Search Results (8,924)

Background: This study aimed to explore the synergistic hypoglycemic effect and mechanism of 1-deoxynojirimycin (DNJ) in mulberry leaves and theaflavins (TFs) in black tea. Methods: The synergistic inhibition of α-glucosidase and α-amylase by DNJ-TFs was evaluated using enzyme assays and the Chou–Talalay model. Insulin-resistant (IR) HepG2 cells and high-fat diet (HFD)-induced type 2 diabetes mellitus mice were treated with DNJ, TFs, or DNJ-TFs, determining the efficacy of drug combinations by measuring glycolipids and inflammatory factors. Network pharmacology and molecular docking were used to identify key target genes and signaling pathways, and CETSA experiments were used to verify the binding of drugs to targets. Key genes were further verified by immunofluorescence, Western blot, and Real-time PCR. Results: DNJ-TFs synergistically suppressed α-glucosidase (CI = 0.85) and α-amylase (CI = 0.76). In HepG2 cells, DNJ-TFs ameliorated palmitic acid-induced IR by promoting glucose uptake, attenuating lipid accumulation, and regulating glycolipid metabolism. In HFD mice, DNJ-TFs counteracted hyperglycemia, dyslipidemia, systemic inflammation and oxidative stress, elevated HOMA-IR, and hepatic steatosis. Network pharmacology integrated with experimental validation identified PTGS2 and MMP9 as key binding targets of DNJ and TFs. Furthermore, DNJ-TFs could inhibit the increase in liver TNFα protein and the decrease in p-AKT, p-GSKα, p-GSKβ, and GLUT2 protein caused by high fat, both in vivo and in vitro. Conclusion: DNJ and TFs exert synergistic glucose-lowering effects by targeting PTGS2/MMP9 and regulating the TNFα/AKT/GSK3/GLUT2 axis, providing a promising natural therapeutic strategy for diabetes management. Full article

Background: Electrochemotherapy (ECT) is a clinically validated local ablative treatment increasingly recognized for its ability to induce immunogenic cell death and stimulate antitumor immunity. Its combination with immune checkpoint inhibitors, such as anti-PD-1 antibodies, may enhance systemic immune responses and improve therapeutic efficacy, particularly in poorly immunogenic tumors. Methods: We evaluated the antitumor effectiveness of ECT combined with a murine analog of the anti-PD-1 antibody in four syngeneic murine tumor models with differing histology and immune status: WEHI fibrosarcoma, CT26 and MC38 colorectal carcinoma, and 4T1 mammary carcinoma. In vitro cytotoxicity assays assessed tumor cell sensitivity to ECT, while in vivo experiments evaluated complete response (CR) rates, immune cell infiltration, and long-term immune memory through secondary tumor challenge. Immunohistochemical analysis of CD4⁺, CD8⁺, and granzyme B⁺ effector cells. Results: In vitro, WEHI cells exhibited the highest sensitivity to ECT. In vivo, ECT monotherapy induced CRs in 100% of WEHI tumors, 60% of CT26, 17% of 4T1, and 15% of MC38. The addition of anti-PD-1 significantly enhanced outcomes in less responsive models, increasing CRs to 90% in CT26, 91% in MC38, and 53% in 4T1. Combination therapy promoted pronounced infiltration of CD4⁺, CD8⁺, and granzyme B⁺ T cells and the formation of tertiary lymphoid structure, particularly in MC38 tumors. Secondary challenge experiments confirmed long-term immune memory in CT26 and MC38 models and induced memory in 4T1, which was absent following monotherapy. Conclusions: ECT synergizes with PD-1 blockade to potentiate local and systemic antitumor immunity, overcoming immune resistance in poorly immunogenic tumors. These findings support further clinical development of ECT in combination with immune checkpoint inhibitors as a component of personalized cancer immunotherapy. Full article

