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Glutaraldehyde is the conventionally used cross‐linker for the activation and cross‐linking of support matrices used in enzyme immobilization. However, the toxic nature of glutaraldehyde makes it unsafe for food applications, propelling the need for nontoxic cross‐linkers. Genipin reacts with the primary and secondary amines generating a dark‐blue colored pigment and is an attractive alternative to glutaraldehyde as a cross‐linker for enzyme immobilization. Apart from its excellent cross‐linking properties, genipin possesses added advantages over glutaraldehyde such as proven health benefits, biocompatibility, and biodegradability. The present study explores the application of chitosan beads cross‐linked with the natural and nontoxic agent, genipin, for immobilizing l‐asparaginase, aimed at its subsequent use in mitigating acrylamide formation in food products. The immobilized l‐asparaginase exhibited improved functionalities such as stability, reusability, and reduction in acrylamide formation in deep‐fried cassava chips. One of the limitations observed during application in the food process was the mechanical fragility of the chitosan beads during speedy stirring. This can be overcome by increasing the concentration and time of contact of the coagulant bath during the formation of chitosan beads. The drying of the enzyme‐bound chitosan beads will also lead to shrinkage and prevent breakage during stirring. This study conclusively demonstrated the applicability of immobilizing l‐asparaginase on genipin cross‐linked chitosan beads in food‐related processes. [ABSTRACT FROM AUTHOR]

This comprehensive review delves into the multifaceted aspects of genipin, a bioactive compound derived from medicinal plants, focusing on its anti-cancer potential. The review begins by detailing the sources and phytochemical properties of genipin, underscoring its significance in traditional medicine and its transition into contemporary cancer research. It then explores the intricate relationship between genipin's chemical structure and its observed anti-cancer activity, highlighting the molecular underpinnings contributing to its therapeutic potential. This is complemented by a thorough analysis of preclinical studies, which investigates genipin's efficacy against various cancer cell lines and its mechanisms of action at the cellular level. A crucial component of the review is the examination of genipin's bioavailability and pharmacokinetics, providing insights into how the compound is absorbed, distributed, metabolized, and excreted in the body. Then, this review offers a general and updated overview of the anti-cancer studies of genipin and its derivatives based on its basic molecular mechanisms, induction of apoptosis, inhibition of cell proliferation, and disruption of cancer cell signaling pathways. We include information that complements the genipin study, such as toxicity data, and we differentiate this review by including commercial status, disposition, and regulation. Also, this review of genipin stands out for incorporating information on proposals for a technological approach through its load in nanotechnology to improve its bioavailability. The culmination of this information positions genipin as a promising candidate for developing novel anti-cancer drugs capable of supplementing or enhancing current cancer therapies. [ABSTRACT FROM AUTHOR]

Cross-linkers are pivotal to meliorate the attributes of the biopolymers, which are exploited in the biomedical industries, and also those intended as packaging supplies. Genipin (GN) is an efficacious cross-linker. Moreover, being naturally procured, biodegradable and biocompatible makes it an auspicious candidate for the biomedical and food industries. Accordingly, we attempted to provide a comprehensive review on GN as an efficient cross-linker for biopolymers. Initially, we presented the chief botanical sources of GN. The GN extraction strategies, which adopted safe solvents, were then discussed while highlighting their realized yields. The proposed GN structures, its possible modes of action, and the factors affecting its interactions, such as pH, temperature, and GN concentration were also reviewed. Afterward, the GN applications that mainly involved cross-linking biopolymers and biopolymers containing materials were discussed. These included tissue engineering, wound dressings, drug delivery, and packaging applications. GN capability to activate biopolymers, such as chitosan and gelatin, into covalently reactive enzyme immobilizers was also discussed. Moreover, other important GN applications, such as exploiting it as a colorant for foods and textiles and incorporating it in altered biosensors, were discussed. [ABSTRACT FROM AUTHOR]

