Wound-healing potential of citrus lemon honey: Antioxidant, anti-inflammatory, antibacterial, and immunomodulatory effects on keratinocyte proliferation and migration

Abstract

Honey from citrus limon is of interest for wound healing due to its antimicrobial, antioxidant, and immunomodulatory properties. This in vitro study investigates whether citrus lemon honey (CLH) can modulate key cellular and biochemical processes involved in wound healing, in comparison with Manuka honey. Antioxidant, anti-inflammatory, and antibacterial activities were evaluated alongside keratinocyte (HaCaT) proliferation and migration assays. Physicochemical analysis showed high water-soluble protein content (480.9 ± 28.4 mg equivalent [Eq] bovine serum albumin/100 g), acidic pH (3.4 ± 0.02), low moisture (19.5%), and moderate electrical conductivity (41.6 ± 2.61 μS/cm). In addition, CLH exhibited notable phenolic (313.6 ± 0.6 mg Eq gallic acid/100 g) and flavonoid contents (6.9 ± 2.6 mg Eq quercetin/100 g), accompanied by strong antioxidant activity (total antioxidant capacity: 3.5 ± 0.05 g Eq ascorbic acid/100 g). CLH demonstrated antibacterial activity against both Gram-positive and Gram-negative bacteria (minimum inhibitory concentrations of 0.8–3.1 w/w%), including clinically relevant wound-associated pathogens, highlighting its potential to reduce microbial burden and prevent infection in wound environments. Furthermore, CLH modulated nitric oxide production in lipopolysaccharide-activated THP-1 macrophages. Functionally, CLH promoted keratinocyte viability and migration in a concentration-dependent manner, with an approximately 20% increase in cell viability at 1–2 mg/mL and a 35% enhancement in migration, without cytotoxicity. These effects were comparable in trend, though less pronounced, to those observed with Manuka honey. Taken together, the combined antibacterial, anti-inflammatory, and pro-regenerative effects suggest that CLH may support wound healing by enhancing tissue repair and limiting infection. From a host–microbe interaction perspective, the antibacterial and immunomodulatory effects of CLH suggest a coordinated role in wound healing, where reduced microbial burden and controlled regulation of inflammation may act synergistically to support tissue repair and restore skin integrity. However, further in vivo and clinical studies are required to confirm its therapeutic relevance.

Keywords

Citrus lemon honey / Immunomodulatory / Antibacterial / Antioxidant activity / Wound healing / HaCaT cells

References

[1] Chylińska N, Maciejczyk M. Hyaluronic Acid and Skin: Its Role in Aging and Wound-Healing Processes. Gels. 2025; 11(4):281. doi: 10.3390/gels11040281

[2] Baker P, Huang C, Radi R, Moll SB, Jules E, Arbiser JL. Skin Barrier Function: The Interplay of Physical, Chemical, and Immunologic Properties. Cells. 2023; 12(23):2745. doi: 10.3390/cells12232745

[3] Summer M, Ali S, Fiaz U, Hussain T, Khan RR, Fiaz H. Revealing the molecular mechanisms in wound healing and the effects of different physiological factors including diabetes, age, and stress. J Mol Histol. 2024; 55(5):637-654. doi: 10.1007/s10735-024-10223-3

[4] Han G, Ceilley R. Chronic wound healing: a review of current management and treatments. Adv Ther. 2017; 34(3):599-610. doi: 10.1007/s12325-017-0478-y

[5] Blackburn J, Ousey K, Rippon M, Cutting K, White R. Applying antimicrobial strategies in wound care practice: a review of the evidence. Int Wound J. 2025; 22(6):e70684. doi: 10.1111/iwj.70684

[6] El-yagoubi K, Masalha M, Abu-Farich B, Touzani S, Lyoussi B, Saad B. Antibacterial Activity, Antioxidant Benefits, Chemical Composition, and Physicochemical Properties of Ziziphus jujuba Honey from Five Distinct Regions in Morocco. J Pure Appl Microbiol. 2025; 19(2):1358-1370. doi: 10.22207/JPAM.19.2.39

