Biomaterial-based combinatorial approach of aescin-comprised zein-coated gelatin nanoparticles alleviates synovial inflammation in experimental inflammatory arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease that mostly affects joints. Although RA therapy has made significant progress, difficulties including extensive medication metabolism and its quick clearance result in its inadequate bioavailability. The anti-inflammatory effect of zein was reported with other medications, but it has certain limitations. There are reports on the anti-oxidant and anti-inflammatory effect of aescin, which exhibits low bioavailability for the treatment of rheumatoid arthritis. Also, the combinatorial effect of zein with other effective drug delivery systems is still under investigation for the treatment of experimental collagen-induced rheumatoid arthritis. The focus of this study was to formulate and define the characteristics of zein-coated gelatin nanoparticles encapsulated with aescin (Ze@Aes-GNPs) and to assess and contrast the therapeutic effectiveness of Ze@Aes-GNPs towards collagen-induced RA in Wistar rats. Nanoprecipitation and the layer-by-layer coating process were used to fabricate Ze@Aes-GNPs and their hydrodynamic diameter was determined to be 182 nm. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) were used to further validate the size, shape, and surface morphology of Ze@Aes-GNPs. When tested against foreskin fibroblasts (BJ), these nanoparticles demonstrated significantly high cytocompatibility. Both Aes and Ze@Aes-GNPs were effective in treating arthritis, as shown by the decreased edoema, erythema, and swelling of the joints, between which Ze@Aes-GNPs were more effective. Further, it was demonstrated that Aes and Ze@Aes-GNPs reduced the levels of oxidative stress (articular elastase, lipid peroxidation, catalase, superoxide dismutase and nitric oxide) and inflammatory indicators (TNF-α, IL-1ß and myeloperoxidase). The histopathology findings further demonstrated that Ze@Aes-GNPs considerably reduced the infiltration of inflammatory cells at the ankle joint cartilage compared to Aes. Additionally, immunohistochemistry examination showed that treatment with Ze@Aes-GNPs suppressed the expression of pro-inflammatory markers (COX-2 and IL-6) while increasing the expression of SOD1. In summary, the experiments indicated that Aes and Ze@Aes-GNPs lowered the severity of arthritis, and critically, Ze@Aes-GNPs showed better effectiveness in comparison to Aes. This suppression of oxidative stress and inflammation was likely driven by Aes and Ze@Aes-GNPs.

Horse Chestnut Saponins-Escins, Isoescins, Transescins, and Desacylescins.

Escins constitute an abundant family of saponins (saponosides) and are the most active components in Aesculum hippocastanum (horse chestnut-HC) seeds. They are of great pharmaceutical interest as a short-term treatment for venous insufficiency. Numerous escin congeners (slightly different compositions), as well as numerous regio-and stereo-isomers, are extractable from HC seeds, making quality control trials mandatory, especially since the structure-activity relationship (SAR) of the escin molecules remains poorly described. In the present study, mass spectrometry, microwave activation, and hemolytic activity assays were used to characterize escin extracts (including a complete quantitative description of the escin congeners and isomers), modify the natural saponins (hydrolysis and transesterification) and measure their cytotoxicity (natural vs. modified escins). The aglycone ester groups characterizing the escin isomers were targeted. A complete quantitative analysis, isomer per isomer, of the weight content in the saponin extracts as well as in the seed dry powder is reported for the first time. An impressive 13% in weight of escins in the dry seeds was measured, confirming that the HC escins must be absolutely considered for high-added value applications, provided that their SAR is established. One of the objectives of this study was to contribute to this development by demonstrating that the aglycone ester functions are mandatory for the toxicity of the escin derivative, and that the cytotoxicity also depends on the relative position of the ester functions on the aglycone.

Escin's Multifaceted Therapeutic Profile in Treatment and Post-Treatment of Various Cancers: A Comprehensive Review.

