Esculin Alleviates Osteoarthritis Progression Through the Sirt1/NF-κB Pathway.

Osteoarthritis (OA), a joint disease associated with inflammatory processes, contributes to joint destruction. Esculin (ESC) extracted from the stem bark of Fraxinus rhynchophylla Hance has been shown to possess anti-inflammatory properties. In this study, we investigated the effect of ESC on chondrocytes treated with IL-1ß and its molecular mechanism. The importance and potential mechanism of ESC in the progression of OA were evaluated. The viability of chondrocytes after exposure to ESC was examined through the CCK-8 assays. The cells were then subjected to quantitative polymerase chain reaction (qPCR), western blot, and enzyme-linked immunosorbent assay (ELISA) techniques to analyze the degradation of the extracellular matrix (ECM) and occurrence of inflammation. The NF-κB mechanism was evaluated by western blot analysis, immunofluorescence (IF), and luciferase reporter assay. Molecular docking was performed to allow for predictions on proteins that interact with ESC. Moreover, the significance of Sirt1 was explored through a knockdown experiment based on siRNA. Micro-computed tomography (CT), H&E, Safranin O-Fast Green (S-O), and immunohistochemical analyses were carried out to assess the treatment efficacy of ESC on OA in destabilization of medial meniscus (DMM) models. ESC treatment effectively inhibited ECM degradation, modulated the levels of pro-inflammatory factors, and regulated the NF-κB signaling in chondrocytes exposed to IL-1ß. Mechanistically, we found that ESCs bound to Sirt1 to inhibit the activity of the NF-κB mechanism. Furthermore, ESC treatment suppressed OA progression in the DMM models. Our findings reveal that ESC ameliorates OA progression via modulating the Sirt1/NF-κB axis. This demonstrates that ESC has the potential to be applied in the treatment of OA.

The possible anti-tumor actions and mechanisms of active metabolites from Cortex Fraxini.

Cortex Fraxini is a traditional Chinese herb that is widely available, inexpensive, and has low toxicity. Modern pharmacological studies have demonstrated that the active metabolites in Cortex Fraxini, including esculin, esculetin, and fraxetin, exert anti-tumor activities by regulating genes and proteins involved in cancer cell proliferation, apoptosis, invasion, and migration. Additionally, these metabolites play a pivotal role in the regulation of several tumor-associated signaling pathways, including the PI3K/Akt, MAPK/ERK, JAK/STAT3, and Wnt/ß-catenin pathways. Due to their pro-apoptotic and anti-proliferative properties in vitro and in vivo, Cortex Fraxini and its active metabolites may be considered as potential candidates for the treatment of tumor. The aim of this review is to highlight the anti-tumor biological activities and underlying mechanisms of action of the active metabolites of Cortex Fraxini, with a view to providing a reference for their further development and application in the treatment of tumors.

Revealing the Mechanism of Esculin in Treating Renal Cell Carcinoma Based on Network Pharmacology and Experimental Validation.

PURPOSE: This study aims to explore the potential mechanisms of esculin in the treatment of renal cell carcinoma (RCC). METHODS: We employed network pharmacology to predict the potential mechanisms and targets of esculin in RCC. Molecular docking techniques were then employed to validate the predicted targets. Additionally, a series of in vitro experiments were conducted to verify the anticancer effects of esculin on RCC cells, including the CCK-8 assay, EdU assay, wound healing assay, apoptosis assay, and Western blot. RESULTS: Network pharmacology and molecular docking results identified GAPDH, TNF, GSK3B, CCND1, MCL1, IL2, and CDK2 as core targets. GO and KEGG analyses suggested that esculin may influence apoptotic processes and target the PI3K/Akt pathway in RCC. Furthermore, the CCK-8 assay demonstrated that esculin inhibited RCC cell viability. Microscopic observations revealed that following esculin treatment, there was an increase in cell crumpling, a reduction in cell density, and an accumulation of floating dead cells. Additionally, with increasing esculin concentrations, the proportion of EdU-positive cells decreased, the wound closure ratio decreased, the proportion of PI-positive cells increased, the expression levels of BAX and cleaved-caspase-3 proteins increased, and the expression level of Bcl2 protein decreased. These findings suggested that esculin inhibits the proliferation and migration of RCC cells while promoting apoptosis. Moreover, esculin was found to target GAPDH and inhibit the PI3K/Akt pathway. CONCLUSIONS: This study is the first to elucidate the therapeutic effects of esculin on RCC cells. The results provide evidence supporting the clinical application of esculin and introduce a promising new candidate for RCC treatment.