Chitosan is a polysaccharide resulting from chitin deacetylation. It has demonstrated interesting characteristics in the field of adhesion but loses most of its adhesive resistance in moist environment. A chemical crosslinking of chitosan with genipin has been implemented to improve its adhesive properties in the presence of water. The study of crosslinking kinetics using rheology revealed its dependency on crosslinking rate and chitosan concentration. The capacity of the chitosan-genipin systems for water absorption has been quantified as their Free Swelling Capacity. The cross-linkage of chitosan by genipin (1% w/w) decreased significantly its ability for water absorption. Finally, the adhesive strength of several chitosans be they supplemented or not with genipin and/or glycerol used as plasticizer have been performed on Thick Adherent Shear Test samples of beech wood according to standard EN 204:2016 and EN 205:2016. The chitosan adhesive formulation containing genipin and glycerol exhibits the better adhesive properties on wood and presents higher water resistance compared to the native chitosan formulation. [ABSTRACT FROM AUTHOR]

Objectives: Bile acids (BAs), as signaling molecules to regulate metabolism, have received considerable attention. Genipin is an iridoid compound extracted from Fructus Gradeniae, which has been shown to relieve adiposity and metabolic syndrome. Here, we investigated the mechanism of genipin counteracting obesity and its relationship with BAs signals in diet-induced obese (DIO) rats. Methods: The DIO rats were received intraperitoneal injections of genipin for 10 days. The body weight, visceral fat, lipid metabolism in the liver, thermogenic genes expressions in brown fat, BAs metabolism and signals, and key enzymes for BAs synthesis were determined. Key findings: Genipin inhibited fat synthesis and promoted lipolysis in the liver, and upregulated thermogenic gene expressions in brown adipose tissue of DIO rats. Genipin increased bile flow rate and upregulated the expressions of aquaporin 8 and the transporters of BAs in liver. Furthermore, genipin changed BAs composition by promoting alternative pathways and inhibiting classical pathways for BAs synthesis and upregulated the expressions of bile acid receptors synchronously. Conclusions: These results suggest that genipin ameliorate obesity through BAs-mediated signaling pathways. [ABSTRACT FROM AUTHOR]

Natural and synthetic colorants present in food can modulate hemostasis, which includes the coagulation process and blood platelet activation. Some colorants have cardioprotective activity as well. However, the effect of genipin (a natural blue colorant) and synthetic blue colorants (including patent blue V and brilliant blue FCF) on hemostasis is not clear. In this study, we aimed to investigate the effects of three blue colorants—genipin, patent blue V, and brilliant blue FCF—on selected parameters of hemostasis in vitro. The anti- or pro-coagulant potential was assessed in human plasma by measuring the following coagulation times: thrombin time (TT), prothrombin time (PT), and activated partial thromboplastin time (APTT). Moreover, we used the Total Thrombus formation Analysis System (T-TAS, PL-chip) to evaluate the anti-platelet potential of the colorants in whole blood. We also measured their effect on the adhesion of washed blood platelets to fibrinogen and collagen. Lastly, the cytotoxicity of the colorants against blood platelets was assessed based on the activity of extracellular lactate dehydrogenase (LDH). We observed that genipin (at all concentrations (1–200 µM)) did not have a significant effect on the coagulation times (PT, APTT, and TT). However, genipin at the highest concentration (200 µM) and patent blue V at the concentrations of 1 and 10 µM significantly prolonged the time of occlusion measured using the T-TAS, which demonstrated their anti-platelet activity. We also observed that genipin decreased the adhesion of platelets to fibrinogen and collagen. Only patent blue V and brilliant blue FCF significantly shortened the APTT (at the concentration of 10 µM) and TT (at concentrations of 1 and 10 µM), demonstrating pro-coagulant activity. These synthetic blue colorants also modulated the process of human blood platelet adhesion, stimulating the adhesion to fibrinogen and inhibiting the adhesion to collagen. The results demonstrate that genipin is not toxic. In addition, because of its ability to reduce blood platelet activation, genipin holds promise as a novel and valuable agent that improves the health of the cardiovascular system and reduces the risk of cardiovascular diseases. However, the mechanism of its anti-platelet activity remains unclear and requires further studies. Its in vivo activity and interaction with various anti-coagulant and anti-thrombotic drugs, including aspirin and its derivatives, should be examined as well. [ABSTRACT FROM AUTHOR]