[7] Jarar D, Kmail A, Masalha M, et al. Wound Healing Mechanisms of Medicago sativa Honey: Antioxidant, Anti-inflammatory, Antibacterial and Phytochemical Characterisation. J Nat Remedies. 2025:2381-2395. doi: 10.18311/jnr/2025/50943

[8] Jarar D, Imtara H, Kmail A, et al. Experimental Evaluation of Avocado Honey’s Wound Healing Properties: Physicochemical, Antibacterial, Anti-inflammatory, and Proliferative Effects Combined with In Silico Toxicity and Molecular Docking Analysis. J Ethnopharmacol. 2025; 358:121038. doi: 10.1016/j.jep.2025.121038

[9] Saad, B. Immunomodulatory and Anti-Inflammatory Properties of Honey and Bee Products. Immuno. 2025; 5(2):19. doi: 10.3390/immuno5020019

[10] Wang H, Li L, Lin X, Bai W, Xiao G, Liu G. Composition, functional properties and safety of honey: a review. J Sci Food Agric. 2023; 103(14):6767-6779. doi: 10.1002/jsfa.12720

[11] Mărgăoan R, Topal E, Balkanska R, et al. Monofloral honeys as a potential source of natural antioxidants, minerals and medicine. Antioxidants. 2021; 10(7):1023. doi: 10.3390/antiox10071023

[12] Fratianni F, Amato G, d’Acierno A, Panfili G, Coppola R, Nazzaro F. In vitro prospective healthy and nutritional benefits of different Citrus monofloral honeys. Sci Rep. 2023; 13(1):1088. doi: 10.1038/s41598-023-27802-1

[13] Abu-Farich B, Masalha M, Hamarshi H, et al. In vitro evaluation of Hyphaene thebaica honey as a multitarget therapeutic product. Microbes Immun. 2025; 2(1):78-91. doi: 10.36922/mi.4994

[14] Prieto P, Pineda M, Aguilar M. Spectrophotometric quantitation of antioxidant capacity through the formation of a phosphomolybdenum complex: specific application to the determination of vitamin E. Anal Biochem. 1999; 269(2):337-341. doi: 10.1006/abio.1999.4019

[15] Abu-Farich B, Masalha M, Egbaria E, et al. Physicochemical properties, chemical composition, antioxidant properties, and antibacterial effects of four Palestinian honey varieties. J Pure Appl Microbiol. 2024; 18(4):2315-2327. doi: 10.22207/JPAM.18.4.03

[16] Vargas-Maya NI, Padilla-Vaca F, Romero-González OE, et al. Refinement of the Griess method for measuring nitrite in biological samples. J Microbiol Methods. 2021; 187:106260. doi: 10.1016/j.mimet.2021.106260

[17] Peña OA, Martin P. Cellular and molecular mechanisms of skin wound healing. Nat Rev Mol Cell Biol. 2024; 25(8):599-616. doi: 10.1038/s41580-024-00715-1

[18] Tashkandi H. Honey in wound healing: An updated review. Open Life Sci. 2021; 16(1):1091-1100. doi: 10.1515/biol-2021-0084

[19] Hossain Md. Lokman L, Lim YL, Katherine H, Hettiarachchi D, Locher C. Honey-Based Medicinal Formulations: A Critical Review. Appl Sci. 2021;11(11):5159. doi: 10.3390/app11115159

[20] Sawarkar, A.B. Color intensity and antioxidant properties of honey produced from North Maharashtra, India. J Food Sci. 2025; 62(12):2312-2320. doi: 10.1007/s13197-024-06170-2

[21] Pontis, J.A, Costa, L.A, Silva, S.J, Flach, A. Color, phenolic and flavonoid content, and antioxidant activity of honey from Roraima, Brazil. Food Sci Technol. 2014; 34(1):69-73. doi: 10.1590/S0101-20612014005000015

[22] European Commission. Council Directive 2001/110/EC of December 20, 2001, relating to honey. Off J Eur Communities. 2002; L10:47-52.

