Regulated Self-Folding in Multi-Layered Hydrogels Considered with an Interfacial Layer.

Multi-layered hydrogels consisting of bi- or tri-layers with different swelling ratios are designed to soft hydrogel actuators by self-folding. The successful use of multi-layered hydrogels in this application greatly relies on the precise design and fabrication of the curvature of self-folding. In general, however, the self-folding often results in an undesired mismatch with the expecting value. To address this issue, this study introduces an interfacial layer formed between each layered hydrogel, and this layer is evaluated to enhance the design and fabrication precision. By considering the interfacial layer, which forms through diffusion, as an additional layer in the multi-layered hydrogel, the degree of mismatch in the self-folding is significantly reduced. Experimental results show that as the thickness of the interfacial layer increases, the multi-layered hydrogel exhibits a 3.5-fold increase in its radius of curvature during the self-folding. In addition, the diffusion layer is crucial for creating robust systems by preventing the separation of layers in the muti-layered hydrogel during actuation, thereby ensuring the integrity of the system in operation. This new strategy for designing multi-layered hydrogels including an interfacial layer would greatly serve to fabricate precise and robust soft hydrogel actuators.

A Spike-like Self-Assembly of Polyaspartamide Integrated with Functionalized Nanoparticles.

The integration of nanoparticles (NPs) into molecular self-assemblies has been extensively studied with the aim of building well-defined, ordered structures which exhibit advanced properties and performances. This study demonstrates a novel strategy for the preparation of a spike-like self-assembly designed to enhance UV blocking. Poly(2-hydroxyethyl aspartamide) (PHEA) substituted with octadecyl chains and menthyl anthranilate (C18-M-PHEA) was successfully synthesized by varying the number of grafted groups to control their morphology and UV absorption. The in situ incorporation of polymerized rod-like TiO2 within the C18-M-PHEA self-aggregates generated spike-like self-assemblies (TiO2@C18-M-PHEA) with a chestnut burr structure in aqueous solution. The results showed that the spike-like self-assemblies integrated with TiO2 NPs exhibited a nine-fold increase in UV protection by simultaneous UV absorption and scattering compared with the pure TiO2 NPs formed via a bulk mixing process. This work provides a novel method for UV protection using self-assembling poly(amino acid)s derivatives integrated with functional nanoparticles to tune their morphology and organization.

Implantable Multi-Cross-Linked Membrane-Ionogel Assembly for Reversible Non-Faradaic Neurostimulation.

Neural interfaces play a major role in modulating neural signals for therapeutic purposes. To meet the demand of conformable neural interfaces for developing bioelectronic medicine, recent studies have focused on the performance of electrical neurostimulators employing soft conductors such as conducting polymers and electronic or ionic conductive hydrogels. However, faradaic charge injection at the interface of the electrode and nerve tissue causes irreversible gas evolution, oxidation of electrodes, and reduction of biological ions, thus causing undesired tissue damage and electrode degradation. Here we report a conformable neural interface engineering based on multicross-linked membrane-ionogel assembly (termed McMiA), which enables nonfaradaic neurostimulation without irreversible charge transfer reaction. The McMiA consists of a genipin-cross-linked biopolymeric ionogel coupled with a dopamine-cross-linked graphene oxide membrane to prevent ion exchange between biological and synthetic McMiA ions and to function as a bioadhesive forming covalent bonds with the target tissues. In addition, the demonstration of bioelectronic medicine via the McMiA-based neurostimulation of sciatic nerves shows the enhanced clinical utility in treating the overactive bladder syndrome. As the McMiA-based neural interface is soft, robust for bioadhesion, and stable in a physiological environment, it can offer significant advancement in biocompatibility and long-term operability for neural interface engineering.

Electroactive nano-Biohybrid actuator composed of gold nanoparticle-embedded muscle bundle on molybdenum disulfide nanosheet-modified electrode for motion enhancement of biohybrid robot.