Study Design: Basic research Objective: To prepared 3 DNPM/chitosan hybrid hydrogels and chose the best DNPM/chitosan hybrid hydrogel for NP tissue engineering. Methods: Three DNPM/chitosan hybrid hydrogels were fabricated by changing the ratio of the decellularized NP matrix to chitosan and crosslinking with genipin. NP stem cells (NPSCs) were cultured on the hybrid hydrogels and their proliferation, morphology, and gene expression were evaluated. Finally, an in vivo experiment was performed to evaluate the immune response to the hydrogels. Results: The adhered NPSCs proliferated well on the hybrid hydrogel. The gene expression of NP-related collagen type II, aggrecan, and Sox-9 from NPSCs cultured on DNPM/chitosan hybrid hydrogel-1 was greater than from cells cultured on DNPM/chitosan hybrid hydrogel-2 and DNPM/chitosan hybrid hydrogel-3. Few inflammatory cells were observed during the in vivo experiment with DNPM/chitosan hybrid hydrogel-1. Conclusions: DNPM/chitosan hybrid hydrogel-1 is a potential candidate scaffold for NP tissue engineering. [ABSTRACT FROM AUTHOR]

Adipose tissue remodeling and plasticity are dynamically regulated by the coordinated functions of adipocytes, macrophages, and endothelial cells and extracellular matrix (ECM) that provides stiffness networks in adipose tissue component cells. Inflammation and fibrosis are crucial exogenous factors that dysregulate adipose tissue functions and drastically change the mechanical properties of the ECM. Therefore, communication among the ECM and adipose tissue component cells is necessary to understand the multifaceted functions of adipose tissues. To obtain in vivo stiffness, we used genipin as a crosslinker for collagen gels. Meanwhile, we isolated primary adipocytes, macrophages, and endothelial cells from C57BL/6J mice and incubated these cells in the differentiation media on temperature-responsive culture dishes. After the differentiation, these cell sheets were transferred onto genipin-crosslinked collagen gels with varying matrix stiffness. We found that inflammatory gene expressions were induced by hard matrix, whereas antiinflammatory gene expressions were promoted by soft matrix in all three types of cells. Interestingly, the coculture experiments of adipocytes, macrophages, and endothelial cells showed that the effects of soft or hard matrix stiffness stimulation on adipocytes were transmitted to the distant adipose tissue component cells, altering their gene expression profiles under normal matrix conditions. Finally, we identified that a hard matrix induces the secretion of CXCL13 from adipocytes, and CXCL13 is one of the important transmitters for stiffness communication with macrophages and endothelial cells. These findings provide insight into the mechanotransmission into distant cells and the application of stiffness control for chronic inflammation in adipose tissues with metabolic dysregulation.

Aim: Endodontic irrigants may affect the mechanical and chemical properties of dentine. This study evaluated the effects of various final irrigation protocols including the use of chitosan nanoparticle (CSnp) and cross‐linking with genipin on the (1) mechanical and (2) chemical properties of dentine against enzymatic degradation. Methodology: CSnp was synthesized and characterized considering physiochemical parameters and stability. The root canals of 90 single‐rooted teeth were prepared and irrigated with NaOCl. Dentine discs were obtained and divided into groups according to the following irrigation protocols: Group NaOCl+EDTA, Group NaOCl+CSnp, Group NaOCl+EDTA+CSnp, Group NaOCl+CSnp+Genipin, Group NaOCl+EDTA+CSnp+Genipin and Group distilled water. (1) Mechanical changes were determined by microhardness analysis using Vickers‐tester. (2) Chemical changes were determined by evaluating molecular and elemental compositions of dentine using Fourier transform infrared spectroscopy (FTIR) analysis and scanning electron microscope (SEM)/energy dispersive X‐ray spectroscopy (EDS) analysis, respectively. All analyses were repeated after the discs were kept in collagenase for 24 h. Data were analysed with repeated measures analysis of variance and Bonferroni correction for microhardness analysis, and Kruskal–Wallis and Wilcoxon tests for FTIR and SEM/EDS analyses (p =.05). Results: (1) Collagenase application did not have a negative effect on microhardness only in Group NaOCl+EDTA+CSnp+Genipin when compared with the post‐irrigation values (p >.05). Post‐collagenase microhardness of Group NaOCl+EDTA+CSnp and Group NaOCl+CSnp+Genipin was similar to the initial microhardness (p >.05). (2) After collagenase, Amide III/PO43− ratio presented no change in Group NaOCl+EDTA+CSnp, Group NaOCl+CSnp+Genipin and Group NaOCl+EDTA+CSnp+Genipin (p >.05), while decreased in other groups (p <.05). Collagenase did not affect CO32−/PO43− ratio in the groups (p >.05). There were no changes in the groups in terms of elemental level before and after collagenase application (p >.05). Conclusions: CSnp and genipin positively affected the microhardness and molecular composition of dentine. This effect was more pronounced when CSnp was used after EDTA. [ABSTRACT FROM AUTHOR]

