Current Pharmaceutical Design - Online First

The delivery of healthcare services via information and communication technology, or telemedicine, has grown to be an essential part of modern medicine. This study explores the evolving role of telemedicine, focusing on its expansion into the Metaverse, and evaluates its potential to improve healthcare accessibility, patient engagement, and medical outcomes.

A comprehensive analysis of the literature was conducted, evaluating studies investigating the efficacy of telemedicine in different medical fields, notably mental health, chronic disease management, and post-surgical follow-ups. This study assessed the impact of emerging technologies, specifically virtual reality (VR) and augmented reality (AR), on telemedicine, emphasizing their applications within the Metaverse. Furthermore, ethical considerations, insurance limitations, and technological disparities were assessed.

Telemedicine has significantly enhanced healthcare access, especially in remote and underserved regions. Patient satisfaction and purpose to continue with telemedicine services are elevated, particularly in specialized areas like Tele-stroke and mental health counseling.

The Metaverse has the potential to transform telemedicine through the establishment of immersive and interactive healthcare settings. VR and AR have the potential to facilitate virtual consultations, enhancing the interaction between patients and healthcare professionals. Additionally, the integration of data may lead to improvements in diagnostic accuracy and treatment planning. However, issues such as data privacy, cybersecurity hazards, and the digital gap must be addressed to provide adequate access.

Telemedicine has demonstrated significant utility within modern healthcare, and its incorporation with the Metaverse offers novel prospects for improving patient care, advancing medical education, and facilitating collaborative research. Despite the promising benefits, it is crucial to address technological, ethical, and regulatory challenges to ensure widespread adoption and successful implementation.

Salvianolic acid B (SAB), as one of the major water-soluble compounds of Salvia miltiorrhiza, has proved to effectively reduce elevated portal pressure in cirrhotic rats. However, the short half-life and in vivo retention time of SAB affect its pharmacodynamics. Therefore, in this study, we prepared albumin nanoparticles loaded with SAB (SAB-ALB-NPs) to improve the in vivo retention time of the drug and enhance bioavailability.

We prepared and characterized SAB-ALB-NPs, including particle size, polydispersity index (PDI), zeta potential, stability, EE, in-vitro release, and pharmacokinetics. Subsequently, we investigated the effects and potential mechanisms of SAB-ALB-NPs in CCl4-induced portal hypertension (PHT) mice models, and it was found that angiotensin-II (Ang-II) induced proliferation and contraction in hepatic stellate cells (HSCs). The CCl4 (0.3:1 in corn oil, 1mL/kg) was injected repeatedly, leading to the PHT mice model. The effect of SAB-ALB-NPs on PHT mice was evaluated by hematoxylin-eosin, Sirius red staining, immunohistochemistry, and Western blot.

We successfully prepared SAB-loaded albumin nanoparticles with smaller-sized particles, lower PDI and zeta potential with stable properties, and higher EE. Importantly, the SAB-ALB-NPs notably prolonged the in vitro release of SAB. SAB-ALB-NPs significantly reduced portal pressure, inhibited inflammation (decrease the concentration of TNF-α and IL-6) and hepatotoxicity of the liver (down-regulated the level of ALT and AST) against fibrous tissue hyperplasia, and reduced collagen deposition in the liver. Afterward, we used Ang-II to facilitate the proliferation of HSCs and induce HSC cell contraction. Cotreatment of SAB-ALB-NPs markedly inhibited Ang II-induced effects on cell proliferation and contraction and improved apoptosis. Importantly, SAB-ALB-NPs were preliminarily found to inhibit the expression of RhoA and ROCK II in Ang-II-treated HSC and CCl4-induced PHT mice, suggesting that SAB-ALB-NPs may participate in the regulation of RhoA/ROCK II pathway.

SAB-ALB-NPs improved portal hypertension by suppressing inflammation and inhibiting HSCs activation and proliferation to attenuate liver fibrosis. This therapeutic function of SAB-ALB-NPs may be owing to SAB-ALB-NPs regulating the RhoA/ROCK2 pathway, which may be one of its molecular mechanisms for reducing portal hypertension.

Recent studies indicate that niclosamide demonstrates considerable promise as both an anthelmintic agent and a possible anticancer medication. Given the increasing interest in nano-sized drug delivery methods for cancer therapy, lipid nanocapsules (LNCs) have emerged as a viable approach to enhance the bioavailability of poorly soluble pharmaceuticals due to their beneficial properties. This research intends to develop niclosamide-loaded lipid nanocapsules (NIC-LNCs) using the phase inversion technique, followed by the optimization of these formulations via the Box-Behnken experimental design.

A reverse-phase high-performance liquid chromatography (RP-HPLC) method was devised and validated for quantifying niclosamide in the LNC formulations. Optimal chromatographic separation was attained utilizing an Agilent Eclipse XDB-C18 column (150×4.6 mm, 5 µm i.d.) with a mobile phase of a 50:50 (v/v) mixture of acetonitrile and 0.1% H3PO4 phosphate buffer, at a flow rate of 1.2 mL/min. The detection wavelength was set at 335 nm, and the analysis was performed at 35°C. The developed analytical methodology was validated through a comprehensive evaluation of accuracy, linearity, precision, limit of detection, limit of quantitation, specificity, and stability.

The optimization of the NIC-LNC formulation through the Box-Behnken design resulted in an optimal formulation labeled LNC5, consisting of 4% niclosamide, 20% lipid, and 20% surfactant. The proven RP-HPLC method enables accurate quantification of NIC in the LNC formulations. The refined NIC-LNC formulation exhibited developed attributes as assessed by the design.

NIC-LNCs were successfully prepared with particle sizes below 100 nm, narrow size distributions (PDI<0.2), and negative zeta potential values in accordance with the literature. All formulations exhibited high encapsulation efficiency and sustained drug release profiles. The optimum formulation revealed a particle size of 43.29 ± 0.32 nm, encapsulation efficiency of 99.99 ± 0.02%, and drug release at one week of 68.85 ± 1.76%. The formulation maintained stability throughout the short-term study period.

The findings indicate that LNC systems are a promising method for drug administration, especially for anticancer drugs with limited solubility in water.

Cervical cancer (CC) is among the most prevalent cancers affecting women globally, with a substantial number of deaths reported annually. Despite advancements in treatment, the persistently high mortality rate underscores the urgent need for novel and effective therapeutic strategies.

