel extract exhibited the strongest antioxidant activity, with DPPH and ABTS IC₅₀ values of 1.04 and 0.53 mg/mL, respectively. The 95% ethanol lime peel extract also showed a lower NO level (55.36 µM) than the LPS control while maintaining moderate cell viability (65.39%). The 95% ethanol lemon peel extract showed a lower NO level (61.41 µM), but this was accompanied by lower cell viability (43.65%), indicating that reduced cellular metabolic activity may have contributed to the lower measured NO level. Targeted UHPLC–ESI–QTOF–MS analysis revealed species- and ethanol concentration-dependent differences in the measured phenolic and flavonoid profiles. Hesperidin and rutin were detected at higher concentrations in lime peel extracts, particularly in the 95% ethanol extract, whereas gallic acid was detected only in lemon peel extracts. Lime peel extracts also generally produced greater reductions in cellular metabolic activity than lemon peel extracts, particularly the 50 and 70% ethanol extracts under selected treatment conditions. Overall, both citrus species and ethanol concentration influenced the chemical composition, antioxidant activity, NO responses, and cellular metabolic activity of the peel extracts. These findings support further investigation of citrus peel extracts as sources of bioactive compounds, while additional analytical and biological studies are required to clarify the contributions of individual constituents and their potential applications.

References

Aktan, F. (2004). iNOS-mediated nitric oxide production and its regulation. Life Sciences, 75(6), 639–653. https://doi.org/10.1016/j.lfs.2003.10.042

Alaqeel, N.K. (2024). Antioxidants from different citrus peels provide protection against cancer. Brazilian Journal of Biology, 84, e271619. https://doi.org/10.1590/1519-6984.271619

Andrade, M.A., Barbosa, C.H., Shah, M.A., Ahmad, N., Vilarinho, F., Khwaldia, K., ... Ramos, F. (2023). Citrus by-products: Valuable source of bioactive compounds for food applications. Antioxidants, 12(1), 38. https://doi.org/10.3390/antiox12010038

Ashraf, H., Iahtisham-Ul-Haq, Butt, M.S., Nayik, G.A., Ramniwas, S., Damto, T., ... Ansari, M.J. (2024). Phytochemical and antioxidant profile of citrus peel extracts in relation to different extraction parameters. International Journal of Food Properties, 27(1), 286–299. https://doi.org/10.1080/10942912.2024.2304274

Ballistreri, G., Fabroni, S., Romeo, F.V., Timpanaro, N., Amenta, M., & Rapisarda, P. (2019). Anthocyanins and other polyphenols in Citrus genus: Biosynthesis, chemical profile, and biological activity. In R.R. Watson (Ed.), Polyphenols in plants: Isolation, purification and extract preparation (2nd ed., pp. 191–215). Academic Press. https://doi.org/10.1016/b978-0-12-813768-0.00014-1

Barreca, D., Bellocco, E., Leuzzi, U., & Gattuso, G. (2014). First evidence of C- and O-glycosyl flavone in blood orange (Citrus sinensis (L.) Osbeck) juice and their influence on antioxidant properties. Food Chemistry, 149, 244–252. https://doi.org/10.1016/j.foodchem.2013.10.096

Benavente-García, O., & Castillo, J. (2008). Update on uses and properties of citrus flavonoids: New findings in anticancer, cardiovascular, and anti-inflammatory activity. Journal of Agricultural and Food Chemistry, 56(15), 6185–6205. https://doi.org/10.1021/jf8006568

Bocco, A., Cuvelier, M.-E., Richard, H., & Berset, C. (1998). Antioxidant activity and phenolic composition of citrus peel and seed extracts. Journal of Agricultural and Food Chemistry, 46(6), 2123–2129. https://doi.org/10.1021/jf9709562

Chemat, F., Abert Vian, M., & Cravotto, G. (2012). Green extraction of natural products: Concept and principles. International Journal of Molecular Sciences, 13(7), 8615–8627. https://doi.org/10.3390/ijms13078615

Cirmi, S., Ferlazzo, N., Lombardo, G.E., Maugeri, A., Calapai, G., Gangemi, S., & Navarra, M. (2016). Chemopreventive agents and inhibitors of cancer hallmarks: May Citrus offer new perspectives? Nutrients, 8(11), 698. https://doi.org/10.3390/nu8110698

Dai, J., & Mumper, R.J. (2010). Plant phenolics: Extraction, analysis and their antioxidant and anticancer properties. Molecules, 15(10), 7313–7352. https://doi.org/10.3390/molecules15107313

Fotakis, G., & Timbrell, J.A. (2006). In vitro cytotoxicity assays: Comparison of LDH, neutral red, MTT and protein assay in hepatoma cell lines following exposure to cadmium chloride. Toxicology Letters, 160(2), 171–177. https://doi.org/10.1016/j.toxlet.2005.07.001