[23] Bogdanov S, Ruoff K, Oddo LP. Physico-chemical methods for the characterisation of unifloral honeys: a review. Apidologie. 2004; 35(Suppl. 1):S4-17. doi: 10.1051/apido:2004047

[24] Martinez-Morales F, Alonso-Castro A., Zapata-Morales JR, Carranza-Álvarez C, Aragon-Martinez OH. Use of standardized units for a correct interpretation of IC50 values obtained from the inhibition of the DPPH radical by natural antioxidants. Chem Pap. 2020; 74(10):3325-3334. doi: 10.1007/s11696-020-01161-x

[25] Tananaki C, Rodopoulou MA, Dimou M, Kanelis D, Liolios V. The total phenolic content and antioxidant activity of nine monofloral honey types. Appl Sci. 2024; 14(10):4329. doi: 10.3390/app14104329

[26] Imtara H, Elamine Y, Lyoussi B. Physicochemical characterization and antioxidant activity of Palestinian honey samples. Food Sci Nutr. 2018; 6(8):2056-2065. doi: 10.1002/fsn3.754

[27] Rodrigues M, Kosaric N, Bonham CA, Gurtner GC. Wound healing: a cellular perspective. Physiol Rev. 2018; 99(1):665-706. doi: 10.1152/physrev.00067.2017

[28] Smith J, Rai V. Novel Factors Regulating Proliferation, Migration, and Differentiation of Fibroblasts, Keratinocytes, and Vascular Smooth Muscle Cells during Wound Healing. Biomedicines . 2024; 12(9):1939. doi: 10.3390/biomedicines12091939

[29] Pastar I, Stojadinovic O, Yin NC, et al. Epithelialization in wound healing: a comprehensive review. Adv Wound Care. 2014; 3(7):445-464. doi: 10.1089/wound.2013.0473

[30] Almasaudi SB, Al-Nahari AA, El Sayed M, et al. Antimicrobial effect of different types of honey on Staphylococcus aureus. Saudi J Biol Sci. 2017; 24(6):1255-1261. doi: 10.1016/j.sjbs.2016.08.007

[31] Kassim M, Achoui M, Mustafa MR, Mohd MA, Yusoff KM. Ellagic acid, phenolic acids, and flavonoids in Malaysian honey extracts demonstrate in vitro anti-inflammatory activity. Nutr Res. 2010; 30(9):650-659. doi: 10.1016/j.nutres.2010.08.008

[32] Alvarez-Suarez JM, Giampieri F, Battino M. Honey as a source of dietary antioxidants: structures, bioavailability and evidence of protective effects against human chronic diseases. Curr Med Chem. 2013; 20(5):621-638. doi: 10.2174/092986713804999358

[33] Gushiken LF, Beserra FP, Bastos JK, Jackson CJ, Pellizzon CH. Cutaneous wound healing: An update from physiopathology to current therapies. Life. 2021; 11(7):665. doi: 10.3390/life11070665

[34] Ebadi P, Fazeli M. Evaluation of the potential in vitro effects of propolis and honey on wound healing in human dermal fibroblast cells. S Afr J Bot. 2021; 137:414-422. doi: 10.1016/j.sajb.2020.10.003

[35] Scepankova H, Combarros-Fuertes P, Fresno JM, et al. Role of honey in advanced wound care. Molecules. 2021; 26(16):4784. doi: 10.3390/molecules26164784

[36] Ranzato E, Martinotti S, Burlando B. Epithelial mesenchymal transition traits in honey-driven keratinocyte wound healing: Comparison among different honeys. Wound Repair Regen. 2012; 20(5):778-785. doi: 10.1111/j.1524-475X.2012.00825.x

[37] Tlak Gajger I, Dar SA, Ahmed MM, Aly MM, Vlainić J. Antioxidant capacity and therapeutic applications of honey: Health benefits, antimicrobial activity and food processing roles. Antioxidants. 2025; 14(8):959. doi: 10.3390/antiox14080959

[38] Majtan J. Honey: an immunomodulator in wound healing. Wound Repair Regen. 2014; 22(2):187-192. doi: 10.1111/wrr.12117

[39] Jull AB, Cullum N, Dumville JC, Westby MJ, Deshpande S, Walker N. Honey as a topical treatment for wounds. Cochrane Database Syst Rev. 2015; 3:CD005083. doi: 10.1002/14651858.CD005083.pub4