This study screened a library of 240 flavonoids against maternal embryonic leucine zipper kinase (MELK) and LYN using molecular docking methods to achieve precise calculations. These proteins play critical roles in CC progression, and their simultaneous inhibition could mark a significant step forward in multitargeted drug design.

Molecular docking revealed binding affinities ranging from -10.0649 to -8.14296 kcal/mol for MELK and -10.2748 to -8.5237 kcal/mol for LYN. The screening process was complemented by pharmacokinetics and interaction fingerprinting analyses, which confirmed that the flavonoids effectively bound to optimal sites, forming stable complexes through multiple interactions. Molecular dynamics simulations extended to 100 ns further validated the stability of these protein-ligand complexes.

The findings indicate that the top-ranked compounds exhibit strong binding affinities and stable interactions, highlighting their potential as multitargeted therapeutic agents against CC.

These findings set the stage for future experimental and clinical studies to validate our results and facilitate the development of novel, flavonoid-based therapeutic strategies against cervical cancer, potentially revolutionizing the treatment landscape of this disease.

Ovarian cancer (OC) is a malignancy of the female reproductive system for which cisplatin chemotherapy is one of the first-line treatments. Despite the initial response to chemotherapy, such patients eventually develop resistance, which poses a major obstacle to treatment, along with potential side effects. Phytochemicals function as chemosensitizers, offering novel therapies in OC patients by targeting drug resistance, and are perceived to be less toxic. Plumbagin has emerged as an anticancer compound, with some findings suggesting its anti-ovarian cancer activity. However, there is no study on the potential of plumbagin to target cisplatin resistance in non-high-grade OC. The current study aimed to determine the antitumor activity of plumbagin for cisplatin resistance in OC cells in vitro, and to identify its potential molecular target for therapeutic benefit using in silico studies.

Plumbagin was used for in vitro cytotoxic effects on cisplatin-resistant (A2780-CR) and sensitive (A2780-CS) isogenic cell lines using a crystal violet cell viability assay. The binding of plumbagin to the nine selected molecular targets was estimated by molecular docking and their binding energies were compared. The stabilities of the selected docked complexes were confirmed by molecular dynamics simulation (MDS) and molecular mechanics generalized born surface area (MM-GBSA) calculations, and conclusions were drawn to predict the inhibition potential of plumbagin to its best targets.

Plumbagin demonstrated the potential to kill A2780-CR cells, and, expectedly, the cell death effect on A2780-CS ovarian cancer cells demonstrated its anti-tumor activity in vitro. It was found to be non-effective in killing normal non-tumorigenic RPE cells, even at higher doses. Docking analysis suggested that it potentially inhibits through various pharmacological targets with high affinity for binding to Chk1 (PDB ID=1ia8) and Aurora Kinase (PDB ID=5ORL). Molecular dynamic simulation data revealed strong and stable protein-ligand complex formation, which was measured in terms of root mean square deviation (RMSD), root mean square fluctuation (RMSF), and radius of gyration (Rg). On the other hand, the MM-GBSA study revealed that the binding free energy of the CT1019-1ia8 complex (-84.26 ± 2.99 Kcal/mol) and CT1019-5ORL (-67.04 ± 2.63 Kcal/mol) was better when compared to other complexes.

Plumbagin showed anti-ovarian cancer benefits in cisplatin-resistant ovarian cells, and the potential pharmacological targets identified were Chk1 and Aurora kinase.

Our study offers promising insights into plumbagin, particularly in combating cisplatin-resistance OC. However, further in vivo and mechanistic studies are required to validate plumbagin's potential as a therapeutic candidate for OC treatment.

Ibrutinib is a selective tyrosine kinase inhibitor used to treat chronic lymphocytic leukemia (CLL). However, it has low oral bioavailability (2.9%), which is attributed to low solubility (0.002 mg/mL) and a first-pass effect. Ibrutinib-loaded nanostructured lipid carriers (IBR-NLCs) were prepared and investigated in this study to overcome the solubility and presystemic metabolism issues. The goal of the current study was to formulate IBR-NLCs for enhanced bioavailability. IBR-NLCs were optimized using a 3² factorial design and evaluated using various in vitro and in vivo parameters.

IBR interaction with solid lipid (Glyceryl monostearate) and liquid lipid (oleic acid) was studied using molecular docking. The hot-melt ultrasonication method was used to formulate IBR-NLCs, and a 3² factorial design was used for optimization. Particle size, PDI, zeta potential, entrapment efficiency, DSC, XRD, FTIR, SEM, and in vitro study were used to evaluate the NLCs. HepG2 cell lines were used to study the in vitro cytotoxicity of IBR-NLCs and IBR suspension. IBR-NLCs were administered to male Wistar rats in the presence and absence of cycloheximide (CXI) to compare pharmacokinetic parameters.

Molecular docking confirmed good interaction between IBR-GMS and IBR-oleic acid. The optimized IBR-NLCs exhibited particle sizes, PDI, zeta potentials, and %EE of 154.5 ± 0.7 nm, 0.2 ± 0.0, -25.8 ± 1.1 mV, and 84.0 ± 1.2%, respectively. Differential Scanning Calorimetry (DSC) reveals the development of molecular dispersion of IBR in the melted lipid matrix, and X-Ray Diffraction (XRD) studies show a decline in the crystalline drug peaks in the formulation's diffractogram. SEM images showed uniformity distributed spherical-shaped particles. According to an in vitro investigation, IBR-NLCs exhibited a sustained release pattern of 98.0 ± 0.5% with a Korsmeyer-Peppas model mechanism (R² = 0.9615). The IC50 values of IBR suspension and IBR-NLCs were 4.155 µg/mL and 3.03 µg/mL. The AUC0-24 of IBR-NLCs administered in the absence of CXI was 1.60 times higher than the AUC0-24 values in the presence of CXI, indicating lymphatic transport.

IBR-NLCs appear to be promising as a novel innovative nanocarrier for the management of CLL.

Ovarian cancer (OC) is a common malignant tumor of the female reproductive system and is usually found at an advanced stage. However, the treatment of OC with conventional the efficacy of surgery and chemotherapy is limited. Brusatol (BRU) is a unique nuclear factor erythroid 2-related factor 2 (Nrf2) pathway inhibitor with significant anti-cancer effects. At the same time, the Nrf2 system also plays a vital role in ferroptosis, which can be used as a new way to treat tumors. This study investigated the mechanism of action of BRU as a novel ferroptosis inducer to inhibit OC cells.