Background: Resveratrol (3,5,4′-trihydroxy-trans-stilbene, RVT) is one of the most extensively studied natural polyphenols, with numerous health benefits documented in the literature. One of its most characterized biological properties is the strong antioxidant capacity. However, its poor biopharmaceutical properties limit its in vivo applicability. In this study, we conducted a detailed comparative analysis of the antioxidant and protective capacity of pure and loaded into Pluronic micelles resveratrol. Methods: Various in vitro antioxidant assays, such as DPPH, ABTS, superoxide anion radical scavenging, ferric (FRAP), and copper-reducing power assay (CUPPRAC), and iron-induced lipid peroxidation were performed. In addition, the in vitro 6-OHDA model of neurotoxicity in brain synaptosomes and the in vivo scopolamine (Sco)-induced model of cognitive impairment in rats were also employed. The main antioxidant biomarkers—the levels of lipid peroxidation (LPO) and total glutathione (GSH), as well as activities of superoxide dismutase, catalase, and glutathione peroxidase—were measured in the cortex and hippocampus. Results: The results from the in vitro tests demonstrated better ferric-reducing power activity and better neuroprotective capacity of the micellar resveratrol (mRVT), as evidenced by preserved synaptosomal viability and maintained GSH levels in a concentration-dependent manner in 6-OHDA-induced neurotoxicity. Regarding the in vivo results, mRVT (10 µM concentration) was the most effective treatment in supporting recognition memory formation in dementia rats. Further, mRVT demonstrated better LPO protective capacity in the hippocampus and GSH preserving activity in the cortex than the pure drug. Conclusions: The incorporation of resveratrol in polymeric micelles could enhance its antioxidant and neuroprotective effects. Full article

Epigallocatechin gallate (EGCG)—the most abundant catechin in green tea—is a promising component of advanced composite biomaterials. The pharmacological activity of EGCG is typically attenuated upon thermal processing, although the exact effects of heating free and modified EGCG in air and vacuum are unknown. To bridge this gap, we herein examined the effects of heating free and modified (in gelatin containing beta-tricalcium phosphate granules) EGCG in vacuum and air (100–220 °C, 1–16 h) on its physicochemical and antioxidant properties using water and ethanol solubility measurements, discoloration and antioxidant activity (2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2′-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid) assays, ultraviolet–visible spectroscopy, mass spectrometry, nuclear magnetic resonance spectroscopy, and attenuated total reflectance Fourier transform infrared spectroscopy. The antioxidant activity of EGCG-modified gelatin sponges was assessed in vitro using the DPPH assay and in vivo using a calvarial bone defect model in eight-week-old male Sprague–Dawley rats. Free and modified EGCG showed antioxidant activities, which were largely retained after heating in vacuum at 150 °C. These findings show that appropriate heating procedures preserve the antioxidant activity of EGCG and provide insights for the development of EGCG-based biomaterials. Full article

Botulinum neurotoxin serotype A (BoNT/A) is the most potent known neurotoxin. While its light chain (LC) catalytic domain is a prime target for next-generation vaccines and therapeutics, the functional differences among BoNT/A subtype LCs (A1, A2, A3) remain to be definitively characterized, despite notable sequence variation. This work aimed to systematically compare the proteolytic activity and immunoprotective efficacy of recombinant BoNT/A1-LC, A2-LC, and A3-LC. Recombinant A1-LC-His, A2-LC-His, A3-LC-His, and A3-LC-Twin-Strep proteins were expressed in Escherichia coli (E. coli) and purified with affinity chromatography. Their proteolytic activity was assessed via in vitro SNAP-25 cleavage assays. The protective potency of these antigens was evaluated in a mouse model. In vitro cleavage assays revealed a substrate cleavage efficiency order of A2-LC > A1-LC > A3-LC. In vivo, both A1-LC and A2-LC immunization conferred robust, broad protection against high-dose challenges with all three toxin subtypes. In stark contrast, A3-LC provided only minimal protection against its homologous toxin and none against heterologous subtypes. Crucially, the functional deficit of A3-LC was confirmed to be an intrinsic property, as the A3-LC-TS variant, designed to exclude tag-specific interference, exhibited comparable low efficacy. According to structural research, A3-LC’s compromised function may be caused by a four-amino-acid loss. The inferior performance of A3-LC is inherent to its primary structure. This work identified A1-LC or A2-LC as the potential proteolytic activity molecule and vaccine antigen by demonstrating functional differences among BoNT/A subtype LCs. These findings provide crucial insights for developing subtype-specific countermeasures against botulism. Full article