Although modern medicine is advancing at an unprecedented rate, basic challenges in cancer treatment and drug resistance remain. Exploiting natural-product-based drugs is a strategy that has been proven over time to provide diverse and efficient approaches in patient care during treatment and post-treatment periods of various diseases, including cancer. Escin-a plant-derived triterpenoid saponin-is one example of natural products with a broad therapeutic scope. Initially, escin was proven to manifest potent anti-inflammatory and anti-oedematous effects. However, in the last two decades, other novel activities of escin relevant to cancer treatment have been reported. Recent studies demonstrated escin's efficacy in compositions with other approved drugs to accomplish synergy and increased bioavailability to broaden their apoptotic, anti-metastasis, and anti-angiogenetic effects. Here, we comprehensively discuss and present an overview of escin's chemistry and bioavailability, and highlight its biological activities against various cancer types. We conclude the review by presenting possible future directions of research involving escin for medical and pharmaceutical applications as well as for basic research.

Structure and Undulations of Escin Adsorption Layer at Water Surface Studied by Molecular Dynamics.

The saponin escin, extracted from horse chestnut seeds, forms adsorption layers with high viscoelasticity and low gas permeability. Upon deformation, escin adsorption layers often feature surface wrinkles with characteristic wavelength. In previous studies, we investigated the origin of this behavior and found that the substantial surface elasticity of escin layers may be related to a specific combination of short-, medium-, and long-range attractive forces, leading to tight molecular packing in the layers. In the current study, we performed atomistic molecular dynamics simulations of 441 escin molecules in a dense adsorption layer with an area per molecule of 0.49 nm2. We found that the surfactant molecules are less submerged in water and adopt a more upright position when compared to the characteristics determined in our previous simulations with much smaller molecular models. The number of neighbouring molecules and their local orientation, however, remain similar in the different-size models. To maintain their preferred mutual orientation, the escin molecules segregate into well-ordered domains and spontaneously form wrinkled layers. The same specific interactions (H-bonds, dipole-dipole attraction, and intermediate strong attraction) define the complex internal structure and the undulations of the layers. The analysis of the layer properties reveals a characteristic wrinkle wavelength related to the surface lateral dimensions, in qualitative agreement with the phenomenological description of thin elastic sheets.

Molecular docking analyses of Escin as regards cyclophosphamide-induced cardiotoxicity: In vivo and in Silico studies.

This study aims to investigate whether Escin (ES) can protect against Cyclophosphamide (CPM)-induced cardiac damage. The experimental rats were categorized as Control, CPM (200 mg/kg), ES (10 mg/kg), and CPM + ES Groups, each having 6 members. Their heart tissues were stained with Hematoxylin and Eosin and the structural changes were investigated under the light microscope. The biochemical markers of ischemia modified albumin (IMA), creatine kinase (CK-MB), antioxidant activity indicators Catalase (CAT), and superoxide dismutase (SOD) activities were measured using blood samples. Besides, the effects of CPM, ES, and CPM + ES upon CAT and SOD activities were shown via molecular docking studies. In the Single-Dose CPM group, CK-MB and IMA levels significantly increased while SOD and CAT levels significantly decreased. However, the heart tissues were damaged. CK-MB and IMA levels significantly decreased in CP+ ES Group. On the other hand, SOD, and CAT levels significantly increased and reduced the damage remarkably. Our findings showed that ES treatment successfully reduced the toxic effects upon the rats. The conclusion is that ES treatment can help protect the heart tissue against CPM-induced toxicity. Both in-vivo results and molecular modeling studies showed that the negative effects of CPM upon SOD activity were bigger than that of CAT.

Aescin-Induced Conversion of Gel-Phase Lipid Membranes into Bicelle-like Lipid Nanoparticles.

Mixtures of the phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and the saponin ß-aescin spontaneously form monodisperse, bilayered discoidal micelles (also known as "bicelles" or "nanodisks") in aqueous solution. Such bicelles form below the melting temperature of DMPC when the phospholipids are in the rigid Lß' state and are precursors of spontaneously formed vesicles. The aescin concentration must be far above the cmcaescin (≈0.3-0.4 mM). It was found that the shape and size of the bicelles are tunable by composition. High amounts of aescin decrease the size of the bicelles from diameters of ∼300 Å at 7 mol % to ∼120 Å at 30 mol % ß-aescin. The structures are scrutinized by complementary small-angle X-ray and neutron scattering experiments. The scattering curves are subsequently analyzed by a model-independent (indirect Fourier transform analysis) and a model-based approach where bicelles are described as polydisperse bilayer disks encircled by a ß-aescin rim. Moreover, the monomodal distribution and low polydispersity of the samples were confirmed by photon correlation spectroscopy. The discoidal structures were visualized by transmission electron microscopy.