Using bioinformatics to screen for key targets and pathways that act on OC in BRU, and then the effects of BRU on OC cells were examined by cell viability assay, clone formation assay, wound healing assay, and apoptosis assay. The intracellular levels of ROS (Reactive Oxygen Species), Fe²⁺, glutathione (GSH), and malondialdehyde (MDA) were also quantified. Western blotting analysis was then performed to verify ferroptosis marker proteins and pathways. In addition, the combination of Ferrostatin-1 (Fer-1) and BRU was further tested for ferroptosis-related markers.

By obtaining BRU and OC targets, 171 potential BRU-OC action targets were screened to the core target NQO1. KEGG enrichment analysis showed that the anticancer effects of IBC were mediated through multiple pathways, including the PI3K-AKT and Ras signaling pathways. In vitro results showed that IBC inhibited the proliferation, invasion, and migration of OC cells and induced ferroptosis in OC cells.

We demonstrated that BRU increased intracellular ROS, Fe^2+, and MDA levels. It also significantly reduced intracellular GSH level and the expression of two marker proteins for ferroptosis, GPX4 and SLC7A11. Meanwhile, BRU could inhibit the Nrf2/HO-1/NQO1 and AKT/mTOR dual signaling pathways in OC cells. Furthermore, the combination of Ferrostatin-1 (Fer-1) and BRU reversed BRU-induced ferroptosis in OC cells.

In this study, we demonstrated for the first time through bioinformatics, molecular docking technology, and experimental validation that BRU acts as a novel inducer of ferroptosis in ovarian cancer cells by targeting the Nrf2/HO-1/NQO1 and AKT/mTOR dual signaling pathways, and may have great potential in the treatment of ovarian cancer cells.

Crizotinib, an inhibitor of the epidermal growth factor receptor (EGFR) tyrosine kinase, holds significant potential for the treatment of lung cancer. However, its toxicities present a major challenge to its clinical use. To enhance the targeted delivery of Crizotinib to lung tumors, polymeric-based nanoparticles were developed.

Crizotinib-loaded polymeric nanoparticles were prepared using a nano-precipitation method, incorporating stearic acid as the lipid, polyethylene glycol as the polymer, and Tween 80 as the surfactant. Key formulation parameters were optimized to achieve high-quality nanoparticles.

The optimized formulation exhibited a mean particle size of 142 nm, a zeta potential of -31.9 mV, an entrapment efficiency of 82.35%, and an in vitro drug release of 60.69%. These nanoparticles were then tested on lung cancer cell lines to assess their cytotoxicity, apoptosis induction, and anti-proliferative effects on the cell cycle. In vitro studies confirmed that the Crizotinib-loaded nanoparticles exerted targeted effects on non-small cell lung carcinoma (NSCLC) cell lines, showing maximum inhibitory effects. One year of storage at 4°C, stability testing demonstrated that the lyophilized nanoparticles maintained their effectiveness.

crizotinib nano-formulations were assessed for a variety of physicochemical and in vitro characterization. Five different formulations were designed and optimized on the basis of Particle size, Zeta potential, %EE, and in vitro drug release. Optimum formulation also showed maximum inhibitory effect on the cancer cell line.

This nanotechnology approach offers a promising targeted drug delivery system for Crizotinib, characterized by small particle size, high encapsulation efficiency (EE), and optimal in vitro drug release.

Cholesterol is considered a major factor contributing to cardiovascular diseases. Statins, the most commonly prescribed cholesterol-lowering drugs, are known to have various limitations. Inhibition of Adenosine Triphosphate-Citrate Lyase (ACLY) has been proposed as an alternative therapeutic strategy for managing hypercholesterolemia by lowering cholesterol levels. This has led to the discovery of a cell-permeable small molecule ACLY inhibitor.

ACLY enzyme activity was assessed using an ACLY Assay Kit with the ADP-Glo Kinase Assay Kit. HepG2 cells were treated with test compounds to demonstrate cholesterol and fatty acid synthesis. Pharmacokinetic studies were performed on CD-1 mice following a single oral dose of the compounds. Hypercholesterolemia was induced in mice through a High-Fat and High Cholesterol Diet (HFHCD), and drugs were administered orally for six weeks. Serum and hepatic lipid profiles were subsequently analyzed.

To increase the pharmacochemical properties, four analogues of BMS-303141, ID0018, ID0023, ID0085, and ID0106, were designed and synthesized. These compounds showed superior ACLY inhibitory activity and dose-dependent suppression of cholesterol and fatty acid synthesis in HepG2 cells. Among the analogues, ID0085 exhibited the most potent ACLY inhibition (IC50: 45 nM, 10-fold lower than BMS-303141) and achieved near-complete suppression in cholesterol and fatty acid synthesis at the highest concentration. Pharmacokinetic studies revealed improved half-lives and systemic exposures for all analogues. 
In hypercholesterolemic mouse models, test compounds significantly reduced serum total cholesterol 
(32.0-57.3%) and low-density lipoprotein cholesterol (67.5-80.2%) levels compared to the vehicle group. Notably, ID0085 also increased high-density lipoprotein cholesterol levels.

Among the synthesized analogues, ID0085 exhibited the most potent ACLY inhibition, superior pharmacokinetic properties, and significant improvements in both serum and hepatic cholesterol profiles compared to BMS-303141.

Based on the results, ID0085 appears to be the most promising therapeutic candidate for the treatment of hypercholesterolemia.

The combination of Astragalus membranaceus and Safflower (AS) is known for its efficacy in benefiting Qi and activating blood circulation, making it a frequently used empirical combination in traditional Chinese medicine. Numerous reports have highlighted the interventional effect of this combination in treating ischemic stroke (IS). However, the active ingredients and potential mechanisms underlying its treatment of stroke have not been fully elucidated.

Ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS), along with various data processing methods, were utilized to identify and assess the chemical constituents in rat serum following AS gavage administration. Chemical constituent targets were predicted using the SEA and Swiss Target Prediction databases, while IS-related targets were sourced from the GeneCards, OMIM, and TTD databases. The intersecting targets of constituents and diseases were screened, and a core target network map was constructed using the String database and Cytoscape software. KEGG pathway enrichment of core targets was analyzed using DAVID and Metascape databases. The middle cerebral artery occlusion (MCAO) rat model was established to evaluate the cerebroprotective effects of AS. The accuracy of predicted pathways was validated using immunofluorescence (IF) and Western blot (WB) analyses.