Acne vulgaris is a prevalent chronic inflammatory skin disorder affecting over 85% of adolescents. Emerging evidence indicates that Cutibacterium acnes phylotype IA₁ contributes to acne initiation and progression, yet its precise mechanisms in epidermal keratinocytes remain unclear. This study investigated C. acnes IA1’s effects on keratinocyte behavior using an in vitro HaCaT cell model. Cells were co-cultured with live C. acnes IA₁ (CICC 10864) for 24 h. Transcriptomic profiling identified 769 differentially expressed genes (DEGs; adjusted p < 0.05, |log2FC| > 1), including 392 upregulated and 377 downregulated. The protein–protein interaction network analysis via Cytoscape revealed key hub genes (HNRNPA2B1, HNRNPM, RBM39). Enrichment analyses (GO, KEGG, Reactome, DO) highlighted significant involvement of the C-type lectin receptor (CLR) signaling pathway. Validation experiments showed cellular morphological changes, altered structure, and markedly elevated interleukin-6 (IL-6; p < 0.01), underscoring its role in inflammation. These findings suggest C. acnes IA₁ drives acne pathogenesis by regulating hub genes that influence sebaceous gland inflammation, immune activity, and keratinocyte proliferation, positioning them as potential biomarkers for microbiome-targeted therapies. Limitations include the in vitro model’s lack of in vivo skin microenvironment complexity and use of only one representative IA₁ strain. Full article

The widespread use of glyphosate-based herbicides (GBHs) has raised concerns about their potential role in hormone-sensitive cancers such as breast cancer. This systematic review aimed to evaluate preclinical evidence on the effects of glyphosate (pure compound) or glyphosate-based herbicide formulations (GBHs) exposure on breast cancer cell proliferation and related molecular pathways. A structured search was conducted across PubMed, ScienceDirect, and Springer Nature Link, Web of Science databases, covering studies published up to 9 November 2025, following a PROSPERO-registered protocol (ID: CRD42021238350). Eligible studies included original in vitro and in vivo preclinical research using human breast cancer cell lines (e.g., MCF-7, T47D, MDA-MB-231, MCF-12A, and MCF-10A) or relevant animal models. Outcomes assessed included cell viability, proliferation, tumor growth, apoptosis, cell cycle regulation, and molecular markers associated with endocrine signaling. Two reviewers independently screened and extracted data, resolving disagreements via discussion or third-party adjudication. From an initial pool of 699 articles, seven in vitro studies met the inclusion and quality criteria. Glyphosate exposure demonstrated weak estrogenic activity in ER-positive breast cancer cells, primarily via ERα modulation and altered gene expression related to proliferation and DNA repair. GBHs showed greater cytotoxic and epigenetic effects in non-tumorigenic cells, often independent of ER signaling. No included study employed in vivo breast cancer models. Overall, preclinical evidence suggests glyphosate may act as a weak endocrine disruptor under specific conditions, but findings are limited by the short-term in vitro designs, heterogeneous methodologies, and lack of chronic or in vivo data. Further research using long-term exposure and animal models is needed to clarify potential risks and inform regulatory and public health decisions. Full article

Background: d-Limonene (LIM) is a food-derived monoterpenoid phytocompound predominantly found in citrus peels, endowed with potent antioxidant and anti-inflammatory properties, and has been reported to inhibit xanthine oxidase (XO) activity in vitro. This study investigated the dose-sparing efficacy of this dietary bioactive compound in combination with low-dose allopurinol (ALP) using a dual rat model combining potassium oxonate (PO)-induced hyperuricemia and monosodium urate (MSU)-triggered gouty arthritis, thereby capturing both metabolic and inflammatory dimensions of gout. Methods: Female Wistar rats were PO-primed and MSU-challenged, then treated with LIM (50 mg/kg), ALP (5 or 10 mg/kg), or LIM + ALP. Outcomes included paw thickness, dysfunction and inflammation indices, serum uric acid, urea, creatinine, AST/ALT, cytokines (IL-1β, TNF-α, IL-6), oxidative stress markers (MDA, SOD, catalase, GSH), and NLRP3 immunoreactivity, supported by radiographic and histopathological analyses. Data were analyzed by one-way ANOVA with Tukey’s post hoc test. Results: LIM improved clinical and biochemical outcomes versus monotherapies. However, LIM + low-dose ALP exhibited the greatest overall efficacy. On Day 30, paw thickness was significantly lower with LIM + ALP than with LIM alone (3.25 ± 0.31 vs. 3.98 ± 0.72 mm; p < 0.001). Serum uric acid and hepatic transaminases declined most with the combination (p < 0.0001 vs. LIM), accompanied by improved renal indices (p < 0.001). Pro-inflammatory cytokines were markedly reduced, NLRP3 immunostaining was minimal, and oxidative balance shifted toward homeostasis (↓ MDA; ↑ SOD, catalase, GSH). Radiographic and histological evaluations corroborated attenuation of joint inflammation and tissue damage. Conclusions: In the PO + MSU gout model, co-administration of the food-derived compound LIM with low-dose ALP achieved additive, dose-sparing benefits across metabolic, inflammatory, and histological endpoints. While in vivo XO activity was not directly assessed, the findings are consistent with XO-pathway modulation, NLRP3–IL-1β suppression, and redox restoration. These results highlight the potential of dietary bioactives such as d-Limonene to complement standard urate-lowering therapy, warranting further pharmacokinetic and safety validation. Full article