Topical hydrogels with escin ß-sitosterol phytosome and escin: Formulation development and in vivo assessment of antihyperalgesic activity.

The physicochemical properties, stability, in vivo antihyperalgesic activity, and skin irritation potential of the carbomer hydrogels with the new chemical entity escin ß-sitosterol (ES) phytosome were characterized and compared with those containing escin. Physicochemical characterization of the hydrogels (performed 48 hr after preparation) included organoleptic examination, pH measurement, light microscopy, differential scanning calorimetry analysis and rheological tests. The obtained results showed that increasing concentration of the active substances within 1-5% affected the appearance (color and transparency) of the hydrogels, their pH, consistency, and rheological behavior. Unlike acidic escin, which was dissolved in the liquid phase of the pseudoplastic hydrogels E1-E5 and reduced their maximal apparent viscosity (ηmax ), minimal apparent viscosity (ηmin ), and hysteresis area (H) in comparison to the plain carbomer hydrogel, amphiphilic ES-enhanced ηmax , ηmin , and thixotropy of the hydrogels ES1-ES5, which is favorable for prolonged retention at skin surface. Evaluation of in-use stability of the hydrogels showed that organoleptic characteristics, flow behavior, and pH values could be preserved for 3 months under ambient conditions. The rat ear test results suggested that the hydrogels are safe to be used on human skin. Both escin and ES-loaded hydrogels exerted significant, concentration-dependent antihyperalgesic effect in inflammatory pain model in rats. ES-loaded hydrogels were significantly more effective than those loaded with escin. This is a first report on the antihyperalgesic effect of topically applied escin as well as ES in a model of inflammatory pain.

Aescin-Cholesterol Complexes in DMPC Model Membranes: A DSC and Temperature-Dependent Scattering Study.

The saponin aescin, a mixture of triterpenoid saponins, is obtained from the seeds of the horse chestnut tree Aesculus hippocastanum. The ß-form employed in this study is haemolytically active. The haemolytic activity results from the ability of aescin to form strong complexes with cholesterol in the red blood cell membrane. In this study, we provide a structural analysis on the complex formation of aescin and cholesterol when embedded in a phospholipid model membrane formed by 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). In this work, the temperatures investigated extend from DMPC's Lß' to its Lα phase in dependence of different amounts of the saponin (0-6 mol% for calorimetric and 0-1 mol% for structural analyses) and the steroid (1-10 mol%). At these aescin contents model membranes are conserved in the form of small unilamellar vesicles (SUVs) and major overall structural modifications are avoided. Additionally, interactions between aescin and cholesterol can be studied for both phase states of the lipid, the gel and the fluid state. From calorimetric experiments by differential scanning calorimetry (DSC), it could be shown that both, the steroid and the saponin content, have a significant impact on the cooperative phase transition behaviour of the DMPC molecules. In addition, it becomes clearly visible that the entire phase behaviour is dominated by phase separation which indeed also depends on the complexes formed between aescin and cholesterol. We show by various methods that the addition of cholesterol alters the impact of aescin on structural parameters ranging from the acyl chain correlation to vesicle-vesicle interactions. While the specific saponin-phospholipid interaction is reduced, addition of cholesterol leads to deformation of SUVs. The analyses of the structures formed were performed by wide-angle X-ray scattering (WAXS), small-angle X-ray scattering (SAXS), and small-angle neutron scattering (SANS).

Temperature dependent self-organization of DMPC membranes promoted by intermediate amounts of the saponin aescin.