Fraga, C.G., Galleano, M., Verstraeten, S.V., & Oteiza, P.I. (2010). Basic biochemical mechanisms behind the health benefits of polyphenols. Molecular Aspects of Medicine, 31(6), 435–445. https://doi.org/10.1016/j.mam.2010.09.006

García-Nicolás, M., Ledesma-Escobar, C.A., & Priego-Capote, F. (2023). Spatial distribution and antioxidant activity of extracts from citrus fruits. Antioxidants, 12(4), 781. https://doi.org/10.3390/antiox12040781

Hajialyani, M., Farzaei, M.H., Echeverría, J., Nabavi, S.M., Uriarte, E., & Sobarzo-Sánchez, E. (2019). Hesperidin as a neuroprotective agent: A review of animal and clinical evidence. Molecules, 24(3), 648. https://doi.org/10.3390/molecules24030648

Itsarasook, K., Prompamorn, P., Tanghiranrat, J., & Panichakul, T. (2021). Investigation of in vitro antioxidant activity and cytotoxicity on human skin fibroblasts of Pluchea indica (L.) Less. leaves extract and development to microemulsion for skin cosmetic product. Asian Journal of Pharmaceutical and Clinical Research, 14(6), 86–93. https://doi.org/10.22159/ajpcr.2021.v14i6.39880

Khan, M.K., Abert-Vian, M., Fabiano-Tixier, A.-S., Dangles, O., & Chemat, F. (2010). Ultrasound-assisted extraction of polyphenols (flavanone glycosides) from orange (Citrus sinensis L.) peel. Food Chemistry, 119(2), 851–858. https://doi.org/10.1016/j.foodchem.2009.08.046

Khan, M.K., Zill-e-Huma, & Dangles, O. (2014). A comprehensive review on flavanones, the major citrus polyphenols. Journal of Food Composition and Analysis, 33(1), 85–104. https://doi.org/10.1016/j.jfca.2013.11.004

Kim, H.P., Son, K.H., Chang, H.W., & Kang, S.S. (2004). Anti-inflammatory plant flavonoids and cellular action mechanisms. Journal of Pharmacological Sciences, 96(3), 229–245. https://doi.org/10.1254/jphs.crj04003x

Lagha-Benamrouche, S., & Madani, K. (2013). Phenolic contents and antioxidant activity of orange varieties (Citrus sinensis L. and Citrus aurantium L.) cultivated in Algeria: Peels and leaves. Industrial Crops and Products, 50, 723–730. https://doi.org/10.1016/j.indcrop.2013.07.048

Ledesma-Escobar, C.A., Priego-Capote, F., & Luque de Castro, M.D. (2016). Comparative study of the effect of sample pretreatment and extraction on the determination of flavonoids from lemon (Citrus limon). PLOS ONE, 11(1), e0148056. https://doi.org/10.1371/journal.pone.0148056

Li, C., & Schluesener, H. (2017). Health-promoting effects of the citrus flavanone hesperidin. Critical Reviews in Food Science and Nutrition, 57(3), 613–631. https://doi.org/10.1080/10408398.2014.906382

Middleton, E., Jr., Kandaswami, C., & Theoharides, T.C. (2000). The effects of plant flavonoids on mammalian cells: Implications for inflammation, heart disease, and cancer. Pharmacological Reviews, 52(4), 673–751. https://doi.org/10.1016/s0031-6997(24)01472-8

Multari, S., Licciardello, C., Caruso, M., & Martens, S. (2020). Monitoring the changes in phenolic compounds and carotenoids occurring during fruit development in the tissues of four citrus fruits. Food Research International, 134, 109228. https://doi.org/10.1016/j.foodres.2020.109228

Naczk, M., & Shahidi, F. (2004). Extraction and analysis of phenolics in food. Journal of Chromatography A, 1054(1–2), 95–111. https://doi.org/10.1016/j.chroma.2004.08.059

Nogata, Y., Sakamoto, K., Shiratsuchi, H., Ishii, T., Yano, M., & Ohta, H. (2006). Flavonoid composition of fruit tissues of citrus species. Bioscience, Biotechnology, and Biochemistry, 70(1), 178–192. https://doi.org/10.1271/bbb.70.178

Papoutsis, K., Pristijono, P., Golding, J.B., Stathopoulos, C.E., Bowyer, M.C., Scarlett, C.J., & Vuong, Q.V. (2017). Effect of vacuum-drying, hot air-drying and freeze-drying on polyphenols and antioxidant capacity of lemon (Citrus limon) pomace aqueous extracts. International Journal of Food Science & Technology, 52(4), 880–887. https://doi.org/10.1111/ijfs.13351