There have been several trials to develop the bioactuator using skeletal muscle cells for controllable biobybird robot. However, due to the weak contraction force of muscle cells, the muscle cells could not be used for practical applications such as biorobotic hand for carrying objects, and actuator of biohybrid robot for toxicity test and drug screening. Based on reported hyaluronic acid-modified gold nanoparticles (HA@GNPs)-embedded muscle bundle on PDMS substrate, in this study for augmented actuation, we developed the electroactive nano-biohybrid actuator composed of the HA@GNP-embedded muscle bundle and molybdenum disulfide nanosheet (MoS2 NS)-modified electrode to enhance the motion performance. The MoS2 NS-modified Au-coated polyimide (PI) electrode to be worked in mild pH condition for viable muscle cell was utilized as supporting- and motion enhancing- substrate since it was electrochemically active, which caused the movement of flexible PI electrode. The motion performance of this electroactive nano-biohybrid actuator by electrical stimulation was increased about 3.18 times compared with that of only HA@GNPs embedded-muscle bundle on bare PI substrate. The proposed electroactive nano-biohybrid actuator can be applied to the biorobotic hand and biohybrid robot.

An integrative multiomics approach to characterize anti-adipogenic and anti-lipogenic effects of Akkermansia muciniphila in adipocytes.

The cellular components of Akkermansia muciniphila are considered potential biotherapeutics for the improvement of obesity, diabetes, and metabolic diseases. However, the molecular-based mechanism of A. muciniphila for treatment of obesity, which can provide important evidence for human research, has rarely been explored. Here, we applied integrative multiomics approaches to investigate the underlying molecular mechanism involved in obesity treatment by A. muciniphila. First, the treatment with a cell lysate of A. muciniphila reduced lipid accumulation in 3T3-L1 cells and downregulated the mRNA expression of proteins involved in adipogenesis and lipogenesis. Our proteomic results revealed that A. muciniphila decreased the expression of proteins involved in fat cell differentiation, fatty acid metabolism, and energy metabolism in adipocytes. Moreover, A. muciniphila significantly reduced the level of metabolites related to glycolysis, the TCA cycle, and ATP in adipocytes. Interestingly, serine protease inhibitor A3 (SERPINA3) homologs were overexpressed in the 3T3-L1 cells treated with A. muciniphila. Small interfering RNA (siRNA) transfection demonstrated that A. muciniphila upregulates SERPINA3G expression and inhibits lipogenesis in adipocytes. Taken together, our multiomics-based approaches enabled to uncover the molecular mechanism of A. muciniphila for treatment of obesity and provide potent anti-lipogenic agents.

Engineering Tissue-Specific, Multiscale Microvasculature with a Capillary Network for Prevascularized Tissue.

Although there are various pre-existing technologies for engineering vasculatures, multiscale modeling of the architecture of human vasculature at a capillary scale remains a challenge. In this study, a novel technology is developed for the production of a functional, multiscale microvasculature comprising of endothelialized channels and tissue-specific capillary networks. Perfusable, endothelialized channels are bioprinted, after which angiogenic sprouts are grown into user-designed capillary networks. The induction of branched and liver-lobule-like capillary networks confirm that the technology can produce various types of tissue-specific multiscale microvasculatures. Further, the channels and capillaries are deemed to be functional when evaluated in vitro. An ex vivo assay demonstrates that the microvasculature can induce neovessel ingrowth, integrate with host vessels, and facilitate blood flow. Remarkably, blood flows through the implanted capillary network without any change in its morphology. Finally, the technology is applied to produce a vascularized liver tissue; it significantly improves its hepatic function. It is believed that this new technology will create new possibilities in the development of highly vascularized and functional tissues/organs on a clinically relevant scale.

Hydrogel Surface-Modified Polyurethane Copolymer Film with Water Permeation Resistance and Biocompatibility for Implantable Biomedical Devices.

To use implantable biomedical devices such as electrocardiograms and neurostimulators in the human body, it is necessary to package them with biocompatible materials that protect the internal electronic circuits from the body's internal electrolytes and moisture without causing foreign body reactions. Herein, we describe a hydrogel surface-modified polyurethane copolymer film with concurrent water permeation resistance and biocompatibility properties for application to an implantable biomedical device. To achieve this, hydrophobic polyurethane copolymers comprising hydrogenated poly(ethylene-co-butylene) (HPEB) and aliphatic poly(carbonate) (PC) were synthesized and their hydrophobicity degree and mechanical properties were adjusted by controlling the copolymer composition ratio. When 10 wt% PC was introduced, the polyurethane copolymer exhibited hydrophobicity and water permeation resistance similar to those of HPEB; however, with improved mechanical properties. Subsequently, a hydrophilic poly(vinyl pyrrolidone) (PVP) hydrogel layer was formed on the surface of the polyurethane copolymer film by Fenton reaction using an initiator and crosslinking agent and the effect of the initiator and crosslinking agent immobilization time, PVP concentration and crosslinking agent concentration on the hydrogel properties were investigated. Finally, MTT assay showed that the hydrogel surface-modified polyurethane copolymer film displays excellent biocompatibility.