The plant-derived biosurfactant aescin is naturally present in many plants and is used for treatment of disorders such as varicose veins and inflammation of veins. The hemolytic activity of this saponin is attributed to its interaction with cholesterol in the red blood cell membrane. This work investigates the phase and aggregation behavior of saponin-containing model membranes consisting of the phospholipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). The aescin concentrations studied range from 1 mol% to 7 mol% with respect to the total lipid content. The methods of choice to elucidate the structural picture are small-angle scattering of X-rays (SAXS) and neutrons (SANS) and cryogenic transmission electron microscopy (cryo-TEM). SANS and SAXS revealed that at lower aescin contents vesicular structures are conserved and vesicles tend to aggregate already at aescin contents of around 1 mol%. Aggregation and vesicle deformation effects are found to be stronger when the phospholipids are in the L [Formula: see text] phase. With increasing aescin content, mixed structures, i.e. aggregated and deformed vesicles and solubilized bilayer fragments, are present. This was proven for a sample with 4 mol% aescin by cryo-TEM. An increasing aescin amount leads to membrane decomposition and free standing bilayers which tend to build stacks at high temperature. These stacks are characterized by SAXS using the modified Caillé theory. Analyses and model dependent fitting reveal formation of well-defined structures beginning at 7 mol% aescin.

The Biosurfactant ß-Aescin: A Review on the Physico-Chemical Properties and Its Interaction with Lipid Model Membranes and Langmuir Monolayers.

This review discusses recent progress in physicochemical understanding of the action of the saponin ß -aescin (also called ß -escin), the biologically active component in the seeds of the horse chestnut tree Aesculus hippocastanum. ß -Aescin is used in pharmacological and cosmetic applications showing strong surface activity. In this review, we outline the most important findings describing the behavior of ß -aescin in solution (e.g., critical micelle concentration ( c m c ) and micelle shape) and special physicochemical properties of adsorbed ß -aescin monolayers at the air-water and oil-water interface. Such monolayers were found to posses very special viscoelastic properties. The presentation of the experimental findings is complemented by discussing recent molecular dynamics simulations. These simulations do not only quantify the predominant interactions in adsorbed monolayers but also highlight the different behavior of neutral and ionized ß -aescin molecules. The review concludes on the interaction of ß -aescin with phospholipid model membranes in the form of bilayers and Langmuir monolayers. The interaction of ß -aescin with lipid bilayers was found to strongly depend on its c m c . At concentrations below the c m c , membrane parameters are modified whereas above the c m c , complete solubilization of the bilayers occurs, depending on lipid phase state and concentration. In the presence of gel-phase phospholipids, discoidal bicelles form; these are tunable in size by composition. The phase behavior of ß -aescin with lipid membranes can also be modified by addition of other molecules such as cholesterol or drug molecules. The lipid phase state also determines the penetration rate of ß -aescin molecules into lipid monolayers. The strongest interaction was always found in the presence of gel-phase phospholipid molecules.

Interaction of the Saponin Aescin with Ibuprofen in DMPC Model Membranes.

In the present work, we study the interaction of the saponin aescin with the nonsteroidal anti-inflammatory drug (NSAID) ibuprofen at concentrations of 1.2-2.5 mM. These amounts are higher than those usually used for medication (10-300 µM) to show possible structures and formulations for orally absorbed drug delivery systems. It is shown how the interaction of both substances, separately or together, alters the thermotropic phase behavior of the 1,2-dimyristoyl- sn-glycero-3-phosphocholine (DMPC) bilayer in the presence of different amounts of aescin, ranging from 20 µM to 1 mM. The methods of choice are differential scanning calorimetry (DSC), and additionally wide-angle (WAXS) and small-angle X-ray scattering (SAXS). We found that these two additives, aescin and ibuprofen, alter the temperature-dependent structural appearance of the DMPC membrane depending on the aescin and drug content. The presence of the saponin and the drug become visible on different length scales, i.e., ranging from a global structural change to inner-membrane interactions. DSC reveals that the drug and saponin alter the cooperativity of the DMPC phase transition in a concentration-dependent manner. Furthermore, there is a significant difference between the drug-containing compared to the drug-free systems. By WAXS, we could resolve that aescin reverses the strong impact of ibuprofen on the diffraction peak of DMPC. Both molecules interact strongly with the phospholipid headgroups. This becomes visible in a changing area per lipid and shifting phase transition to higher temperatures. SAXS experiments reveal that the addition of ibuprofen leads to major morphological changes in the phospholipid bilayer. SAXS experiments performed on representative samples do not only cover the drug-saponin interaction within the bilayer from the structural perspective but also confirm the visually observed macroscopic concentration and temperature-dependent phase behavior. Vesicular shape of extruded samples is conserved at low aescin contents. At intermediate aescin content, aggregation between vesicles occurs, whereby the strength of aggregation is reduced by ibuprofen. At high aescin contents, DMPC bilayers are solubilized. The kind of formed structures depends on temperature and drug content. At low temperature, separated bilayer sheets are formed. Their size increases with ibuprofen in a concentration-dependent manner. At high temperature, the drug-free system reorganizes into stacked sheets. Whereas sheets at 5 mol % ibuprofen close to vesicles, the ones with 10 mol % of the drug increase massively in size. Altogether, ibuprofen was found to rather enhance than inhibit structural and thermotropic membrane modifications induced by the aescin on the DMPC model membrane.