Esculin alleviates lipopolysaccharide (LPS)-induced pneumonia by regulating the USP7/MAPK14 axis.

Pneumonia is a serious and life-threatening lung inflammation with high morbidity and mortality. Accumulating evidence has suggested that esculin, a derivative of coumarin, possesses potent anti-inflammatory effects. This study is designed to explore the pharma role and underlying mechanism of esculin against lipopolysaccharides (LPS)-induced pneumonia. TC-1 cells were stimulated by LPS to mimic the inflammatory injury model in vitro. Cell viability, proliferation, and apoptosis were determined using MTT assay, 5-ethynyl-2'-deoxyuridine assay, and flow cytometry. Interleukin-1ß and tumor necrosis factor α levels were analyzed using an enzyme-linked immunosorbent assay. Reactive oxygen species and superoxide dismutase were examined using special assay kits. Macrophage polarization was detected using flow cytometry. Mitogen-activated protein kinase 14 (MAPK14) level was detected by real-time quantitative polymerase chain reaction. MAPK14 and ubiquitin-specific protease 7 (USP7) protein levels were determined using western blot assay. After Ubibrowser database prediction, the interaction between USP7 and MAPK14 was verified using a Co-immunoprecipitation assay. The biological role of esculin was verified in LPS-challenged ALI mice in vivo. Here, we found that esculin significantly relieved LPS-induced TC-1 cell proliferation inhibition, and apoptosis, inflammatory response, oxidative stress, and M1-type macrophage polarization promotion. MAPK14 and USP7 expressions were enhanced in LPS-treated TC-1 cells, which was partly abolished by esculin treatment. Overexpressing MAPK14 attenuated the repression of esculin on LPS-triggered TC-1 cell injury. At the molecular level, USP7 interacted with MAPK14 and maintained its stability by removing ubiquitin. Moreover, esculin repressed the progression of pneumonia in vivo by regulating MAPK14. Taken together, esculin exposure could mitigate LPS-induced TC-1 cell injury partly by targeting the USP7/MAPK14 axis, providing a better understanding of the role of esculin in the anti-inflammatory therapeutics for pneumonia.

Esculin suppresses the PERK-eIF2α-CHOP pathway by enhancing SIRT1 expression in oxidative stress-induced rat chondrocytes, mitigating osteoarthritis progression in a rat model.

OBJECTIVE: Osteoarthritis (OA) is a degenerative disease characterized by the gradual degeneration of chondrocytes, involving endoplasmic reticulum (ER) stress. Esculin is a natural compound with antioxidant, anti-inflammatory and anti-tumor properties. However, its impact on ER stress in OA therapy has not been thoroughly investigated. We aim to determine the efficiency of Esculin in OA treatment and its underlying mechanism. METHODS: We utilized the tert-butyl hydroperoxide (TBHP) to establish OA model in chondrocytes. The expression of SIRT1, PERK/eIF2α pathway-related proteins, apoptosis-associated proteins and ER stress-related proteins were detected by Western blot and Real-time PCR. The apoptosis was evaluated by flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining. X-ray imaging, Hematoxylin & Eosin staining, Safranin O staining and immunohistochemistry were used to assess the pharmacological effects of Esculin in the anterior cruciate ligament transection (ACLT) rat OA model. RESULTS: Esculin downregulated the expression of PERK/eIF2α pathway-related proteins, apoptosis-associated proteins and ER stress-related proteins, while upregulated the expression of SIRT1 and Bcl2 in the TBHP-induced OA model in vitro. It was coincident with the results of TUNEL staining and flow cytometry. We further confirmed the protective effect of Esculin in the rat ACLT-related model. CONCLUSION: Our results suggest the potential therapeutic value of Esculin on osteoarthritis. It probably inhibits the PERK-eIF2α-ATF4-CHOP pathway by upregulating SIRT1, thereby mitigating endoplasmic reticulum stress and protecting chondrocytes from apoptosis.