Thirty-five ingredients in serum were identified, and 437 targets and 3748 IS-related targets were identified, 291 of which overlapped. Protein-protein interaction (PPI) analysis predicted 15 major targets, including TNF and MAPK3. KEGG pathway analysis indicated that the MAPK/NF-κB and VEGF/Notch1 signaling pathways may play pivotal roles in the therapeutic effects of AS in IS. Moreover, AS significantly ameliorated neurological and motor function impairments, as well as brain histopathological damage, in MCAO rats. AS treatment led to reduced levels of the inflammatory cytokines IL-6 and TNF-α, inhibited astrocyte hyperactivation, decreased nuclear translocation of NF-κB p65, reduced expression of p-MAPK (Erk1/2)/ MAPK (Erk1/2) and p-NF-κB (p65)/NF-κB (p65) proteins, increased the number of CD31⁺/Ki67⁺ and VEGF⁺/ Ki67⁺-positive vessels, and upregulated the expression of VEGF, VEGFR-2, Notch1, and DLL4 proteins.

AS may regulate MAPK/NF-κB and VEGF/Notch1 pathways to reduce inflammation and promote post-ischemic neovascularization, providing a promising method for the treatment of ischemic stroke.

The progressive neurodegenerative disease known as Alzheimer's disease (AD) is typified by neuroinflammation, amyloid-beta buildup, and cognitive impairment. Current pharmacological treatments merely alleviate symptoms, despite extensive research, which underscores the need for innovative, multi-target medicines. Since apolipoprotein E4 (ApoE4) is a significant genetic risk factor linked to the development of AD, it is a potentially effective treatment target. With their neuroprotective qualities, natural substances like Ginkgolide may help treat some diseases. This study investigates Ginkgolide's potential as a multi-target treatment for AD, with a particular emphasis on how it interacts with the ApoE4 N-terminal domain.

The interaction between Ginkgolide and ApoE4 (PDB ID: 8AX8) was assessed using pharmacokinetic profiling, molecular docking, and molecular dynamics (MD) simulations. MD simulations were used to determine stability, and AutoDock Vina was used to obtain the binding affinity. To predict pharmacokinetics and toxicity, SwissADME and PkCSM were employed. The effectiveness of ginkgolide was contextualized using comparative docking with curcumin and resveratrol.

Ginkgolide formed sustained hydrophobic contacts with important sites and demonstrated a substantial binding affinity (-7.1 kcal/mol) to ApoE4. MD simulations verified negligible fluctuations and complex stability over 100 ns. Pharmacokinetics showed no significant toxicity risks, good gastrointestinal absorption, and favorable blood-brain barrier permeability. In terms of binding affinity and stability, ginkgolide fared better than curcumin and resveratrol, indicating its greater therapeutic potential.

The results indicate that ginkgolide effectively binds and stabilizes the ApoE4 N-terminal domain, supporting its potential role in modulating a key pathological factor in Alzheimer’s disease. Its superior pharmacokinetic profile and interaction dynamics compared to curcumin and resveratrol suggest a broader therapeutic relevance. These in silico insights provide a mechanistic basis for further investigation into ginkgolide’s neuroprotective effects.

The results demonstrated ginkgolide as a potentially effective multi-target treatment for AD through ApoE4 regulation. It is a better option than other natural chemicals because of its potent binding affinity, stability, and pharmacokinetics. These findings highlight the value of in silico methods in the early stages of drug discovery and the need for additional experimental support before they can be used in clinical settings.

The marine ecosystem, known for its diverse biochemistry and organisms adapted to harsh environments, contains numerous plants with promising anticancer potential. Halodule uninervis, a seagrass, contains a variety of bioactive compounds that provide various pharmacological properties. However, its potential anticancer effects against breast cancer remain largely unexplored.

HRLC-MS analysis was conducted to identify the phytochemicals in the ethanolic extract of H. uninervis leaves. Several publicly available databases, including SEA, STP, MALACARDS, DISGENET, and OMIM, were used to identify target genes. Protein-protein interaction (PPI) networks, gene ontology, and pathway analysis were carried out through the STRING and DAVID databases. Molecular docking was performed by Autodock Vina, while molecular dynamics (MD) simulations and MMPBSA analyses were conducted using GROMACS, demonstrating the stability of the complexes up to 200 ns.

The top five therapeutically active phytochemicals were Quercetin, Arborinine, Methyl 3,4,5-trimethoxycinnamate, Citreorosein, and Scopolin. The five hub genes, AKT1, EGFR, TNF, ESR1, and GAPDH, were found by network analyses. Molecular docking and MD simulation demonstrate that Quercetin and Citreorosein are the best phytochemicals exhibiting the highest affinities to breast cancer targets AKT1, EGFR, and ESR1.

For the first time, this in-silico study investigates the potential of citreorosein and quercetin, two phytochemicals predominantly found in H. uninervis leaves, to inhibit the activity of AKT1, EGFR, and ESR1. However, as these results are based on predictive computational analyses, further experimental validation is necessary to confirm their precise mechanisms of action.

Phytochemicals, namely Quercetin and Citreorosein, may have an impact on the progression of breast cancer by binding to the key targets AKT1, ESR1, and EGFR.

Genetic factors play a significant role in the development of leukemia. The overexpression of MDM2 is associated with the progression of certain leukemias. This meta-analysis investigates the relationship between the MDM2 SNP 309T>G and various forms of leukemia across global populations.

A comprehensive literature search was conducted to retrieve genotyping data from twenty case-control studies related to MDM2 SNP 309T>G polymorphism and leukemia. A random-effects model was used to calculate the pooled odds ratio (OR) and 95% confidence interval (95% CI) for the association analysis. MetaGenyo software was utilized to conduct statistical analyses in this meta-analysis.

The findings indicate a significant association between MDM2 309 SNPT>G polymorphism and leukemia in Asian and Caucasian populations. Additionally, this polymorphism is associated with an increased risk of Acute Myeloid Leukemia (AML) and Chronic Myeloid Leukemia (CML), implying that MDM2 may play a role in the pathogenesis of these specific forms of leukemia.

This meta-analysis suggests that MDM2 may represent a susceptibility gene for leukemia risk.

Glioblastoma Multiforme (GBM) is a highly aggressive and fatal brain malignancy, with Temozolomide (TMZ) serving as the first-line chemotherapeutic treatment. However, over 50% of patients do not respond to TMZ, and the underlying mechanisms remain unclear. This study utilized the GeCKO library to identify novel genes involved in TMZ resistance and to explore their functions.

