Topical Application of Sepia Ink Extract-Loaded Phytosome on Mouse Skin Injury Accelerates Wound Healing: A Histopathological,Immunohistochemical,and Biochemical Study
Restricted accessResearch articleFirst published online 2026
Topical Application of Sepia Ink Extract-Loaded Phytosome on Mouse Skin Injury Accelerates Wound Healing: A Histopathological,Immunohistochemical,and Biochemical Study
Sepia officinalis ink contains bioactive secondary metabolites abundant in melanin granules, has extensive medicinal properties and is commonly utilized in the prevention of different diseases. This research examined the topical wound healing efficacy of sepia ink extract (SIE) and SIE- Nanoparticle-loaded phytosome (SIE-NP) in a murine model of excisional wound healing. Mice were divided into five groups, all groups were anesthetized, and excisional skin wounds were established using a biopsy punch, except the control group. Both SIE and SIE-NP significantly improved the wound healing rate; nevertheless, the effect of SIE-NP was more pronounced. Treatment with SIE and SIE-NP accelerated the wound healing process by mitigating oxidative stress, evidenced by a reduction in malondialdehyde levels and an enhancement in antioxidants levels, including catalase and glutathione. SIE and SIE-NP attenuated oxidative stress-induced DNA fragmentation in wound tissue. Additionally, they reduced the levels of inflammatory markers, such as TNF-α and IL-6, myeloperoxidase activity, and the infiltration of inflammatory cells in the skin tissue. Furthermore, SIE and SIE-NP reduced histopathological abnormalities and concentration of fragmented DNA and elevated nitric oxide levels and collagen content. Moreover, SIE and SIE-NP decreased apoptosis by lowering caspase 3 expression. The data indicate that SIE and SIE-NP expedited wound healing via their antioxidant, anti-inflammatory, and anti-apoptotic characteristics.
SariBR, Yesilot S, Ozmen O, Aydin Acar C. Superior in vivo wound-healing activity of biosynthesized silver nanoparticles with Nepeta cataria (catnip) on excision wound model in rat. Biol Trace Elem Res. 2025;203(3):1502–1517.
3.
SenCK. Human wounds and its burden: An updated compendium of estimates. Adv Wound Care. 2019;8(2):39–48.
4.
RathnaRPKulandhaivelM. Advancements in wound healing: Integrating biomolecules, drug delivery carriers, and targeted therapeutics for enhanced tissue repair. Arch Microbiol. 2024;206(4):199.
5.
Sakr HIKhowailedAAKamelMMFarghalyMEFaridEZ. Endothelial-platelet dysfunction as an indicator of pre-eclampsia and its severity. Med J Cairo Univ. 2019;87:1775–1782.
6.
BukatukaCFMbituyimana B, Xiao L, et al.Recent trends in the application of cellulose-based hemostatic and wound healing dressings. J Funct Biomater. 2025;16(5):151. doi:10.3390/jfb16050151
7.
ChinkoBCPrecious-AbrahamAD. Wound healing activity of hydromethanolic Dioscorea bulbifera extract on male Wistar rat excision wound models. Pharmacol Res Mod Chin Med. 2024;11:100425.
8.
AlsareiiSA, Ahmad J, Umar A, Ahmad MZ, Shaikh IA. Enhanced in vivo wound healing efficacy of a novel piperine-containing bioactive hydrogel in excision wound rat model. Molecules. 2023;28(2):545. doi:10.3390/molecules28020545
9.
KhalilH, Cullen M, Chambers H, McGrail M.Medications affecting healing: An evidence-based analysis. Int Wound J. 2017;14(6):1340–1345.
10.
NqakalaZB, Sibuyi NRS, Fadaka AO, Meyer M, Onani MO, Madiehe AM. Advances in nanotechnology towards development of silver nanoparticle-based wound-healing agents. Int J Mol Sci. 2021;22(20):11272. doi:10.3390/ijms222011272
11.
Fath-All AA, Atia T, Mohamed AS, et al.Efficacy of yeast-mediated SeNPs on gastric ulcer healing and gut microbiota dysbiosis in male albino rats. Tissue Cell. 2025;96:102953. doi:10.1016/j.tice.2025.102953
12.