Antioxidant and anti­inflammatory effects of esculin and esculetin (Review).

Fraxinus chinensis Roxb is a deciduous tree, which is distributed worldwide and has important medicinal value. In Asia, the bark of Fraxinus chinensis Roxb is a commonly used traditional Chinese medicine called Qinpi. Esculetin is a coumarin compound derived from the bark of Fraxinus chinensis Roxb and its glycoside form is called esculin. The aim of the present study was to systematically review relevant literature on the antioxidant and anti-inflammatory effects of esculetin and esculin. Esculetin and esculin can promote the expression of various endogenous antioxidant proteins, such as superoxide dismutase, glutathione peroxidase and glutathione reductase. This is associated with the activation of the nuclear factor erythroid-derived factor 2-related factor 2 signaling pathway. The anti-inflammatory effects of esculetin and esculin are associated with the inhibition of the nuclear factor κ-B and mitogen-activated protein kinase inflammatory signaling pathways. In various inflammatory models, esculetin and esculin can reduce the expression levels of various proinflammatory factors such as tumor necrosis factor-α, interleukin (IL)-1ß and IL-6, thereby inhibiting the development of inflammation. In summary, esculetin and esculin may be promising candidates for the treatment of numerous diseases associated with inflammation and oxidative stress, such as ulcerative colitis, acute lung and kidney injury, lung cancer, acute kidney injury.

Preparation, characterisation, pharmacokinetics and distribution of esculin microspheres administered via intravitreal injection into rabbit brain.

This study explored the distribution of esculin microspheres in rabbit brain tissue following intravitreal injection and investigated the possibility of direct entry of the drug into the brain through the eye, to develop a formulation with enhanced therapeutic efficacy against Parkinson's disease.Chitosan microspheres of esculin were prepared via an emulsification cross-linking method and their characteristics were evaluated, including angle of repose, bulk density, and swelling ratio. Furthermore, the pharmacokinetic parameters and brain tissue distribution in rabbits were compared among groups administered esculin eye drops, intravitreal esculin solution, and intravitreal esculin microspheres, to determine whether esculin could enter the brain through an ocular route.The results showed that the prepared esculin microspheres were spherical and had good fluidity. Notably, intravitreal administration enhanced the area under the curve (AUC) of esculin in the thalamus. Delivery through microspheres prolonged the drug retention time in both rabbit plasma and brain tissues, as well as the brain-targeting efficiency of esculin.The collective findings indicated that there may be a direct eye-brain pathway facilitating enter of esculin microspheres into brain tissue after intravitreal injection, supporting the utility of intravitreal esculin microspheres as an effective therapeutic formulation for Parkinson's disease, a long-term chronic condition.

Esculin induces endoplasmic reticulum stress and drives apoptosis and ferroptosis in colorectal cancer via PERK regulating eIF2α/CHOP and Nrf2/HO-1 cascades.