Peripheral nerve injuries often recover poorly. Hyaluronic acid (HA) biomaterials, with regenerative and anti-fibrotic properties, may augment repair. We performed a PRISMA-guided systematic review of PubMed, Scopus, Web of Science, and Embase (January 2000–December 2023), capturing in vitro, in vivo, and clinical investigations of HA in peripheral nerve repair; data on study context, interventions, and outcomes were extracted. Screening and extraction were performed in duplicate. Forty-eight studies met inclusion criteria. Across in vitro and in vivo models, HA-based biomaterials consistently reduced perineural fibrosis, enhanced axonal regeneration, and improved SFI, CMAP, and NCV compared with conventional repair. Several HA hydrogels and composite conduits achieved functional outcomes approaching autografts, particularly when combined with exosomes, neurotrophic factors, or mechanobiologically tuned scaffolds. Early clinical studies demonstrated safety but remain limited by size and short follow-up. Overall, HA-containing biomaterials appear anti-fibrotic, neuroprotective, and pro-regenerative, supporting their promise as adjuncts for peripheral nerve reconstruction. For this to translate into clinical practice, future work should standardize formulations and dosing, employ rigorous, clinically relevant animal models with long-term endpoints, and advance well-powered, controlled trials to test effectiveness and durability in patients. Clinically, HA platforms show promise as anti-adhesion barriers after neurolysis and as biofunctional fillers/coatings for nerve conduits, but standardized formulations and adequately powered trials are needed to define indications and dosing. Full article

Background: Frostbite injury creates an ischemic, hypoxic, and acidic microenvironment that often triggers severe oxidative stress and inflammation. Current therapeutic approaches are limited by low drug delivery efficiency and an inability to adequately regulate multiple pathological pathways. Although oxyresveratrol (OR) exhibits excellent antioxidant and anti-inflammatory activities, its application is hampered by poor aqueous solubility and low stability. Methods: We constructed Oxyresveratrol@Zeolitic Imidazolate Framework-8 nanoparticles (OR@ZIF-8) and further embedded them in a sodium hyaluronate (HA) matrix to form an OR@ZIF-8@HA composite hydrogel. The physicochemical properties and pH-responsive drug release behavior of the system were characterized. Its antioxidant activity, ability to promote cell migration, and capacity to modulate macrophage polarization were evaluated in cellular assays. The therapeutic efficacy was further investigated using a mouse frostbite model, with wound repair analyzed via histological staining. Results: The OR@ZIF-8 nanoparticles achieved a cumulative release rate of 75.46 ± 3.68% under acidic conditions within 36 h. In vitro experiments demonstrated that the formulation significantly scavenged TNF-α and IL-6, by 161.85 ± 19.43% and 125.37 ± 12.65%, respectively, and increased the level of IL-10 by 44.97 ± 4.57%. In a scratch assay, it promoted wound healing, achieving a closure rate of 97.55 ± 2.77% after 36 h. In vivo studies revealed that the OR@ZIF-8@HA treatment group achieved a wound healing rate of 96.14 ± 4.12% on day 14. Conclusions: The OR@ZIF-8@HA composite hydrogel effectively overcomes the limitations of OR application via intelligent pH-responsive delivery. Through synergistic multi-mechanistic actions, it significantly accelerates frostbite wound healing, offering a novel and efficient therapeutic strategy for frostbite management. Full article