Loss-of-function genes related to TMZ resistance in GBM cells were identified using the GeCKO library and Next-Generation Sequencing (NGS), validated by qPCR and CCK-8 assays. CD99L2 function was assessed through proliferation, migration, and EdU assays in U251 and U87 cells. Tumor samples from 55 stage IV GBM patients were analyzed to explore the correlation between CD99L2 expression and Progression-Free Survival (PFS).

GeCKO library screening identified seven genes associated with TMZ resistance. After validation, CD99L2 was confirmed as a key contributor to TMZ resistance. Knockdown of CD99L2 increased the IC50 of U251 and U87 cells by 1.39- and 1.54-fold, respectively. Conversely, CD99L2 overexpression reduced the IC50 by 0.52- and 0.58-fold. CD99L2 knockdown also promoted tumor proliferation and aggressiveness. Additionally, higher CD99L2 expression was associated with longer PFS in GBM patients (median PFS: 7.87 months vs. 2.7 months, P=0.0003).

The functions of CD99L2 remain poorly understood. A few studies have reported that CD99L2 may serve as an adhesion molecule modulating inflammatory responses. One study has shown that CD99L2 is highly expressed in the brain and affects neuronal excitability. These findings suggest that CD99L2 may play a positive role in the body’s defense against glioma.

This study demonstrated that CD99L2 knockdown promotes TMZ resistance and tumorigenesis in GBM, suggesting its potential as a novel biomarker for TMZ resistance.

Impaired intestinal immune function is commonly observed in neonates with intrauterine growth retardation (IUGR), yet its underlying mechanisms and regulatory pathways remain poorly understood. Therefore, we aimed to investigate gene regulatory patterns and microbiota alterations in IUGR piglets.

Three newborn IUGR piglets and three normal littermates were selected from the same sow and sacrificed at seven days of age. Ileal digesta was collected for 16S rRNA amplicon sequencing (16S-seq), and ileum segments were dissociated for single-cell RNA sequencing (scRNA-seq).

The scRNA-seq results revealed a reduced proportion of plasma B cells in IUGR piglets, along with alterations in the distribution of various T cell subsets. KEGG pathway analysis further indicated a downregulation of the B cell receptor signaling pathway in B cells from IUGR piglets. In contrast, both the T cell receptor signaling pathway and antigen processing and presentation were attenuated in T cells. Pseudotime trajectory analysis suggested that the differentiation of B cells was impaired in IUGR piglets. SCENIC analysis revealed that GATA3, IRF2, and BCL11A were downregulated in T cells of IUGR piglets. The 16S-seq results revealed that α-diversity was lower in IUGR piglets. At the genus level, the relative abundance of Prevotella was significantly lower in IUGR piglets.

Significant changes were identified in the proportions of B and T cells, their associated signaling pathways, and intestinal microbiota composition in IUGR piglets, suggesting underlying immune dysfunction and dysbiosis.

We identified novel immune-related transcription factors and key microbes as potential therapeutic targets, shedding light on strategies for preventing and treating IUGR.

Parkinson’s disease (PD) is characterized by the degeneration of dopaminergic neurons within the substantia nigra, leading to progressive motor dysfunction. There are still limited disease-modifying options that counteract the process of disease progression. This study aimed to evaluate the neuroprotective effects of thymol, both in its free form and when loaded onto selenium nanoparticles (SeNPs), in a 6-hydroxydopamine (6-OHDA)-induced PD model using SH-SY5Y cells.

SeNPs were synthesized using a chemical reduction method with ascorbic acid, achieving a 68% entrapment efficiency for thymol. FTIR analysis suggested an interaction between thymol and selenium, which was confirmed by EDX analysis. Nano-Se-thymol particles were observed to be spherical, with a mean size of 135.7 nm and a negative surface charge.

Nano-Se-thymol exhibited low toxicity in normal fibroblast cells and demonstrated greater neuroprotective effects against 6-OHDA-induced cytotoxicity compared to thymol. Nano-Se-thymol significantly reduced ROS generation and increased cell viability compared to 6-OHDA. Furthermore, Nano-Se-thymol decreased the expression of NF-κB inflammatory markers and caspase-3 apoptotic proteins, which were elevated by 6-OHDA, compared to thymol alone.

Nano-Se-Thymol significantly attenuates 6-OHDA-induced cytotoxicity in an established in vitro model of PD. The neuroprotective efficacy of Nano-Se-Thymol is attributed to its enhanced antioxidant capacity, as evidenced by a significant reduction in ROS levels, along with its ability to inhibit apoptosis and modulate cell cycle progression.

Nano-Se-thymol is a potential disease-modifying agent for the treatment of PD; however, further studies and long-term safety assessments are essential to confirm these benefits and understand the underlying mechanisms.

The aim of the study was to prepare polyelectrolyte complex stabilized piperine adjuvant simvastatin nanoformulations and evaluate the antimicrobial effect. Simvastatin has antimicrobial properties but low therapeutic efficacy due to rapid metabolism, with only 12% oral bioavailability. Piperine, a CYP3A4 inhibitor, enhances bioavailability by inhibiting drug-metabolizing enzymes. This study developed chitosan-neem gum polyelectrolyte complex (Ch-NG PEC) nanoparticles combining piperine and simvastatin and evaluated their antimicrobial efficacy compared to simvastatin alone.

A flower-shaped nanoparticles of piperine adjuvant simvastatin were prepared by using chitosan (Ch)-neem gum (NG) as a polyelectrolyte complex (PEC) forming agent, and the anti-microbial effect of nanoformulations with and without piperine was evaluated. A solvent-anti-solvent method was used to form the nanoparticles, and a 3²-factorial design was employed to analyze the impact of chitosan and neem gum concentration on the size of the nanoparticles and entrapment efficiency of simvastatin and piperine followed by their release profile and kinetics.

Nanoparticles showed high drug entrapment efficiency (simvastatin: 96.4-99.7%, piperine: 64.8-99.4%) with sizes ranging from 341.3-629.1 nm. Drug release exceeded 50% in 3 hours and 99% in 8 hours, following Hixon-Crowell and Baker’s Lonsdale models. Antimicrobial assays revealed activity against Staphylococcus aureus but not Candida albicans. The results of the anti-microbial assay indicated that the PEC-based NPs stabilized piperine adjuvant simvastatin showed anti-microbial activity against Staphylococcus aureus but did not exhibit anti-fungal activity against Candida albicans.