[40] Ling Y, Feng Y, Li Z. Cheng L, Zhang X, Ren D. Anti-Inflammatory Mechanism of Walnut Meal Peptide/Ellagic Acid Nanoparticles on LPS-Stimulated RAW264.7 Cells by Inhibition of NF-κB and MAPK Activation. Int J Pept Res Ther. 2023; 29(99). doi: 10.1007/s10989-023-10571-w

[41] Hu WH, Dai DK, Zheng BZ, et al. The binding of kaempferol-3-O-rutinoside to vascular endothelial growth factor potentiates anti-inflammatory efficiencies in lipopolysaccharide-treated mouse macrophage RAW264.7 cells. Phytomedicine. 2021; 80:153400. doi: 10.1016/j.phymed.2020.153400

[42] Ramos-Torrecillas J, González-Acedo A, Melguizo-Rodríguez L, et al. Anti-Inflammatory and Antimicrobial Effect of Ellagic Acid and Punicalagin in Dermal Fibroblasts. Int J Mol Sci. 2025; 26(17):8681. doi: 10.3390/ijms26178681

[43] Ardah MT, Eid N, Kitada T, Haque, M.E. Ellagic acid prevents α-synuclein aggregation and protects sh-sy5y cells from aggregated α-synuclein-induced toxicity via suppression of apoptosis and activation of autophagy. Int J Mol Sci. 2021; 22(24):13398. doi: 10.3390/ijms222413398

[44] Alam M, Ahmed S, Elasbali AM, et al. Therapeutic implications of caffeic acid in cancer and neurological diseases. Front Oncol. 2022; 12:860508. doi: 10.3389/fonc.2022.860508

[45] Speisky H, Arias-Santé MF, Fuentes J. Oxidation of quercetin and kaempferol markedly amplifies their antioxidant, cytoprotective, and anti-inflammatory properties. Antioxidants. 2023; 12(1):155. doi: 10.3390/antiox12010155

[46] Wasihun AG, Kasa BG. Evaluation of antibacterial activity of honey against multidrug resistant bacteria in Ayder Referral and Teaching Hospital, Northern Ethiopia. SpringerPlus. 2016; 5(1):842. doi: 10.1186/s40064-016-2493-x

[47] Almasaudi S. The antibacterial activities of honey. Saudi J Biol Sci. 2021; 28(4):2188-96. doi: 10.1016/j.sjbs.2020.10.017

[48] Machado A, Toubarro D, Baptista J, Tejera E, Álvarez-Suárez JM. Selected honey as a multifaceted antimicrobial agent: review of compounds, mechanisms, and research challenges. Future Microbiol. 2025; 20(7-9):589-610. doi: 10.1080/17460913.2025.2498233

[49] Sekar M, Zuraini NZ, Rani NN, Lum PT, Gan SH. Antimicrobial properties of honey. In: Honey: Composition and Health Benefits . John Wiley & Sons, Ltd. 2023:186-196. doi: 10.1002/9781119113324.ch13

[50] Ogwu MC, Izah SC. Honey as a natural antimicrobial. Antibiotics. 2025; 14(3):255. doi: 10.3390/antibiotics14030255

[51] Liu M, Lu J, Müller P, et al. Antibiotic-specific differences in the response of Staphylococcus aureus to treatment with antimicrobials combined with manuka honey. Front Microbiol. 2015; 5:779. doi: 10.3389/fmicb.2014.00779

[52] Kwakman PH, Te Velde AA, de Boer L, Vandenbroucke-Grauls CM, Zaat SA. Two major medicinal honeys have different mechanisms of bactericidal activity. PloS ONE. 2011; 6(3):e17709. doi: 10.1371/journal.pone.0017709

[53] Majtan J, Bohova J, Prochazka E, Klaudiny J. Methylglyoxal may affect hydrogen peroxide accumulation in manuka honey through the inhibition of glucose oxidase. J Med Food. 2014; 17(2):290-293. doi: 10.1089/jmf.2012.0201

[54] Kaźmierczak-Barańska J, Karwowski BT. The Antioxidant Potential of Commercial Manuka Honey from New Zealand-Biochemical and Cellular Studies. Curr Issues Mol Biol. 2024; 46(7):6366-6376. doi: 10.3390/cimb46070380