ETHNOPHARMACOLOGICAL RELEVANCE: Cortex fraxini (also known as Qinpi), the bark of Fraxinus rhynchophylla Hance and Fraxinus stylosa Lingelsh, constitutes a crucial component in several traditional Chinese formulas (e.g., Baitouweng Tang, Jinxiao Formula, etc.) and has demonstrated efficacy in alleviating intestinal carbuncle and managing diarrhea. Cortex fraxini has demonstrated commendable anticancer activity in the realm of Chinese ethnopharmacology; nevertheless, the underlying mechanisms against colorectal cancer (CRC) remain elusive. AIM OF THE STUDY: Esculin, an essential bioactive compound derived from cortex fraxini, has recently garnered attention for its ability to impede viability and induce apoptosis in cancer cells. This investigation aims to assess the therapeutic potential of esculin in treating CRC and elucidate the underlying mechanisms. MATERIALS AND METHODS: The impact of esculin on CRC cell viability was assessed using CCK-8 assay, Annexin V/PI staining, and Western blotting. Various cell death inhibitors, along with DCFH-DA, ELISA, biochemical analysis, and Western blotting, were employed to delineate the modes through which esculin induces HCT116 cells death. Inhibitors and siRNA knockdown were utilized to analyze the signaling pathways influenced by esculin. Additionally, an azomethane/dextran sulfate sodium (AOM/DSS)-induced in vivo CRC mouse model was employed to validate esculin's potential in inhibiting tumorigenesis and to elucidate its underlying mechanisms. RESULTS: Esculin significantly suppressed the viability of various CRC cell lines, particularly HCT116 cells. Investigation with diverse cell death inhibitors revealed that esculin-induced cell death was associated with both apoptosis and ferroptosis. Furthermore, esculin treatment triggered cellular lipid peroxidation, as evidenced by elevated levels of malondialdehyde (MDA) and decreased levels of glutathione (GSH), indicative of its propensity to induce ferroptosis in HCT116 cells. Enhanced protein levels of protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK) and p-eIF2α suggested that esculin induced cellular endoplasmic reticulum (ER) stress, subsequently activating the Nrf2/ARE signaling pathway and initiating the transcriptional expression of heme oxygenase (HO)-1. Esculin-induced excessive expression of HO-1 could potentially lead to iron overload in HCT116 cells. Knockdown of Ho-1 significantly attenuated esculin-induced ferroptosis, underscoring HO-1 as a critical mediator of esculin-induced ferroptosis in HCT116 cells. Furthermore, utilizing an AOM/DSS-induced colorectal cancer mouse model, we validated that esculin potentially inhibits the onset and progression of colon cancer by inducing apoptosis and ferroptosis in vivo. CONCLUSIONS: These findings provide comprehensive insights into the dual induction of apoptosis and ferroptosis in HCT116 cells by esculin. The activation of the PERK signaling pathway, along with modulation of downstream eIF2α/CHOP and Nrf2/HO-1 cascades, underscores the mechanistic basis supporting the clinical application of esculin on CRC treatment.

Esculin inhibits hepatic stellate cell activation and CCl4-induced liver fibrosis by activating the Nrf2/GPX4 signaling pathway.

BACKGROUND: Liver fibrosis (LF) is a pathological process of the liver that threatens human health. Currently, effective treatments are still lacking. Esculin, a prominent constituent found in the Fraxinus rhynchophylla. (bark), Aesculus hippocastanum. (bark), and Cichorium intybus. (herb), has been shown to possess significant anti-inflammatory, antioxidant, and antibacterial properties. However, to date, there have been no studies investigating its potential efficacy in the treatment of LF. OBJECTIVE: The study aims to investigate the therapeutic effect of esculin on LF and elucidate its potential molecular mechanism. METHODS: Carbon tetrachloride (CCl4) was injected intraperitoneally to induce LF in mice, and transforming growth factor ß1 (TGF-ß1) was injected to induce LX-2 cells to investigate the improvement effect of esculin on LF. Kit, histopathological staining, immunohistochemistry (IHC), immunofluorescence (IF), polymerase chain reaction (PCR), and western blot (WB) were used to detect the expression of fiber markers and nuclear factor erythroid 2-related factor 2 (Nrf2)/glutathione peroxidase 4 (GPX4) signaling pathway in liver tissue and LX-2 cells. Finally, molecular docking, cellular thermal shift assay (CETSA), and drug affinity responsive target stability (DARTS) were used to verify the targeting between Nrf2 and esculin. RESULTS: Esculin significantly inhibited CCl4-induced hepatic fibrosis and inflammation in mice. This was evidenced by the improvement of liver function indexes, fibrosis indicators, and histopathology. Additionally, esculin treatment prominently reduced the levels of pro-inflammatory factors, oxidative stress, and liver Fe2+ in CCl4-induced mice. In vitro studies also showed that esculin treatment significantly inhibited TGF-ß1-induced LX-2 cell activation and decreased alpha-smooth muscle actin (α-SMA) and collagen I expression. Mechanism experiments proved that esculin can activate the Nrf2/GPX4 signaling pathway and inhibit liver ferroptosis. However, when LX-2 cells were treated with the Nrf2 inhibitor (ML385), the therapeutic effect of esculin significantly decreased. CONCLUSION: This study is the first to demonstrate that esculin is a potential natural active ingredient in the treatment of LF, which can inhibit the activation of hepatic stellate cells (HSC) and improve LF. Its therapeutic effect is related to the activation of the Nrf2/GPX4 signaling pathway.