Saponin Adsorption at the Air-Water Interface-Neutron Reflectivity and Surface Tension Study.

Saponins are a large group of glycosides present in many plant species. They exhibit high surface activity, which arises from a hydrophobic scaffold of triterpenoid or steroid groups and attached hydrophilic saccharide chains. The diversity of molecular structures, present in various plants, gives rise to a rich variety of physicochemical properties and biological activity and results in a wide range of applications in foods, cosmetics, medicine, and several other industrial sectors. Saponin surface activity is a key property in such applications and here the adsorption of three triterpenoid saponins, escin, tea saponins, and Quillaja saponin, is studied at the air-water interface by neutron reflectivity and surface tension. All these saponins form adsorption layers with very high surface visco-elasticity. The structure of the adsorbed layers has been determined from the neutron reflectivity data and is related to the molecular structure of the saponins. The results indicate that the structure of the saturated adsorption layers is governed by densely packed hydrophilic saccharide groups. The tight molecular packing and the strong hydrogen bonds between the neighboring saccharide groups are the main reasons for the unusual rheological properties of the saponin adsorption layers.

Novel Colloidal Microstructures of ß-Escin and the Liposomal Components Cholesterol and DPPC.

The discovery of immunostimulating complex formation by the saponin Quil A from the plant Quillaja saponaria with cholesterol and a phospholipid opened up new avenues for the development of drug delivery systems for vaccine application with additional adjuvant properties. In this study, ß-escin, a monodesmosidic triterpene saponin from horse chestnut, was investigated in terms of its interaction with liposomal components (cholesterol, dipalmitoylphosphatidylcholine) by Langmuir film balance studies and with regard to particle formation visualized by transmission electron microscopy. A strong interaction of ß-escin with cholesterol was observed by Langmuir isotherms due to the intercalation of the saponin into the monolayer, whereas no interaction occurred with dipalmitoylphosphatidylcholine. Transmission electron microscopy studies also confirmed the strong interaction of ß-escin with cholesterol. In aqueous pseudo-ternary systems (ß-escin, dipalmitoylphosphatidylcholine, cholesterol) and in pseudo-binary systems (ß-escin, cholesterol), new colloidal structures built up from ring-like and worm-like subunits were observed with a size of about 100 - 200 nm. These colloidal structures are formed in pseudo-binary systems by aggregation of the subunits, whereas in pseudo-ternary systems, they are formed among others by attacking the liposomal membrane. The rehydration of the liposomal dispersions in NANOpure water or Tris buffer pH 7.4 (140 mM) resulted in the same particle formation. In contrast, the sequence of the dispersions' production process affected the particle formation. Unless adding the saponin to the other components from the beginning, just a liposomal dispersion was formed without any colloidal aggregates of the subunits mentioned above.

DMPC vesicle structure and dynamics in the presence of low amounts of the saponin aescin.