Parhiz, H., Roohbakhsh, A., Soltani, F., Rezaee, R., & Iranshahi, M. (2015). Antioxidant and anti-inflammatory properties of the citrus flavonoids hesperidin and hesperetin: An updated review of their molecular mechanisms and experimental models. Phytotherapy Research, 29(3), 323–331. https://doi.org/10.1002/ptr.5256

Peng, Z., Zhang, H., Li, W., Yuan, Z., Xie, Z., Zhang, H., ... Xu, J. (2021). Comparative profiling and natural variation of polymethoxylated flavones in various citrus germplasms. Food Chemistry, 354, 129499. https://doi.org/10.1016/j.foodchem.2021.129499

Pietta, P.-G. (2000). Flavonoids as antioxidants. Journal of Natural Products, 63(7), 1035–1042. https://doi.org/10.1021/np9904509

Prior, R.L., Wu, X., & Schaich, K. (2005). Standardized methods for the determination of antioxidant capacity and phenolics in foods and dietary supplements. Journal of Agricultural and Food Chemistry, 53(10), 4290–4302. https://doi.org/10.1021/jf0502698

Rangel-Huerta, O.D., Aguilera, C.M., Martin, M.V., Soto, M.J., Rico, M.C., Vallejo, F., ... Mesa, M.D. (2015). Normal or high polyphenol concentration in orange juice affects antioxidant activity, blood pressure, and body weight in obese or overweight adults. The Journal of Nutrition, 145(8), 1808–1816. https://doi.org/10.3945/jn.115.213660

Re, R., Pellegrini, N., Proteggente, A., Pannala, A., Yang, M., & Rice-Evans, C. (1999). Antioxidant activity applying an improved ABTS radical cation decolorization assay. Free Radical Biology and Medicine, 26(9–10), 1231–1237. https://doi.org/10.1016/s0891-5849(98)00315-3

Rice-Evans, C.A., Miller, N.J., & Paganga, G. (1996). Structure-antioxidant activity relationships of flavonoids and phenolic acids. Free Radical Biology and Medicine, 20(7), 933–956. https://doi.org/10.1016/0891-5849(95)02227-9

Russo, G.L., Russo, M., & Spagnuolo, C. (2014). The pleiotropic flavonoid quercetin: From its metabolism to the inhibition of protein kinases in chronic lymphocytic leukemia. Food & Function, 5(10), 2393–2401. https://doi.org/10.1039/C4FO00413B

Saini, R.K., Ranjit, A., Sharma, K., Prasad, P., Shang, X., Gowda, K.G.M., & Keum, Y.-S. (2022). Bioactive compounds of citrus fruits: A review of composition and health benefits of carotenoids, flavonoids, limonoids, and terpenes. Antioxidants, 11(2), 239. https://doi.org/10.3390/antiox11020239

Seoka, M., Ma, G., Zhang, L., Yahata, M., Yamawaki, K., Kan, T., & Kato, M. (2020). Expression and functional analysis of the nobiletin biosynthesis-related gene CitOMT in citrus fruit. Scientific Reports, 10(1), 15288. https://doi.org/10.1038/s41598-020-72277-z

Singh, B., Singh, J.P., Kaur, A., & Singh, N. (2020). Phenolic composition, antioxidant potential and health benefits of citrus peel. Food Research International, 132, 109114. https://doi.org/10.1016/j.foodres.2020.109114

Tabtimmai, L., Choowongkomon, K., Kittakoop, P., Lekphrom, R., Schevenels, F.T., Jadsadajerm, S., & Wisetsai, A. (2025). Lanostane triterpenes, flavanones and acetogenins from the roots of Uvaria siamensis and their cytotoxic activity. ACS Omega, 10(2), 2253–2259. https://doi.org/10.1021/acsomega.4c09267

Zhang, H., Tian, G., Zhao, C., Han, Y., DiMarco-Crook, C., Lu, C., Bao, Y., Li, C., Xiao, H., & Zheng, J. (2019). Characterization of polymethoxyflavone demethylation during drying processes of citrus peels. Food & Function, 10(9), 5707–5717. https://doi.org/10.1039/c9fo01053j

Zhang, H.-W., Hu, J.-J., Fu, R.-Q., Liu, X., Zhang, Y.-H., Li, J., ... Gao, N. (2018). Flavonoids inhibit cell proliferation and induce apoptosis and autophagy through downregulation of PI3Kγ-mediated PI3K/AKT/mTOR/p70S6K/ULK signaling pathway in human breast cancer cells. Scientific Reports, 8(1), 11255. https://doi.org/10.1038/s41598-018-29308-7

Zou, Z., Xi, W., Hu, Y., Nie, C., & Zhou, Z. (2016). Antioxidant activity of Citrus fruits. Food Chemistry, 196, 885–896. https://doi.org/10.1016/j.foodchem.2015.09.072