Esculin targets TLR4 to protect against LPS-induced septic cardiomyopathy.

BACKGROUND: Esculin, a main active ingredient from Cortex fraxini, possesses biological activities such as anti-thrombosis, anti-inflammatory, and anti-oxidation effects. However, the effects of Esculin on septic cardiomyopathy remains unclear. This study aimed to explore the protective properties and mechanisms of Esculin in countering sepsis-induced cardiac trauma and dysfunction. METHODS AND RESULTS: In lipopolysaccharide (LPS)-induced mice model, Esculin could obviously improve heart injury and function. Esculin treatment also significantly reduced the production of inflammatory and apoptotic cells, the release of inflammatory cytokines, and the expression of oxidative stress-associated and apoptosis-associated markers in hearts compared to LPS injection alone. These results were consistent with those of in vitro experiments based on neonatal rat cardiomyocytes. Database analysis and molecular docking suggested that TLR4 was targeted by Esculin, as shown by stable hydrogen bonds formed between Esculin with VAL-308, ASN-307, CYS-280, CYS-304 and ASP-281 of TLR4. Esculin reversed LPS-induced upregulation of TLR4 and phosphorylation of NF-κB p65 in cardiomyocytes. The plasmid overexpressing TLR4 abolished the protective properties of Esculin in vitro. CONCLUSION: We concluded that Esculin could alleviate LPS-induced septic cardiomyopathy via binding to TLR4 to attenuate cardiomyocyte inflammation, oxidative stress and apoptosis.

In vivo comprehensive metabolite profiling of esculetin and esculin derived from chicory in hyperuricemia rats using ultra-high-performance liquid chromatography coupled with quadrupole-orbitrap high-resolution mass spectrometry.

Chicory, renowned for its multifaceted benefits, houses two vital coumarins, esculetin and esculin, both instrumental in reducing uric acid. This study emphasizes the metabolic pathways of esculetin and esculin under both standard and hyperuricemia conditions. Hyperuricemia was induced in Sprague-Dawley rats using oxonic acid potassium salt (300 mg·kg-1 ) and a 10% fructose water regimen over 21 days. Leveraging the ultra-high-performance liquid chromatography-Q Exactive hybrid quadrupole-orbitrap high resolution mass spectrometry, we analyzed the fragmentation behaviors of esculetin and esculin in rat bio-samples. Post oral-intake of esculetin or esculin, a notable dip in serum uric acid levels was observed in hyperuricemia rats. The investigation unveiled 24 esculetin metabolites and 14 for esculin. The metabolic pathways of both compounds were hydrolysis, hydroxylation, hydrogenation, dehydroxylation, glucuronidation, sulfation, and methylation. Interestingly, certain metabolites presented variations between standard and hyperuricemia rats, indicating that elevated levels of uric acid may affect enzyme activity linked to these metabolic reactions. This is the first systematic study on comparison of metabolic profiles of esculetin and esculin in both normal and hyperuricemia states, which was helpful to enrich our understanding of the complicated structure-activity relationships between esculin and esculetin and shed light to their action mechanism.

Tracing the opposing assimilate and nutrient flows in live conifer needles.