Hamdan S, Pastar I, DrakulichS, et al.Nanotechnology-driven therapeutic interventions in wound healing: Potential uses and applications. ACS Cent Sci. 2017;3(3):163–175.
13.
BaraniM, Sangiovanni E, Angarano M, et al.Phytosomes as innovative delivery systems for phytochemicals: A comprehensive review of literature. Int J Nanomedicine. 2021;16:6983–7022.
14.
SaleemiMALimV. Phytosomes used for herbal drug delivery. In: BarabadiHMostafaviESaravananM, eds. Pharmaceutical Nanobiotechnology for Targeted Therapy. Springer; 2022:255–279.
15.
TafishAM, El-Sherbiny M, Al-Karmalawy AA, Soliman OAE, Saleh NM.Carvacrol-loaded phytosomes for enhanced wound healing: Molecular docking, formulation, DoE-aided optimization, and in vitro/in vivo evaluation. Int J Nanomedicine. 2023;18:5749–5780.
16.
AbdelmawgoodIA, Mahana NA, Badr AM, Mohamed AS.Echinochrome exhibits anti-asthmatic activity through suppression of airway inflammation, oxidative stress, and histopathological alterations in ovalbumin-induced asthma in BALB/c mice. Naunyn Schmiedebergs Arch Pharmacol. 2024;397(3):1803–1815.
17.
DerbyCD. Cephalopod ink: Production, chemistry, functions and applications. Mar Drugs. 2014;12(5):2700–2730. doi:10.3390/md12052700
18.
SadekSA. Sepia officinalis ink mitigates gastric ulcer via modulation of antioxidant/anti-inflammatory pathways. Beni-Suef Univ J Basic Appl Sci. 2022;11(1):63.
19.
MetwallyAA, Shanab O.Cuttlebone extract (Sepia officinalis) exerts its anti-inflammatory effects via suppression of the NF-κB signaling pathway. Aswan Univ J Sci Technol. 2025;5(1):197–205.
20.
FahmySRSolimanAMAliEM. Antifungal and antihepatotoxic effects of sepia ink extract against oxidative stress in invasive pulmonary aspergillosis in neutropenic mice. Afr J Tradit Complement Altern Med. 2014;11(3):148–159.
21.
Jeevana JyothiBMary RagalathaP. Development and in vitro evaluation of phytosomes of naringin. Asian J Pharm Clin Res. 2019;15(1):280–289.
EbeedBWAbdelmawgood IA, KotbMA, et al.Β-glucan nanoparticles alleviate acute asthma by suppressing ferroptosis and DNA damage in mice. Apoptosis. 2025;30(1-2):35–54.
24.
MielkeH, Strickland J, Jacobs MN, Mehta JM.Biometrical evaluation of the performance of the revised OECD test guideline 402 for assessing acute dermal toxicity. Regul Toxicol Pharmacol. 2017;89:26–39.
25.
SassP Sosnowski P, KamińskaJ, et al. Examination of epigenetic inhibitor zebularine in treatment of skin wounds in healthy and diabetic mice. J Tissue Eng Regen Med. 2022;16(12):1238–1248.
26.
Xavier-SantosJBPassos JGR, Gomes JAS, et al.Topical gel containing phenolic-rich extract from Ipomoea pes-capre leaf has anti-inflammatory, wound-healing, and antiophidic properties. Biomed Pharmacother. 2022;149:112921.
27.
SchieleARHenze IS, Bettschart-Wolfensberger R, Gent TC. Antinociceptive and cardiorespiratory effects of a single dose of dexmedetomidine in laboratory mice subjected to craniotomy. Animals (Basel).2024;14(6):913. doi:10.3390/ani14060913
28.
SakulAAOkur ME, AylaS, et al.Wound healing activity of Arum maculatum. Medeniyet Med J. 2023;38(1):8–15. doi:10.4274/MMJ
29.
MirhajMTavakoli M, VarshosazJ, et al.Preparation of a biomimetic bi-layer chitosan wound dressing composed of A-PRF/sponge layer and L-arginine/nanofiber. Carbohydr Polym. 2022;292:119648. doi:10.1016/j.carbpol.2022.119648
BeutlerEDuronOKellyBM. Improved method for the determination of blood glutathione. J Lab Clin Med. 1963;61:882–888.