Colorectal cancer (CRC) remains one of the most lethal malignancies worldwide, with 5-fluorouracil (5-Fu) as a mainstay of treatment. However, intrinsic and acquired resistance to 5-Fu significantly limits therapeutic success. Furin, a proprotein convertase, is known to activate multiple substrates critical for tumor progression, yet its precise role in CRC remains unclear. In this study, we examined furin expression in a large cohort of CRC patient samples and performed functional analyses in CRC cell lines and xenograft models. Furin overexpression was seen in 46.9% (530/1131) of CRC cases and was significantly correlated with TGF-β and ERK1/2 activation. In vitro, induced furin overexpression enhanced proliferation and clonogenicity, accompanied by upregulation of TGF-β and ERK1/2 phosphorylation, whereas furin silencing attenuated tumor cell growth and TGF-β/ERK signaling. Manipulation of TGF-β revealed a reciprocal regulatory loop, whereby TGF-β upregulated furin expression, establishing a feed-forward circuit that augmented ERK signaling and tumor growth. Notably, 5-Fu-resistant CRC cell lines displayed elevated furin, TGF-β, and phospho-ERK1/2, while furin knockdown restored drug sensitivity. In vivo, furin overexpression enhanced tumor growth in xenografts, whereas its depletion markedly reduced tumor burden and TGF-β/ERK signaling activity. Collectively, these findings demonstrate that furin promotes CRC progression and chemoresistance through a positive feedback loop with TGF-β that sustains ERK activation. Targeting furin, alone or in combination with TGF-β/ERK inhibitors, may offer a promising therapeutic strategy for CRC. Full article

Background: Epidermal growth factor receptor (EGFR)-targeted tyrosine kinase inhibitors (TKIs) have exhibited efficacy in EGFR-mutant non-small cell lung cancer (NSCLC) patients. However, the response is modest in patients with wild-type (wt)-EGFR, and approximately 30–40% of patients develop TKI resistance. Recently, a role for BRG1 (SMARCA4) in regulating gene expression and its frequent alteration in various cancers, including NSCLC, has been reported. Yet, its specific function in response to EGFR-TKI therapy remains elusive. Herein, we investigated the role of BRG1 in EGFR-TKI response in vitro and in vivo using lung cancer models. Methods: In vitro, A549, H358, and HCC827 cell lines that varied in their EGFR and BRG1 status were assessed for response to EGFR-TKI upon overexpression or gene silencing of BRG1 through cell viability, cell migration, and Western blotting assays. In vivo, A549 and H358 tumor xenografts that overexpressed BRG1 or had BRG1 silenced were investigated for tumor growth response to EGFR-TKI. Results: EGFRwt/BRG1mt (A549) cells were resistant to TKI, and restoration of wt-BRG1 expression reverted them to TKI sensitivity both in vitro and in vivo. In contrast, silencing of BRG1wt in H358 cells showed a tendency toward TKI resistance. Additionally, wt-EGFR and pAKT^Ser473 protein complex formation in A549 cells was disrupted with an AKT inhibitor (MK2206), resulting in enhanced cytotoxicity in vitro. Conclusions: Our study demonstrates that EGFR-TKI response in wt-EGFR cells is dictated by BRG1 status. These findings propose screening of wt-EGFR NSCLC patients for BRG1 status for identifying individuals likely to benefit from EGFR-TKI therapy versus patients who will benefit from AKT inhibitor treatment. Full article

Background/Objectives: Neuroblastoma (NB) is the most common extracranial solid tumor in children and accounts for 12–15% of pediatric cancer-related deaths. Current multimodal therapies lack specific cellular targets, causing systemic toxicity and drug resistance. The development of innovative tumor-targeted nanoformulations might represent a promising approach to enhance NB diagnosis and antitumor efficacy, while decreasing off targets side effects. Fibronectin extra-domain B (FN-EDB) is upregulated in the tumor microenvironment. Methods: FN-EDB expression was evaluated by immunohistochemical staining in cell line-derived and tumor patient-derived animal models of NB. A gold nanoparticle, decorated with an antibody (Ab) recognizing FN-EDB (L19-AuNP) was developed by the company Nano Flow and its tumor binding was tested by ELISA in vitro and in patient-derived xenograft (PDX) models of NB by photoacoustic imaging in vivo. Results: All animal models of NB used have been shown to express FN-EDB. L19 Ab demonstrated excellent binding specificity to FN-EDB both when used in free form and after conjugation to AuNP. Compared to the non-functionalized (no Ab L19-coupled) AuNP, which showed an increase in PDI and zeta potential over time, making them unsuitable for use in in vivo studies, L19-AuNP demonstrated good stability. In vivo, L19-AuNP specifically homed into PDX models of NB, accumulating better in tumors expressing higher levels of FN-EDB. Negligible distribution to healthy organs occurred. Conclusions: In this preliminary study, L19-AuNP was shown to be a novel diagnostic tool specifically for binding NB expressing FN-EDB, paving the way for the development of theranostic nanoformulations co-encapsulating gold moiety and standard-of-care therapy for NB. Full article