The vasculature along conifer needles is fundamentally different from that in angiosperm leaves as it contains a unique transfusion tissue inside the bundle sheath. In this study, we used specific tracers to identify the pathway of photoassimilates from mesophyll to phloem, and the opposing pathway of nutrients from xylem to mesophyll. For symplasmic transport we applied esculin to the tip of attached pine needles and followed its movement down the phloem. For apoplasmic transport we let detached needles take up a membrane-impermeable contrast agent and used micro-X-ray computed tomography to map critical water exchange interfaces and domain borders. Microscopy and segmentation of the X-ray data enabled us to render and quantify the functional 3D structure of the water-filled apoplasm and the complementary symplasmic domain. The transfusion tracheid system formed a sponge-like apoplasmic domain that was blocked at the bundle sheath. Transfusion parenchyma cell chains bridged this domain as tortuous symplasmic pathways with strong local anisotropy which, as evidenced by the accumulation of esculin, pointed to the phloem flanks as the preferred phloem-loading path. Simple estimates supported a pivotal role of the bundle sheath, showing that a bidirectional movement of nutrient ions and assimilates is feasible and emphasizing the role of the bundle sheath in nutrient and assimilate exchange.

How pollen tubes fight for food: the impact of sucrose carriers and invertases of Arabidopsis thaliana on pollen development and pollen tube growth.

Pollen tubes of higher plants grow very rapidly until they reach the ovules to fertilize the female gametes. This growth process is energy demanding, however, the nutrition strategies of pollen are largely unexplored. Here, we studied the function of sucrose transporters and invertases during pollen germination and pollen tube growth. RT-PCR analyses, reporter lines and knockout mutants were used to study gene expression and protein function in pollen. The genome of Arabidopsis thaliana contains eight genes that encode functional sucrose/H+ symporters. Apart from AtSUC2, which is companion cell specific, all other AtSUC genes are expressed in pollen tubes. AtSUC1 is present in developing pollen and seems to be the most important sucrose transporter during the fertilization process. Pollen of an Atsuc1 knockout plant contain less sucrose and have defects in pollen germination and pollen tube growth. The loss of other sucrose carriers affects neither pollen germination nor pollen tube growth. A multiple knockout line Atsuc1Atsuc3Atsuc8Atsuc9 shows a phenotype that is comparable to the Atsuc1 mutant line. Loss of AtSUC1 can`t be complemented by AtSUC9, suggesting a special function of AtSUC1. Besides sucrose carriers, pollen tubes also synthesize monosaccharide carriers of the AtSTP family as well as invertases. We could show that AtcwINV2 and AtcwINV4 are expressed in pollen, AtcwINV1 in the transmitting tissue and AtcwINV5 in the funiculi of the ovary. The vacuolar invertase AtVI2 is also expressed in pollen, and a knockout of AtVI2 leads to a severe reduction in pollen germination. Our data indicate that AtSUC1 mediated sucrose accumulation during late stages of pollen development and cleavage of vacuolar sucrose into monosaccharides is important for the process of pollen germination.

The potential of esculin in ameliorating Type-2 diabetes mellitus induced neuropathy in Wistar rats and probing its inhibitory mechanism of insulin aggregation.

Diabetic neuropathy encompasses multiple pathological disturbances, many of which coincide with the pathophysiological mechanisms of neurodegenerative disorders. In the present study, various biophysical techniques like Rayleigh light scattering assay, Thioflavin T assay, far-UV Circular Dichroism spectroscopy, Transmission electron microscopy have unveiled the anti-fibrillatory effect of esculin upon human insulin fibrillation. MTT cytotoxicity assay demonstrated the biocompatibility of esculin and in-vivo studies such as behavioral tests like hot plate test, tail immersion test, acetone drop test, plantar test were performed for validating diabetic neuropathy. Assessment of levels of serum biochemical parameters, oxidative stress parameters, pro-inflammatory cytokines as well as neuron specific markers was done in the current study. Rat brains were subjected to histopathology and their sciatic nerves were subjected to transmission electron microscopy to analyze myelin structure alterations. All these results reveal that esculin ameliorates diabetic neuropathy in experimental diabetic rats. Conclusively, our study demonstrates the anti-amyloidogenic potential of esculin in the form of inhibition of human insulin fibrillation, making it a promising candidate in combating neurodegenerative disorders in the near future and the results of various behavioral, biochemical, and molecular studies reveal that esculin possesses anti-lipidemic, anti-inflammatory, anti-oxidative and neuroprotective properties which help in ameliorating diabetic neuropathy in streptozotocin induced diabetic Wistar rats.