Vesicle shape and bilayer parameters are studied by small-angle X-ray (SAXS) and small-angle neutron (SANS) scattering in the presence of the saponin aescin. We confirm successful incorporation of aescin molecules by analysis of the radii of gyration RG and study furthermore the impact of aescin incorporation on bilayer thickness parameters from the neutron and X-ray perspective. Additionally, the bending elasticity (κ) of these 1,2-dimyristoyl-sn-glycero-3-phosphocholine vesicle bilayers is studied in the presence of aescin. Neutron spin-echo spectroscopy (NSE) allows to detect subtle changes in the dynamics and κ of lipid membranes. Changes of κ are detectable at temperatures below and above the main phase transition temperature Tm of the lipid. The impact of aescin is much more significant below Tm. It has been found that below Tm the addition of aescin to the vesicles decreases the value of κ and softens the bilayer. Above Tm the value of κ increases with increasing aescin content and the bilayer becomes more rigid. Altogether, we demonstrate by analysis of the structure and dynamics of the vesicles that the impact of aescin strongly depends on the lipid state. Below Tm the membrane becomes fluidized and softer, above Tm solidified and stiffer compared to a DMPC membrane without additive at similar conditions.

Molecular targets and anti-cancer potential of escin.

Escin is a mixture of triterpenoid saponins extracted from the horse chestnut tree, Aesculus hippocastanum. Its potent anti-inflammatory and anti-odematous properties makes it a choice of therapy against chronic venous insufficiency and odema. More recently, escin is being actively investigated for its potential activity against diverse cancers. It exhibits anti-cancer effects in many cancer cell models including lung adenocarcinoma, hepatocellular carcinoma and leukemia. Escin also attenuates tumor growth and metastases in various in vivo models. Importantly, escin augments the effects of existing chemotherapeutic drugs, thereby supporting the role of escin as an adjunct or alternative anti-cancer therapy. The beneficial effects of escin can be attributed to its inhibition of proliferation and induction of cell cycle arrest. By regulating transcription factors/growth factors mediated oncogenic pathways, escin also potentially mitigates chronic inflammatory processes that are linked to cancer survival and resistance. This review provides a comprehensive overview of the current knowledge of escin and its potential as an anti-cancer therapy through its anti-proliferative, pro-apoptotic, and anti-inflammatory effects.

ß-Escin Effectively Modulates HUVECS Proliferation and Tube Formation.

In the present study we evaluated the anti-angiogenic activities of ß-escin (the major active compound of Aesculus hippocastanum L. seeds). Human umbilical-vein endothelial cells (HUVECs) were used as an in vitro model for studying the molecular mechanism underlying the anti-angiogenic effect of ß-escin. We investigated the in vitro effects on proliferation, migration, and tube formation of HUVECs and in vivo anti-angiogenic activity was evaluated in a chick chorioallantoic membrane (CAM) angiogenesis assay. Moreover, the effect on gene expressions was determined by the RT2 ProfilerTM human angiogenesis PCR Array. It was found that ß-escin exerts inhibitory effect on the basic fibroblast growth factor (bFGF)-induced proliferation, migration and tube formation, as well as CAM angiogenesis in vivo. The inhibition of critical steps of angiogenic process observed with ß-escin could be partially explained by suppression of Akt activation in response to bFGF. Moreover, the anti-angiogenic effects of ß-escin could also be mediated via inhibition of EFNB2 and FGF-1 gene expressions in endothelial cells. In conclusion, ß-escin affects endothelial cells as a negative mediator of angiogenesis in vitro and in vivo and may therefore be considered as a promising candidate for further research elucidating its underlying mechanism of action.

Aescin Incorporation and Nanodomain Formation in DMPC Model Membranes.

The saponin aescin from the horse chestnut tree is a natural surfactant well-known to self-assemble as oriented-aggregates at fluid interfaces. Using model membranes in the form of lipid vesicles and Langmuir monolayers, we study the mixing properties of aescin with the phase-segregating phospholipid 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC). The binary membranes are experimentally studied on different length scales ranging from the lipid headgroup area to the macroscopic scale using small-angle X-ray scattering (SAXS), photon correlation spectroscopy (PCS), and differential scanning calorimetry (DSC) with binary bilayer vesicles and Langmuir tensiometry (LT) with lipid monolayers spread on the surface of aescin solutions. The binary interaction was found to strongly depend on aescin concentration in two well differentiated concentration regimes. Below 7 mol %, the results reveal phase segregation of nanometer-sized aescin-rich domains in an aescin-poor continuous bilayer. Above this concentration, aescin-aescin interactions dominate, which inhibit vesicle formation but lead to the formation of new membrane aggregates of smaller sizes. From LT studies in monolayers, the interaction of aescin with DMPC was shown to be stronger in the condensed phase than in the liquid expanded phase. Furthermore, a destructuring role was revealed for aescin on phospholipid membranes, similar to the fluidizing effect of cholesterol and nonsteroidal anti-inflammatory drugs (NSAIDs) on lipid bilayers.