Development of an in vitro coculture device for the investigation of host-microbe interactions via integrative multiomics approaches.

The commensal gut bacterium Akkermansia muciniphila is well known as a promising probiotic candidate that improves host health and prevents diseases. However, the biological interaction of A. muciniphila with human gut epithelial cells has rarely been explored for use in biotherapeutics. Here, we developed an in vitro device that simulates the gut epithelium to elucidate the biological effects of living A. muciniphila via multiomics analysis: the Mimetic Intestinal Host-Microbe Interaction Coculture System (MIMICS). We demonstrated that both human intestinal epithelial cells (Caco-2) and the anaerobic bacterium A. muciniphila can remain viable for 12 h after coculture in the MIMICS. The transcriptomic and proteomic changes (cell-cell junctions, immune responses, and mucin secretion) in gut epithelial cells treated with A. muciniphila closely correspond with those reported in previous in vivo studies. In addition, our proteomic and metabolomic results revealed that A. muciniphila activates glucose and lipid metabolism in gut epithelial cells, leading to an increase in ATP production. This study suggests that A. muciniphila improves metabolism for ATP production in gut epithelial cells and that the MIMICS may be an effective general tool for evaluating the effects of anaerobic bacteria on gut epithelial cells.

An Active and Soft Hydrogel Actuator to Stimulate Live Cell Clusters by Self-folding.

The hydrogels are widely used in various applications, and their successful uses depend on controlling the mechanical properties. In this study, we present an advanced strategy to develop hydrogel actuator designed to stimulate live cell clusters by self-folding. The hydrogel actuator consisting of two layers with different expansion ratios were fabricated to have various curvatures in self-folding. The expansion ratio of the hydrogel tuned with the molecular weight and concentration of gel-forming polymers, and temperature-sensitive molecules in a controlled manner. As a result, the hydrogel actuator could stimulate live cell clusters by compression and tension repeatedly, in response to temperature. The cell clusters were compressed in the 0.7-fold decreases of the radius of curvature with 1.0 mm in room temperature, as compared to that of 1.4 mm in 37 °C. Interestingly, the vascular endothelial growth factor (VEGF) and insulin-like growth factor-binding protein-2 (IGFBP-2) in MCF-7 tumor cells exposed by mechanical stimulation was expressed more than in those without stimulation. Overall, this new strategy to prepare the active and soft hydrogel actuator would be actively used in tissue engineering, drug delivery, and micro-scale actuators.

Tuning the Hydrophobicity of a Hydrogel Using Self-Assembled Domains of Polymer Cross-Linkers.

Hydrogels incorporated with hydrophobic motifs have received considerable attention to recapitulate the cellular microenvironments, specifically for the bio-mineralization of a 3D matrix. Introduction of hydrophobic molecules into a hydrogel often results in irregular arrangement of the motifs, and further phase separation of hydrophobic domains, but limited efforts have been made to resolve this challenge in developing the hydrophobically-modified hydrogel. Therefore, this study presents an advanced integrative strategy to incorporate hydrophobic domains regularly in a hydrogel using self-assembled domains formed with polymer cross-linkers, building blocks of a hydrogel. Self-assemblies formed by polymer cross-linkers were examined as micro-domains to incorporate hydrophobic motifs in a hydrogel. The self-assembled structures in a pre-gelled solution were confirmed with the fluorescence analysis and the hydrophobicity of a hydrogel could be tuned by incorporating the hydrophobic chains in a controlled manner. Overall, the results of this study would greatly serve to tuning performance of a wide array of hydrophobically-modified hydrogels in drug delivery, cell therapies and tissue engineering.

Discovery of glycocholic acid and taurochenodeoxycholic acid as phenotypic biomarkers in cholangiocarcinoma.