33.
AebiH. Catalase in vitro. In: Methods in Enzymology. Academic Press; 1984:121–126.
34.
AllertonFW. The determination of nitrates in water. Analyst. 1947;72(857):349–351.
35.
IoannouYAChenFW. Quantitation of DNA fragmentation in apoptosis. Nucleic Acids Res. 1996;24(5):992–993.
36.
AhmadSUBinti Aladdin NA, Jamal JA, Shuid AN, MohamedIN. Evaluation of wound-healing and antioxidant effects of Marantodes pumilum in an excision wound model. Molecules. 2021;26(1):228. doi:10.3390/molecules26010228
37.
Beheshti AlaghaBDavoodiFMohammadiRMahmoudiSS. Effects of fisetin on cutaneous full-thickness wound healing in a rat model. Iran J Vet Surg. 2026;21(1):9–14. doi:10.30500/ivsa.2025.519077.1444
38.
HauckSZager P, HalfterN, et al.Collagen/hyaluronan-based hydrogels releasing sulfated hyaluronan improve dermal wound healing in diabetic mice via reducing inflammatory macrophage activity. Bioact Mater. 2021;6(12):4342–4359. doi:10.1016/j.bioactmat.2021.04.026
39.
AhmadMTahir M, HongZ, et al.Plant and marine-derived natural products: sustainable pathways for drug discovery and therapeutic development. Front Pharmacol. 2025;15:1497668. doi:10.3389/fphar.2024.1497668
40.
SalaheldinATShehata MR, Sakr HI, Atia T, MohamedAS. Therapeutic potency of ovothiol A on ethanol-induced gastric ulcers in Wistar rats. Mar Drugs. 2022;21(1):25. doi:10.3390/md21010025
41.
AbdelmawgoodIAMahana NA, BadrAM, et al.Echinochrome ameliorates physiological, immunological, and histopathological alterations in ovalbumin-induced asthma. Mar Drugs. 2023;21(8):455. doi:10.3390/md21080455
42.
XieJLi H, Che H, Dong X, Yang X, XieW. Extraction, physicochemical characterisation, and bioactive properties of ink melanin from cuttlefish (Sepia esculenta). Int J Food Sci Technol. 2021;56(7):3627–3640. doi:10.1111/ijfs.14992
43.
CukjatiDReberšekSMiklavčičD. A reliable method of determining wound healing rate. Med Biol Eng Comput. 2001;39(2):263–271.
44.
HuangZWangWZengG, et al.Regulating Sepia melanin agglomerates using tailored kaolinite to accelerate skin wound healing. Colloids Surf B Biointerfaces. 2026;260:115410. doi:10.1016/j.colsurfb.2025.115410
45.
PotekaevNNBorzykh OB, MedvedevGV, et al.The role of extracellular matrix in skin wound healing. J Clin Med. 2021;10(24):5947. doi:10.3390/jcm10245947
46.
YamasakiKEdington HD, McCloskyC, et al.Reversal of impaired wound repair in iNOS-deficient mice by topical adenoviral-mediated iNOS gene transfer. J Clin Invest. 1998;101(5):967–971. doi:10.1172/JCI2067
47.
DonniniSZicheM. Constitutive and inducible nitric oxide synthase: Role in angiogenesis. Antioxid Redox Signal. 2002;4(5):817–823.
48.
PintoRVCarvalhoSAntunesFPiresJPintoML. Emerging nitric oxide and hydrogen sulfide releasing carriers for skin wound-healing therapy. ChemMedChem. 2022;17(1):e202100429. doi:10.1002/cmdc.202100429
49.
YounisNSMohamedMEEl SemaryN. Green synthesis of silver nanoparticles by Synechocystis sp.: characterization and wound-healing actions. Mar Drugs. 2022;20(1):56. doi:10.3390/md20010056
50.
SerraMBBarroso WA, da SilvaNN, et al.From inflammation to current and alternative therapies in wound healing. Int J Inflamm. 2017;2017:3406215. doi:10.1155/2017/3406215
51.