Antiviral escin derivatives from the seeds of Aesculus turbinata Blume (Japanese horse chestnut).

Porcine epidemic diarrhea virus (PEDV) causes severe diarrhea and high fatality of piglets, influencing the swine industry. Japanese horse chestnut (seed of Aesculus turbinata) contains many saponin mixtures, called escins, and has been used for a long time as a traditional medicinal plant. Structure-activity relationship (SAR) studies on escins have revealed that acylations at C-21 and C-22 with angeloyl or tigloyl groups were important for their cytotoxic effects. However, the strong cytotoxicity of escins makes them hard to utilize for other diseases and to develop as nutraceuticals. In this research, we investigated whether escin derivatives 1-7 (including new compounds 2, 3, 5 and 6), without the angeloyl or tigloyl groups and with modified glycosidic linkages by hydrolysis, have PEDV inhibitory effects with less cytotoxicity. Compounds 1-7 had no cytotoxicity at 20µM on VERO cells, while compounds 8-10 showed strong cytotoxicity at similar concentrations on PEDV. Our results suggest that escin derivatives showed strong inhibitory activities on PEDV replication with lowered cytotoxicity. These studies propose a method to utilize Japanese horse chestnut for treating PEDV and to increase the diversity of its bioactive compounds.

Molecular Mechanism for Cellular Response to ß-Escin and Its Therapeutic Implications.

ß-escin is a mixture of triterpene saponins isolated from the horse chestnut seeds (Aesculus hippocastanum L.). The anti-edematous, anti-inflammatory and venotonic properties of ß-escin have been the most extensively clinically investigated effects of this plant-based drug and randomized controlled trials have proved the efficacy of ß-escin for the treatment of chronic venous insufficiency. However, despite the clinical recognition of the drug its pharmacological mechanism of action still remains largely elusive. To determine the cellular and molecular basis for the therapeutic effectiveness of ß-escin we performed discovery and targeted proteomic analyses and in vitro evaluation of cellular and molecular responses in human endothelial cells under inflammatory conditions. Our results demonstrate that in endothelial cells ß-escin potently induces cholesterol synthesis which is rapidly followed with marked fall in actin cytoskeleton integrity. The concomitant changes in cell functioning result in a significantly diminished responses to TNF-α stimulation. These include reduced migration, alleviated endothelial monolayer permeability, and inhibition of NFκB signal transduction leading to down-expression of TNF-α-induced effector proteins. Moreover, the study provides evidence for novel therapeutic potential of ß-escin beyond the current vascular indications.

ß-escin selectively targets the glioblastoma-initiating cell population and reduces cell viability.

Glioblastoma multiforme (GBM) is a highly aggressive tumour of the central nervous system and is associated with an extremely poor prognosis. Within GBM exists a subpopulation of cells, glioblastoma-initiating cells (GIC), which possess the characteristics of progenitor cells, have the ability to initiate tumour growth and resist to current treatment strategies. We aimed at identifying novel specific inhibitors of GIC expansion through use of a large-scale chemical screen of approved small molecules. Here, we report the identification of the natural compound ß-escin as a selective inhibitor of GIC viability. Indeed, ß-escin was significantly cytotoxic in nine patient-derived GIC, whilst exhibiting no substantial effect on the other human cancer or control cell lines tested. In addition, ß-escin was more effective at reducing GIC growth than current clinically used cytotoxic agents. We further show that ß-escin triggers caspase-dependent cell death combined with a loss of stemness properties. However, blocking apoptosis could not rescue the ß-escin-induced reduction in sphere formation or stemness marker activity, indicating that ß-escin directly modifies the stem identity of GIC, independent of the induction of cell death. Thus, this study has repositioned ß-escin as a promising potential candidate to selectively target the aggressive population of initiating cells within GBM.