Although several biomarkers can be used to distinguish cholangiocarcinoma (CCA) from healthy controls, differentiating the disease from benign biliary disease (BBD) or pancreatic cancer (PC) is a challenge. CCA biomarkers are associated with low specificity or have not been validated in relation to the biological effects of CCA. In this study, we quantitatively analyzed 15 biliary bile acids in CCA (n = 30), BBD (n = 57) and PC (n = 17) patients and discovered glycocholic acid (GCA) and taurochenodeoxycholic acid (TCDCA) as specific CCA biomarkers. Firstly, we showed that the average concentration of total biliary bile acids in CCA patients was quantitatively less than in other patient groups. In addition, the average composition ratio of primary bile acids and conjugated bile acids in CCA patients was the highest in all patient groups. The average composition ratio of GCA (35.6%) in CCA patients was significantly higher than in other patient groups. Conversely, the average composition ratio of TCDCA (13.8%) in CCA patients was significantly lower in all patient groups. To verify the biological effects of GCA and TCDCA, we analyzed the gene expression of bile acid receptors associated with the development of CCA in a CCA cell line. The gene expression of transmembrane G protein coupled receptor (TGR5) and sphingosine 1-phosphate receptor 2 (S1PR2) in CCA cells treated with GCA was 8.6-fold and 3.4-fold higher compared with control (untreated with bile acids), respectively. Gene expression of TGR5 and S1PR2 in TCDCA-treated cells was not significantly different from the control. Taken together, our study identified GCA and TCDCA as phenotype-specific biomarkers for CCA.

Functionalized Magnetic PLGA Nanospheres for Targeting and Bioimaging of Breast Cancer.

Superparamagnetic iron oxide nanoparticles (SPIONs) are actively used as highly sensitive imaging probes to provide contrast in MRI. In this study, we propose the use of SPIONs encapsulated with antibody-conjugated poly(lactic-co-glycolic acid) (PLGA) as a potent theragnostic agent. The SPIONs were synthesized by a chemical co-precipitation method of ferric and ferrous ions, and subsequently encapsulated with PLGA by using an emulsification-diffusion method. Herceptin was chemically conjugated to the SPION-encapsulating PLGA nanoparticles to target the human epidermal growth factor receptor 2 (Her2/neu) overexpressing breast cancers. FACS and MR molecular imaging revealed that the Her2/neu overexpressing cell line showed a stronger contrast enhancement than the Her2/neu non-expressing cell lines, and the signal intensity of in vivo MR imaging decreased as the concentration of Herceptin increased. This strategy of encapsulating SPIONs with PLGA will be highly useful in functionalizing magnetic nanoparticles and improving the diagnostic and therapeutic efficacy of a wide array of cancer treatments.

Chemical and mechanical modulation of polymeric micelle assembly.

Recently, polymeric micelles self-assembled from amphiphilic polymers have been studied for various industrial and biomedical applications. This nanoparticle self-assembly typically occurs in a solvent-exchange process. In this process, the quality of the resulting particles is uncontrollably mediated by polymeric solubility and mixing conditions. Here, we hypothesized that improving the solubility of an amphiphilic polymer in an organic solvent via chemical modification while controlling the mixing rate of organic and aqueous phases would enhance control over particle morphology and size. We examined this hypothesis by synthesizing a poly(2-hydroxyethyl)aspartamide (PHEA) grafted with controlled numbers of octadecyl (C18) chains and oligovaline groups (termed "oligovaline-PHEA-C18"). The mixing rate of DMF and water was controlled either by microfluidic mixing of laminar DMF and water flows or through turbulent bulk mixing. Interestingly, oligovaline-PHEA-C18 exhibited an increased solubility in DMF compared with PHEA-C18, as demonstrated by an increase of mixing energy. In addition, increasing the mixing rate between water and DMF using the microfluidic mixer resulted in a decrease of the diameter of the resulting polymeric micelles, as compared with the particles formed from a bulk mixing process. Overall, these findings will expand the parameter space available to control particle self-assembly while also serving to improve existing nanoparticle processing techniques.

Antioxidant and Anti-Inflammatory Effects of Chaenomeles sinensis Leaf Extracts on LPS-Stimulated RAW 264.7 Cells.