The Use of Spectral Imaging to Follow the Iron and pH-Dependent Accumulation of Fluorescent Coumarins.

Plants challenged with iron deficiency produce in their roots and secrete into the rhizosphere several small molecules named coumarins that derive from the phenylpropanoid pathway. Coumarins are biosynthesized in different root cell types and transported to the root epidermis prior to their secretion in the surrounding media. Taking advantage of the natural fluorescence of most coumarins glycosides when exposed to UV light, we developed a method to uncover their individual cellular localization and accumulation. This approach couples spectral imaging acquisition and linear unmixing analysis. In this protocol, we describe guidelines, experimental setup, and conditions for the analysis of coumarins localization and accumulation in Arabidopsis thaliana root seedlings grown in control and iron deficiency conditions, at both acidic and alkaline pH.

Esculin alleviates LPS-induced acute lung injury via inhibiting neutrophil recruitment and migration.

OBJECTIVES: Acute lung injury (ALI) poses a serious threat to human health globally, particularly with the Coronavirus 2019 (COVID-19) pandemic. Excessive recruitment and infiltration of neutrophils is the major etiopathogenesis of ALI. Esculin, also known as 6,7-dihydroxycoumarin, is a remarkable compound derived from traditional Chinese medicine Cortex fraxini. Accumulated evidence indicates that esculin has potent anti-inflammatory effects, but its pharmaceutical effect against ALI and potential mechanisms are still unclear. METHODS: This study evaluated the protective effect of esculin against ALI by histopathological observation and biochemical analysis of lung tissues and bronchoalveolar lavage fluid (BALF) in lipopolysaccharide (LPS)-challenged ALI mice in vivo. The effects of esculin on N-formyl-met-leu-phe (fMLP)-induced neutrophil migration and chemotaxis were quantitatively assessed using a Transwell assay and an automated cell imaging system equipped with a Zigmond chamber, respectively. The drug affinity responsive target stability (DARTS) assay, in vitro protein binding assay and molecular docking were performed to identify the potential therapeutic target of esculin and the potential binding sites and pattern. RESULTS: Esculin significantly attenuated LPS-induced lung pathological injury, reduced the levels of pro-inflammatory cytokines in both BALF and lung, and suppressed the activation of NF-κB signaling. Esculin also significantly reduced the number of total cells and neutrophils as well as myeloperoxidase (MPO) activity in the BALF. Esculin impaired neutrophil migration and chemotaxis as evidenced by the reduced migration distance and velocity. Furthermore, esculin remarkably inhibited Vav1 phosphorylation, suppressed Rac1 activation and the PAK1/LIMK1/cofilin signaling axis. Mechanistically, esculin could interact with ß2 integrin and then diminish its ligand affinity with intercellular adhesion molecule-1 (ICAM-1). CONCLUSIONS: Esculin inhibits ß2 integrin-dependent neutrophil migration and chemotaxis, blocks the cytoskeletal remodeling process required for neutrophil recruitment, thereby contributing to its protective effect against ALI. This study demonstrates the new therapeutic potential of esculin as a novel lead compound.

Esculin protects human blood cells from bioallethrin-induced toxicity: An ex vivo study.

Bioallethrin, a household insecticide, is a member of the pyrethroid family and is known for its adverse effects on human health. Human exposure to pyrethroids is unavoidable due to their widespread use in controlling several fatal vector-borne diseases, mostly in developing nations. Bioallethrin is known to induce oxidative stress in target cells, including erythrocytes. Here we have studied the protective effect of dietary antioxidant esculin on bioallethrin-induced damage in isolated human erythrocytes. The cells were incubated with 200 µM bioallethrin, without or with different concentrations of esculin (200, 400 and 600 µM), and the results compared to the untreated control samples. Bioallethrin-treated erythrocytes showed a significant increase in oxidative stress markers, like protein and lipid oxidation, accompanied by decrease in free amino groups and ratio of reduced to oxidized glutathione. There was enhanced generation of reactive oxygen and nitrogen species with changes in plasma membrane integrity. Bioallethrin oxidized hemoglobin to methemoglobin, which cannot transport oxygen. It altered the activities of antioxidant enzymes and lowered the electron donating and free radical quenching ability of erythrocytes. The cell morphology and redox system of erythrocyte membrane were also altered by bioallethrin. Treatment with esculin, prior to incubation with bioallethrin, led to significant restoration in all these parameters in an esculin concentration-dependent manner. Thus esculin attenuated the biolletherin-induced oxidative damage to erythrocytes. Esculin can, therefore, be an effective chemoprotectant against xenobiotic-induced toxicity in human erythrocytes.