Piperine-adjuvant simvastatin Ch-NG-PEC nanoparticles demonstrate potential as a dual-treatment agent for hypercholesterolemia and bacterial infections.

Rheumatoid arthritis (RA) is a chronic inflammatory condition of the joints and a leading cause of global disability. However, the use of current anti-inflammatory treatments is often limited by serious side effects and multi-organ toxicity, necessitating the exploration of safer alternatives.

This study aims to investigate the anti-rheumatic potential of natural compounds of Cassia angustifolia as small-molecule inhibitors of PTGS2.

The therapeutic potential of C. angustifolia was evaluated through antioxidant and anti-inflammatory assays. Gas chromatography-mass spectrometry (GC-MS) was used to identify its constituents. ADMET profiling (absorption, distribution, metabolism, excretion, and toxicity), network pharmacology, and molecular dynamics simulation were employed to uncover the active compounds against PTGS2 for RA treatment.

C. angustifolia extract contained significant phenolic (18.2 ± 0.008 mg GAE/g DW) and flavonoid (27.57 ± 0.03 mg RE/g DW) content. GC-MS yielded 288 compounds of which four passed the toxicity parameters. Protein-protein interaction analysis revealed 10 RA-related targets, with PTGS2 emerging as the most prominent one. Molecular docking and simulations revealed that compound-2 [2-Benzo [1,3] dioxol-5-yl-8-methoxy-3-nitro-2H-chromene] and compound-4 [alpha-hydroxy-N-[2-methoxyphenyl]-benzene propanamide] binds strongly with PTGS2 (-7.7 kcal/mol and -7.9 kcal/mol, respectively) predicting its stable interaction.

C. angustifolia compounds present a significant potential as PTGS2 inhibitors, warranting further in vitro and in vivo investigations to confirm their therapeutic efficacy against RA.

Sorafenib is a first-line treatment for patients with advanced hepatocellular carcinoma (HCC), but its clinical efficacy is often compromised by the acquisition of drug resistance. Various cancers, including HCC, are affected by long non-coding RNA (lncRNA), but the mechanisms underlying HCC sorafenib resistance have not been extensively studied. This article aims to summarize the recently reported pathways associated with sorafenib resistance and discusses potential applications for the treatment of HCC.

Relevant studies on the resistance of HCC to anti-tumor drugs were retrieved from PubMed. Given the compelling evidence that sorafenib is an effective treatment for advanced HCC, we analyzed the research papers on lncRNA and sorafenib resistance in HCC in the PubMed system in the past decade and found that lncRNA may be involved in sorafenib resistance in HCC through multiple pathways.

lncRNA is widely involved in the resistance mechanism of HCC to sorafenib. Recent studies have revealed that numerous lncRNAs, such as NEAT1, affect the sensitivity of HCC to sorafenib through various mechanisms, including autophagy and AKT signaling pathways.

lncRNAs play a pivotal role in modulating HCC resistance to sorafenib. And lncRNA is expected to become a new solution to the resistance of sorafenib and other targeted drugs.

Bear bile powder (BBP) has been traditionally used in Chinese medicine for calming the liver, pacifying the mind, and relieving convulsions, as recorded in Ben Jing Feng Yuan and Yu Qiu Yao Jie. Although the antidepressant effects of BBP have been previously reported, the underlying neurological mechanisms have yet to be fully elucidated. This study aimed to investigate the antidepressant effects of BBP on corticosterone (CORT)-induced depression-like behaviors in female mice and to explore the involvement of the BDNF/TrkB/CREB signaling pathway.

Female mice received subcutaneous CORT injections to induce depression-like behaviors, followed by oral administration of BBP at doses of 50, 100, and 200 mg/kg. Behavioral assessments, biochemical analyses, UPLC-MS/MS, immunohistochemistry, and Western blotting were conducted to evaluate antidepressant effects. Additionally, a CORT-induced HT22 cell injury model was established to assess the neuroprotective mechanisms of BBP, with or without the TrkB antagonist K252a, focusing on the BDNF/TrkB/CREB pathway.

BBP significantly alleviated depression-like behaviors in CORT-treated female mice. It restored neurotransmitter levels, reduced neuronal necrosis in the hippocampal CA3 region, increased DCX-positive cells in the dentate gyrus, and activated hippocampal BDNF/TrkB/CREB signaling. In vitro, BBP attenuated CORT-induced apoptosis and promoted proliferation in HT22 cells. Applying K252a confirmed that BBP’s neuroprotective and antidepressant effects were mediated via the BDNF/TrkB/CREB pathway.

These findings suggest that BBP exerts notable antidepressant and neuroprotective effects in female depression models by modulating neurotransmitters and enhancing neurogenesis through the BDNF/TrkB/CREB pathway. Using both in vivo and in vitro models strengthens the evidence for BBP’s mechanism of action. However, further studies involving additional brain regions and upstream regulatory mechanisms are warranted.

BBP effectively alleviates CORT-induced depressive-like behaviors in female mice by restoring neurotransmitter balance, protecting hippocampal neurons, and promoting neurogenesis via the BDNF/TrkB/CREB pathway. These results provide a theoretical basis for the potential application of BBP in managing female depression.

The autoimmune inflammatory disease known as rheumatoid arthritis (RA) has a complicated and poorly understood etiology. Fibroblast-like synoviocytes (FLSs) have tumor-like characteristics in RA, including aggressive growth and heightened activation that leads to the release of pro-inflammatory factors. These processes are essential for the gradual deterioration of joint tissues. Kaempferol, with the chemical formula 3,5,7-trihydroxy-2-(4-hydroxyphenyl)-4H-1-benzopyran-4-one, is found in many different types of plants and plant families. The pharmacological effects of this substance have been well-documented. The benefits of this substance encompass protection for the heart and brain, as well as fighting inflammation, bacteria, cancer, osteoporosis, and allergies. It also has properties that can help with anxiety, pain relief, and hormonal balance. However, its precise function in the management of RA is still unclear.

To investigate the effect of kaempferol on apoptosis in RA FLSs and elucidate the underlying mechanisms.