The fruit of Chaenomeles sinensis has been traditionally used in ethnomedicine for the treatment of various human ailments, including pneumonia, bronchitis, and so on, but the pharmacological applications of the leaf part of the plant have not been studied. In this study, we evaluated the various radical scavenging activities and anti-inflammatory effects of different Chaenomeles sinensis leaf (CSL) extracts. The water extract showed a higher antioxidant and radical scavenging activities. However the ethanolic extracts showed higher NO scavenging activity than water extract, therefore the ethanolic extract of CSL was examined for anti-inflammatory effects on lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. The 70% ethanol extract of CSL (CSLE) has higher anti-inflammatory activity and significantly inhibited the production of nitric oxide (NO), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α). In addition, CSLE suppressed LPS-stimulated inducible nitric oxide synthase (iNOS) and NO production, IL-1ß and phospho-STAT1 expression. In this study, we investigated the effect of CSLE on the production of inflammatory mediators through the inhibition of the TRIF-dependent pathways. Furthermore, we evaluated the role of CSLE on LPS-induced expression of pro-inflammatory cytokines, such as TNF-α, IL-1ß and IL-6. Our results suggest that CSLE attenuates the LPS-stimulated inflammatory responses in macrophages through regulating the key inflammatory mechanisms, providing scientific support for its traditional uses in treating various inflammatory diseases.

A MALDI-MS-based quantitative analytical method for endogenous estrone in human breast cancer cells.

The level of endogenous estrone, one of the three major naturally occurring estrogens, has a significant correlation with the incidence of post-menopausal breast cancer. However, it is challenging to quantitatively monitor it owing to its low abundance. Here, we develop a robust and highly sensitive mass-assisted laser desorption/ionization mass spectrometry (MALDI-MS)-based quantitative platform to identify the absolute quantities of endogenous estrones in a variety of clinical specimens. The one-step modification of endogenous estrone provided good linearity (R(2) > 0.99) and significantly increased the sensitivity of the platform (limit of quantitation: 11 fmol). In addition, we could identify the absolute amount of endogenous estrones in cells of the breast cancer cell line MCF-7 (34 fmol/10(6) cells) by using a deuterated estrone as an internal standard. Finally, by applying the MALDI-MS-based quantitative method to endogenous estrones, we successfully monitored changes in the metabolic expression level of estrones (17.7 fmol/10(6) letrozole-treated cells) in MCF-7 cells resulting from treatment with an aromatase inhibitor. Taken together, these results suggest that this MALDI-MS-based quantitative approach may be a general method for the targeted metabolomics of ketone-containing metabolites, which can reflect clinical conditions and pathogenic mechanisms.

Trapa japonica Pericarp Extract Reduces LPS-Induced Inflammation in Macrophages and Acute Lung Injury in Mice.

In this study, we found that chloroform fraction (CF) from TJP ethanolic extract inhibited lipopolysaccharide (LPS)-induced production of nitric oxide (NO) and intracellular ROS in RAW264.7 cells. In addition, expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) genes was reduced, as evidenced by western blot. Our results indicate that CF exerts anti-inflammatory effects by down-regulating expression of iNOS and COX-2 genes through inhibition of MAPK (ERK, JNK and p38) and NF-κB signaling. Similarly we also evaluated the effects of CF on LPS-induced acute lung injury. Male Balb/c mice were pretreated with dexamethasone or CF 1 h before intranasal instillation of LPS. Eight hours after LPS administration, the inflammatory cells in the bronchoalveolar lavage fluid (BALF) were determined. The results indicated that CF inhibited LPS-induced TNF-α and IL-6 production in a dose dependent manner. It was also observed that CF attenuated LPS-induced lung histopathologic changes. In conclusion, these data demonstrate that the protective effect of CF on LPS-induced acute lung injury (ALI) in mice might relate to the suppression of excessive inflammatory responses in lung tissue. Thus, it can be suggested that CF might be a potential therapeutic agent for ALI.

Characterization of the antioxidant fraction of Trapa japonica pericarp and its hepatic protective effects in vitro and in vivo.

The ethanolic extract of Trapa japonica pericarp (TJP) and its various fractions were evaluated for their antioxidant potential. The ethyl acetate fraction (EF) from TJP exhibited significant antioxidant and protective effects against tert-butylhydroperoxide (t-BHP)-induced oxidative damage in vitro and in vivo. In vitro experimental results showed that the EF suppressed t-BHP-induced damage in Chang cells by inhibiting reactive oxygen species generation and regulating the mitochondrial membrane potential. Furthermore, western blot analysis showed that the EF effectively inhibited t-BHP-induced apoptosis by suppressing caspase-3, caspase-7, caspase-8, and caspase-9. In the in vivo study, the EF significantly prevented serum increases in glutamate oxaloacetate transaminase and glutamate pyruvate transaminase and hepatic malondialdehyde levels caused by t-BHP. Furthermore, the EF markedly increased hepatic superoxide dismutase, catalase, and glutathione levels. Histopathological examinations further confirmed that the EF could protect the liver from t-BHP-induced oxidative injury. These findings indicate that the EF could be developed as a therapy or to prevent hepatic injury.