Molecular interactions of esculin with bovine serum albumin and recognition of binding sites with spectroscopy and molecular docking.

Esculin is structurally a hydroxycoumarin found in various medicinal plants. This study investigates the binding mode of esculin with bovine serum albumin by employing numerous spectroscopic studies and molecular docking approaches. Ultraviolet absorption spectroscopy revealed ground state complex formation between esculin and bovine serum albumin. At the same time, steady-state fluorescence studies showed quenching in the fluorescence emission spectra of BSA in the presence of esculin. To get insight into the location of the binding pocket of esculin on BSA, warfarin and ibuprofen site markers were used. Competitive site marker displacement assay revealed that esculin binds to Sudlow's site I (subdomain IIA) in bovine serum albumin. Thermodynamic parameters suggested that hydrogen bonding and van der Waals interaction stabilizes the esculin-BSA complex. Förster's non-radiation energy transfer analysis described the high propensity of energy transfer between bovine serum albumin and esculin. The molecular docking approach facilitated locating the binding pocket, amino acid residues involved, types of interacting forces, and binding energy (ΔG) between esculin and BSA. Circular dichroism revealed the effect of the binding of esculin on the secondary structure and helped understand the thermal unfolding profile of BSA in the presence of esculin.Communicated by Ramaswamy H. Sarm.

Ethnopharmacology of Rubus idaeus Linnaeus: A critical review on ethnobotany, processing methods, phytochemicals, pharmacology and quality control.

ETHNOPHARMACOLOGICAL RELEVANCE: Rubus idaeus Linnaeus (RI) is a Chinese herbal medicine that has been widely used in China for a long time to reinforce the kidney, nourish the liver, improve vision, and arrest polyuria. AIM OF THE STUDY: This work aims to evaluate the recent progress of the chemical composition, pharmacological activity, pharmacokinetics, metabolism, and quality control and of Rubus idaeus, which focuses on the insufficiency of existing research and will shed light on future studies of Rubus idaeus. METHODS: Literatures about "Rubus idaeus","Red raspberry" and "Fupenzi"are retrieved by browsing the database, such as Web of Science (http://www.webofknowledge.com/wos), Pubmed (https://pubmed.ncbi.nlm.nih.gov/), CNKI (http://www.cnki.net/), and Wanfang Data (http://www.wanfangdata.com.cn). In addition, related textbooks and digital documents are interrogated to provide a holistic and critical review of the topic. The period of the literature covered from 1981 to 2022. RESULTS: Approximately 194 compounds have been isolated from Rubus idaeus, which is rich in phenols, terpenoids, alkaloids, steroids, and fatty acids. Numerous investigations have demonstrated that Rubus idaeus exhibits many pharmacological activities, including hypoglycemic and hypolipidemic, anti-Alzheimer effect, anti-osteoporosis, hepatoprotective, anti-cancer, neuroprotective, anti-bacteria and skin care, etc. However, it is worth noting that most of the research is not associated with the conventional effect, such as reducing urination and treating opacity of the cornea. CONCLUSION: The effectiveness of Rubus idaeus has been proved by its long-term clinical application. The research on the pharmacological activity of Rubus idaeus has flourished. In many pharmacological experiments, only the high-dose group can achieve the corresponding efficacy, so the efficacy of Rubus idaeus needs to be further interrogated. Meanwhile, the relationship between pharmacological activity and specific compounds of Rubus idaeus has not been clarified yet. Last but not least, studies involving toxicology and pharmacokinetics are very limited. Knowledge of bioavailability and toxicological behavior of Rubus idaeus can help understand the herb's pharmacodynamic and safety profile.