We used the CCK-8 assay to assess the effects of different kaempferol concentrations on RA FLSs. We also used flow cytometry with Annexin V-FITC/PI staining to analyse cell cycle distribution and quantify apoptotic cells. To verify apoptosis, the TUNEL test was employed. Important proteins associated with apoptosis were verified to be expressed using western blotting. Finally, network pharmacology analysis was used to identify potential kaempferol targets, and their interactions with AKT1, PIK3R1, and HSP90AA1 proteins were studied using molecular docking and molecular dynamics simulations.

Kaempferol treatment significantly increased apoptosis in RA FLSs, up-regulating the pro-apoptotic protein Bax and down-regulating the anti-apoptotic protein Bcl-2. Specifically, kaempferol at 100 and 200 μM increased the apoptosis index to 29.77 ± 6.02% and 55.63 ± 11.05%, respectively, compared to the control. The induction of caspase-9 and caspase-3 cleavage was observed, indicating the activation of the mitochondrial pathway. Kaempferol also inhibited the phosphorylation of PI3K and Akt, with a significant reduction in their activation. Molecular docking studies demonstrated that kaempferol interacted with AKT1, PIK3R1, and HSP90AA1 proteins, with binding energies of -6.51, -4.26, and -6.51 kcal/mol, respectively, suggesting a strong affinity and potential direct impact on these proteins.

Kaempferol induces apoptosis in RA FLSs by inhibiting phosphorylation of the PI3K/Akt signaling pathway, increasing levels of pro-apoptotic proteins, and decreasing levels of anti-apoptotic proteins. Thus, kaempferol, a naturally occurring flavonoid, has great promise in the management of RA.

This research explored the antimicrobial, antifungal, and in vivo anticandidal activities of two herbal extracts: Ocimum basilicum (HEOB) and Ocimum sanctum (HEOS). Additionally, the study analyzed the phytochemical components of these extracts.

To examine the efficacy of HEOB and HEOS extracts in terms of their antimicrobial, antifungal, and anti-candidal activities and analyze their phytochemical composition, antioxidant potential, and immunomodulatory properties in vivo.

Dried flowers and leaves from Ocimum basilicum and Ocimum sanctum were extracted using a cold maceration process with a 1:1 ethanol-water solution. Phytochemical analysis followed established protocols, and the total phenolic and flavonoid contents were measured using colourimetric methods. HPLC was used to determine the concentrations of specific compounds, including rosmarinic acid, rutin, eugenol, and quercetin. Antioxidant activity, specifically nitric oxide (NO) scavenging and antimicrobial properties, was assessed in vitro using the cup plate method. In vivo studies were conducted on immunocompromised mice with systemic candidiasis, treated with plant extracts at 200 and 400 mg/kg or with ketoconazole as a control. Survival rates, tissue histology, and leukocyte counts were evaluated, and statistical analysis was performed using ANOVA.

HEOB and HEOS extracts possess strong antimicrobial and antioxidant activities, largely due to flavonoids such as rutin, quercetin, rosmarinic acid and eugenol. In vivo experiments revealed that both extracts effectively reduced fungal load, increased survival rates, and alleviated immunosuppression in mice with systemic candidiasis. The extracts also exhibited significant immunomodulatory properties by boosting cell-mediated immune responses. At higher concentrations, the antifungal performance of HEOB and HEOS was similar to that of ketoconazole.

HEOB and HEOS exhibited strong antibacterial, antifungal, and anticandidal properties, showing significant effectiveness in treating systemic candidiasis. Their immunomodulatory effects and ability to boost cell-mediated immunity make these extracts promising options for addressing systemic candidiasis, particularly in individuals with weakened immune systems. This research offers valuable insights and sets the stage for future investigations into the treatment of oral and vaginal candidiasis.

Xuefu Zhuyu Decoction (XFZYD) is clinically used in China to promote blood circulation, resolve blood stasis, and alleviate ventricular remodeling (VR). However, its molecular mechanisms remain unclear.

This study investigates the active components and underlying molecular mechanisms of XFZYD in treating VR.

Targets of XFZYD's active components and VR-related targets were identified. A protein-protein interaction (PPI) network and a drug-ingredient-target network were constructed. GO functional annotation and KEGG pathway enrichment analysis were performed to explore biological functions. Hub targets and their corresponding active ingredients were validated through molecular docking and molecular dynamics (MD) simulations.

A total of 1,089 active ingredients with high gastrointestinal absorption (GI) and drug-likeness (DL ≥ 2) were identified. Five hundred and thirty-eight common targets were shared between XFZYD and VR, with 10 core targets, including AKT1, STAT3, TP53, EGFR, SRC, TNF, MAPK3, CTNNB1, IL6, and VEGFA. GO analysis revealed XFZYD's influence on wound healing, oxygen response, epithelial cell proliferation, and receptor signaling. KEGG analysis highlighted key pathways such as PI3K-Akt signaling, lipid and atherosclerosis, and fluid shear stress. Molecular docking revealed that active ingredients display favorable interactions with the hub genes, with binding energies from -9.5 to -6.0 kcal/mol. These interactions were further validated through MD simulations, demonstrating stable binding throughout the 100 ns simulation period.

XFZYD exhibits therapeutic effects on VR through multiple active components and pathways, providing a scientific basis for its clinical application and further research.

This study aimed to formulate and evaluate dimenhydrinate (DMH) as fast-disintegrating tablets (FDTs) complexed with β-cyclodextrin (β-CD) to enhance its solubility, dissolution profile, and pharmacological performance.

A DMH:β-CD inclusion complex was prepared at a 1:1 molar ratio using the kneading method. Characterization was performed through phase solubility studies, FTIR analysis, molecular docking, and in vitro dissolution testing. FDTs were developed using various superdisintegrants and assessed for quality attributes of a tablet, including hardness, friability, wetting time, water absorption ratio, and drug content.

Phase solubility and FTIR analyses confirmed the formation of a stable DMH:β-CD complex. Molecular docking indicated a binding affinity of -4.2 kcal/mol between β-CD and diphenhydramine. Among the FDT formulations, CP3 containing 9% crospovidone showed the best performance, with a disintegration time of 4.3 seconds and the highest drug release rate. In vivo pharmacological tests demonstrated enhanced sedative and antiemetic activities of the optimized FDTs compared to conventional DMH formulations.

The findings suggest that cyclodextrin-based complexation combined with orodispersible tablet technology can significantly enhance DMH's pharmacological efficacy and patient compliance. However, additional investigations on long-term stability, pharmacokinetics, and clinical scalability are warranted.