Thermo-triggerable self-assembly comprising cinnamoyl polymeric ß cyclodextrin and cinnamoyl Pluronic F127.

Thermo-triggerable self-assembly was prepared by co-dissolving cinnamoyl Pluronic F127 (CinPlu) and cinnamoyl polymeric ß cyclodextrin (CinPßCD) in an aqueous phase. On TEM photo, the CinPlu/CinPßCD self-assembly was 100-200nm in diameter. The specific loading of Nile red (NR) in the assembly was calculated to be 5.5% (wt NR/wt polymer), and the molar ratio of NR to ßCD residue in the assembly was about 0.89:1. No significant release of NR from the assembly was observed at 10°C and 20°C. However, when the temperature was raised to 30°C, 40°C, 50°C, and 60°C, the cumulative release amount in 5min was 17%, 25%, 32%, and 52%, respectively. The specific loading of doxorubicin (DOX) in the assembly was about 6.8% (wt DOX/wt polymer) (corresponding to the molar ratio of DOX to ßCD residue was about 0.41:1). The DOX release from the assembly was proportional to the temperature of release medium. NR and DOX were likely to be expelled out of the cavity of ßCD residue by the interaction of the thermally hydrophobicized Pluronic F127 chain (molecular piston) and the cavity of ßCD residue (cylinder). After 4h-incubation with KB cell, DOX loaded in CinPlu/CinPßCD self-assembly was found to be internalized into the cancer cell more than free DOX, observed on a confocal laser scanning microscope and a fluorescence activated cell sorter. CinPlu/CinPßCD self-assembly enhanced the in vitro anti-cancer activity of DOX against KB cell without increasing significantly the in vitro toxicity of DOX against Raw264.7 cell.

Alginate Sulfates Mitigate Binding Kinetics of Proangiogenic Growth Factors with Receptors toward Revascularization.

Ever since proangiogenic growth factors have been used as a vascular medicine to treat tissue ischemia, efforts have been increasingly made to develop a method to enhance efficacy of growth factors in recreating microvascular networks, especially at low dose. To this end, we hypothesized that polysaccharides substituted with sulfate groups would amplify growth factor receptor activation and stimulate phenotypic activities of endothelial cells involved in neovascularization. We examined this hypothesis by modifying alginate with a controlled number of sulfates and using it to derive a complex with vascular endothelial growth factor (VEGF), as confirmed with fluorescence resonance energy transfer (FRET) assay. Compared with the bare VEGF and with a mixture of VEGF and unmodified alginates, the VEGF complexed with alginate sulfates significantly reduced the dissociation rate with the VEGFR-2, elevated VEGFR-2 phosphorylation level, and increased the number of endothelial sprouts in vitro. Furthermore, the VEGF-alginate sulfate complex improved recovery of perfusion in an ischemic hindlimb of a mouse due to the increase of the capillary density. Overall, this study not only demonstrates an important cofactor of VEGF but also uncovers an underlying mechanism by which the cofactor mitigates the VEGF-induced signaling involved in the binding kinetics and activation of VEGFR. We therefore believe that the results of this study will be highly useful in improving the therapeutic efficacy of various growth factors and expediting their uses in clinical treatments of wounds and tissue defects.

Radical scavenging activities of Asterina pectinifera fermented with Cordyceps militaris mycelia.

Asterina pectinifera was fermented with Cordyceps militaris mycelia for improvement of anti-oxidant activities. DPPH, alkyl, hydroxyl, and superoxide radical scavenging activities were evaluated using electron spin resonance. Anti-oxidant activities were also determined based on the ferric reducing anti-oxidant power assays and the ABTS radical scavenging activity. The lipid peroxidation inhibition activity was confirmed using ferric thiocyanate and thiobarbituric acid assays. The free radical scavenging activity and anti-oxidative effects of A. pectinifera fermented with C. militaris mycelia (FACM) extracts were higher than for A. pectinifera and C. militaris mycelia extracts alone. FACM extracts contained different biochemical ingredients due to fermentation of A. pectinifera and provide a beneficial anti-oxidant activity. FACM extracts are a promising source of beneficial antioxidants for use in food industries.