Background/Objectives: Hydroxyapatite (HAp)-based implants and HAp–titanium (HApTi) composites are widely used in orthopedic and dental applications, but their long-term success is limited by peri-implant bone loss. Local delivery of osteoactive molecules from implant surfaces may enhance osseointegration and reduce periprosthetic osteolysis. This study combined in silico modeling and experimental assays to compare calcium fructoborate (CaFb), sodium alendronate, and calcium alendronate as functionalization agents for HAp and HApTi implants. Methods: Molecular docking (AutoDock 4.2.6) and 100 ns molecular dynamics (MD) simulations (AMBER14 force field, SPC water model) were performed to characterize ligand–substrate interactions and to calculate binding free energies (ΔG_binding) and root mean square deviation (RMSD) values for ligand–HAp/HApTi complexes. HAp and HApTi discs obtained by powder metallurgy were subsequently functionalized by surface adsorption with CaFb or alendronate salts. The amount of adsorbed ligand was determined gravimetrically, and in vitro release profiles were quantified by HPTLC–MS for CaFb and by HPLC after FMOC derivatization for alendronates. Results: CaFb–HAp and CaFb–HApTi complexes showed the lowest binding free energies (−1.31 and −1.63 kcal/mol, respectively), indicating spontaneous and stable interactions. For HAp-based complexes, the mean ligand RMSD values over 100 ns were 0.27 ± 0.17 nm for sodium alendronate, 0.72 ± 0.28 nm for calcium alendronate (range 0.35–1.10 nm), and 0.21 ± 0.19 nm for CaFb (range 0.15–0.40 nm). For HApTi-based complexes, the corresponding RMSD values were 0.30 ± 0.15 nm for sodium alendronate, 0.72 ± 0.38 nm for calcium alendronate and 0.26 ± 0.14 nm for CaFb. These distributions indicate that CaFb and sodium alendronate maintain relatively stable binding poses, whereas calcium alendronate shows larger conformational fluctuations, consistent with its less favorable binding energies. Experimentally, CaFb exhibited the greatest chemisorbed amount and percentage on both HAp and HApTi, followed by sodium and calcium alendronate. HApTi supported higher loadings than HAp for all ligands. Release studies demonstrated a pronounced burst and rapid plateau for both alendronate salts, whereas CaFb displayed a slower initial release followed by a prolonged, quasi-linear liberation over 14 days. Conclusions: The convergence between in silico and adsorption–release data highlights CaFb as the most promising candidate among the tested ligands for long-term functionalization of HAp and HApTi surfaces. Its stronger and more stable binding, higher loading capacity and more sustained release profile suggest that CaFb-coated HApTi implants may provide a favorable basis for future in vitro and in vivo studies aimed at improving osseointegration and mitigating periprosthetic osteolysis, although direct evidence for osteolysis prevention was not obtained in the present work. Full article

The liquid egg processing industry generates a significant amount of solid byproduct known as chalaza (CHA), which is rich in sialic acid and exhibits notable biological activity. In this study, the preparation process, N-glycan profile, and skin-whitening activity of CHA-derived glycopeptides (CHAH) were investigated. By comparing the hydrolysis efficiency of trypsin, alcalase, and papain, a dual-enzyme hydrolysis strategy was developed: initial hydrolysis with 1.5% trypsin for 3 h, followed by treatment with 1% papain for 2 h. The resulting CHAH exhibited both a high hydrolysis yield and strong antioxidant activity. The sialic acid content in CHAH reached 1.96% (w/w), and 14 distinct N-glycan chain structures were identified. The whitening effect of CHAH was assessed using a combined approach involving an in vitro B16 cell model and an in vivo zebrafish model. CHAH was found to inhibit tyrosinase activity and reduce melanin production in a concentration-dependent manner. Mechanistic studies revealed that CHAH acts by significantly downregulating the expression of key genes involved in melanin synthesis, including MITF, TYR, TYRP1, and TYRP2. This study establishes an efficient preparation method for CHAH, elucidates its skin-whitening efficacy and underlying mechanism, and provides experimental support for the potential industrial application of CHAH as an active ingredient in skincare products. Full article