The DMH:β-CD FDTs developed in this study offer promising improvements in solubility, dissolution, and therapeutic performance, indicating their potential for better clinical outcomes and patient acceptability.

In this study, the causation between serum metabolites and the risk of Diabetic Nephropathy (DN) was investigated by means of a Mendelian Randomization (MR) analysis.

Our data on diabetic nephropathy were obtained from the IEU OpenGWAS Project database, while serum metabolite data originated came from the GWAS summary statistics by Chen et al. The Inverse Variance Weighted (IVW) method was the main analysis approach, with Weighted Median (WME) and MR-Egger regression serving as supplementary approaches to construing the causalities between serum metabolites and the DN risk. In addition to the MR-Egger regression intercept, Cochran's Q test was utilized for sensitivity analysis, with P values used as the metric to assess the results.

In total, 14 SNPs regarding serum metabolites were chosen as Instrumental Variables (IVs). The IVW results indicated that levels of Behenoylcarnitine (C22), Arachidoylcarnitine (C20), and the ratio of

5-methylthioadenosine (MTA) to phosphate exerted a positive causal effect on the DN risk. Conversely, levels of 5-hydroxylysine, Butyrylglycine, 1-stearoyl-glycerophosphocholine (18:0), Isobutyrylglycine, 1-stearoyl-2-oleoyl-GPE (18:0/18:1), N2,N5-diacetylornithine, 2-butenoylglycine, 3-hydroxybutyroylglycine, N-acetyl-isoputreanine, the ratio of Arginine to Ornithine, and the ratio of Aspartate to Mannose exerted a negative impact of causality on the DN risk. By identifying these serum metabolites, high-risk patients can be recognized in the early stages of diabetic nephropathy, enabling preventive measures or delaying its progression. These findings also provide a solid foundation for further research into the underlying etiology of diabetic nephropathy.

The translation of serum metabolites into clinical applications for DN aims to utilize changes in serum metabolites as biomarkers for early diagnosis, thereby monitoring the progression of DN and providing a foundation for personalized treatment. For instance, the development of serum metabolite diagnostic kits could be used for early detection and prevention of DN. Changes in metabolites can help identify different stages of DN.

Triple-negative breast cancer (TNBC) is an aggressive subtype characterized by

the absence of estrogen and progesterone receptors (ER, PR) and low or absent HER2 expression, limiting treatment options. Quercetin, a flavonoid with anti-cancer properties, has the potential to be a therapeutic

intervention.

The study aimed to explore the potential of Quercetin derivatives as therapeutic agents for TNBC using several computational methods.

The study utilized PASS prediction, molecular docking, ADMET prediction, QSAR models, MD simulations, binding free energy, and DFT calculations to evaluate the efficacy of quercetin derivatives.

ADMET analysis confirmed the solubility, non-carcinogenicity, and low toxicity of four quercetin derivatives: LM01, LM02, LM05, and LM10. These derivatives exhibited strong binding affinity against TNBC protein PPAR1, with binding energies of -10.6, -10.7, -11.4, and -10 kcal/mol, respectively. MD simulations confirmed their stability, with consistent RMSD values and favorable RMSF values. Post-simulation calculations and reduced HOMO-LUMO energy gaps further supported their potential as promising candidates.

Our computational findings suggest that quercetin derivatives, particularly LM01, LM02, and LM10, exhibit strong stability and binding affinity, positioning them as promising candidates for TNBC treatment. Further experimental validation is required to confirm their therapeutic potential.

Colon-targeted drug delivery is a crucial area of research aimed at treating local disorders like IBD, including ulcerative colitis and Crohn's disease. By delivering drugs directly to the colon, this approach enhances therapeutic efficacy and minimizes systemic toxicity. Nanoparticles are an effective vehicle for controlled drug delivery, improving treatment outcomes for colon-specific diseases.

The study aimed to develop an oral nanoparticulate formulation of Satranidazole (STZ) using a solvent evaporation technique for colonic targeting and characterize its physicochemical properties, compatibility, and in vitro drug release profile.

Using a modified solvent evaporation method, STZ-loaded nanoparticles (STZ-NPs) were formulated using Eudragit RS100 and RL100 polymers. Preformulation studies, including FT-IR and DSC, were performed to confirm the compatibility between the drug and polymers. The nanoparticles were evaluated in terms of entrapment efficiency, particle size, zeta potential, polydispersity index, and in vitro drug release study.

The optimized formulation (F3) demonstrated the highest entrapment efficiency (83.55%) with particle sizes ranging from 107.9 nm to 302 nm and a zeta potential between -34.25 mV and +48.8 mV. In vitro drug release studies showed controlled release over 16 hours, with the optimized batch achieving 95.85% drug release, indicating effective accumulation in the inflamed colon.

The Satranidazole-loaded nanoparticles, containing time- and pH-dependent polymers, successfully inhibited premature drug release in acidic environments and provided controlled release at colonic pH. Thus, this delivery system shows promise as an effective treatment for IBD, offering targeted drug release and reduced systemic toxicity.

Dysphania ambrosioides, commonly known as “mastruz,” is a medicinal plant traditionally used for its therapeutic properties, including antimicrobial and anti-inflammatory effects. Previous studies have also suggested its antitumor potential. However, its role in oral squamous cell carcinoma (OSCC) remains unexplored. This study aimed to evaluate the in vitro cytotoxic effects of D. ambrosioides extracts on SCC4 (OSCC) and HaCaT (human keratinocyte) cell lines.

Crude extracts were obtained using different methods, including hexanic, ethanolic, hydroethanolic (7:3), and aqueous extractions, all performed ultrasonic-assisted extraction. The extracts were tested at concentrations ranging from 7.81 µg/mL to 1000 µg/mL using 2-fold serial dilutions. Cell viability was assessed after 48 hours of treatment using the MTT assay, with DMSO as the control.

The extracts exhibited concentration-dependent cytotoxic effects on both cell lines, with HaCaT cells showing greater sensitivity. However, the lack of selectivity toward tumor cells over normal cells suggests a broad-spectrum cytotoxic activity without tumor-specific therapeutic targeting.

These findings highlight the need for further fractionation of the extracts and identification of the bioactive compounds responsible for the observed effects. Although the extracts demonstrated significant cytotoxic activity, their therapeutic potential should not be limited to cytotoxicity alone. Future studies should explore additional biological activities, such as anti-inflammatory or immunomodulatory properties, to fully understand the therapeutic applications of D. ambrosioides.