MahmoudNNHamad K, Al ShibitiniA, et al.Investigating inflammatory markers in wound healing: Understanding implications and identifying artifacts. ACS Pharmacol Transl Sci. 2024;7(1):18–27. doi:10.1021/acsptsci.3c00336
52.
Mahmoud Al-AdwanSSalem Al-Qaisi T, Abduljalal JabbarA, et al.Anchusa officinalis accelerates wound healing via improved TGF-β1 expression, antioxidant levels, and inhibition of TNF-α and IL-6. Cutan Ocul Toxicol. 2025;44(2):147–160. doi:10.1080/15569527.2025.2481145
53.
VaidyanathanLLokeswariTS. Anti-bacterial and anti-inflammatory properties of Vernonia arborea accelerate healing of infected wounds in adult zebrafish. BMC Complement Med Ther. 2024;24(1):95. doi:10.1186/s12906-024-04383-8
54.
NamYKim J, Baek J, KimW. Improvement of cutaneous wound healing via topical application of heat-killed Lactococcus chungangensis CAU 1447 on diabetic mice. Nutrients. 2021;13(8):2666. doi:10.3390/nu13082666
55.
MurthySGautam MK, Goel S, Purohit V, Sharma H, GoelRK. Evaluation of in vivo wound-healing activity of Bacopa monniera. Biomed Res Int. 2013;2013:972028. doi:10.1155/2013/972028
56.
KwonMJKim B, Lee YS, KimTY.Role of superoxide dismutase 3 in skin inflammation. J Dermatol Sci. 2012;67(2):81–87. doi:10.1016/j.jdermsci.2012.06.003
57.
KhalafMLSoliman AM, Fahmy SR, MohamedAS. Echinochrome-A attenuates arterial thrombosis complications in liver and kidney in rats. Curr Chem Biol. 2025;19(1):50–64. doi:10.2174/0122127968334087241210053042
58.
AbdelmawgoodIAKotb MA, EltrasHSH, et al.Protective effect of isopimpinellin on ovalbumin-induced airway inflammation and oxidative stress. Respir Physiol Neurobiol. 2025;337:104470. doi:10.1016/j.resp.2025.104470
59.
AbdelmawgoodIAKotb MA, Waleed EbeedB, et al.2-Hydroxychalcone Attenuates airway inflammation, oxidative stress, and NF-κB activation in ovalbumin-induced asthma. J Asthma. 2025;62(11):1880–1892. doi:10.1080/02770903.2025.2526384
60.
YoshikawaTNaito Y, KishiA, et al.Role of active oxygen, lipid peroxidation, and antioxidants in indomethacin-induced gastric mucosal injury in rats. Gut. 1993;34(6):732–737. doi:10.1136/gut.34.6.732
61.
MusalmahMFairuz AH, Gapor MT, NgahWZ. Effect of vitamin E on plasma malondialdehyde, antioxidant enzyme levels, and wound closure during healing in normal and diabetic rats. Asia Pac J Clin Nutr. 2002;11(7):S448–S451. doi:10.1046/j.1440-6047.11.s.7.6.x
62.
HiguchiY. Chromosomal DNA fragmentation in apoptosis and necrosis induced by oxidative stress. Biochem Pharmacol. 2003;66(8):1527–1535.
63.
HiguchiYMatsukawaS. Appearance of 1-2 mbp giant DNA fragments as an early response leading to cell death by oxidative stress. Free Radic Biol Med. 1997;23(1):90–99.
64.
DavoodiFMohammadi R, Asri-Rezaei S, Behfar M, Dezfoulian O, RaisiA. Effects of pyrroloquinoline quinone (PQQ) on skin wound healing in mice. Surgery. 2025;184:109453. doi:10.1016/j.surg.2025.109453
65.
FahmySRSolimanAM. In vitro antioxidant, analgesic, and cytotoxic activities of Sepia officinalis ink and Coelatura aegyptiaca extracts. Afr J Pharm Pharmacol. 2013;7(22):1512–1522.
66.
GuYPYang XM, LuoP, et al.Inhibition of acrolein-induced autophagy and apoptosis by glycosaminoglycan from Sepia esculenta ink in mouse leydig cells. Carbohydr Polym. 2017;163:270–279. doi:10.1016/j.carbpol.2017.01.081
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