The detection of pollutant dyes in the environment, particularly in waterways, can be extended and potentially simplified using terahertz spectroscopy. The use of hydrogels to absorb these contaminants from water and create solid samples with moderate transparency at terahertz frequencies evidently facilitates spectroscopic analysis. In this study, we demonstrate that chitosan and poly(vinyl alcohol) hydrogels, as well as their cross-linked and nanocomposite hybrid blends, efficiently capture the acid blue 113 azo dye (AB113). We show that terahertz transmittance and refractive index measurements conducted on these hydrogel materials offer an effective alternative method for detecting water contaminants, especially azo dyes. The terahertz transmittance spectra provide evidence of azo dye molecules within the hydrogel membranes. Additionally, considering the alterations in the hydrogels' refractive index due to the sorption of AB113 dye molecules, we derived an analytical model to accurately estimate the amount of dye sorbed by the polymeric networks. The findings of this study establish a practical and promising approach for both qualitative and quantitative terahertz detection of AB113 dye using hybrid hydrogels. A detailed comparison with optical and infrared spectroscopy is also provided for reference. [ABSTRACT FROM AUTHOR]

Chronic wounds are challenging to heal and increase global mortality. The effectiveness of skin graft is limited by rejection, fibrosis, and inadequate donor site. Multifunctionalised-hydrogel skin substitutes promoted higher wound healing by maintaining the moisture microenvironment and permit gas exchange/nourishment in prolong cell viability/activity. The purpose of this study was to evaluate a skin substitute using two strategies; via injectable and 3D bioprinting technique. New hydrogel formulations that composed of gelatin (GE) and polyvinyl-alcohol (PVA) were constructed using a pre-mix crosslinking approach with genipin (GNP) to generate the biodegradable and biocompatible skin substitute with reduced secondary traumatic wound. GPVA5_GNP (6% GE: 5% PVA crosslinked with GNP) was the most stable hydrogel for wound healing application with the longest enzymatic degradation and stable hydrogel for absorption of excess wound exudates. Primary human dermal fibroblasts (HDFs) migrated extensively through 3D bioprinted hydrogels with larger average pore sizes and interconnected pores than injectable hydrogels. Moreover, 3D bioprinted GPVA hydrogels were biocompatible with HDFs and demonstrated > 90% cell viability. HDFs maintained their phenotype and positively expressed collagen type-I, vinculin, short and dense F-actin, alpha-smooth muscle actin, and Ki67. Additionally, the presence of GNP demonstrated antioxidant capacity and high-ability of angiogenesis. The utilisation of the 3D bioprinting (layer-by-layer) approach did not compromise the HDFs' growth capacity and biocompatibility with selected bioinks. In conclusion, it allows the cell encapsulation sustainability in a hydrogel matrix for a longer period, in promoting tissue regeneration and accelerating healing capacity, especially for difficult or chronic wound. [ABSTRACT FROM AUTHOR]

Lactiplantibacillus plantarum SN13T is a probiotic plant-derived lactic acid bacterium that can grow in various medicinal plant extracts. In this study, we fermented an aqueous extract of gardenia fructus, the fruit of a medicinal plant, with SN13T, such that the bioactivity of the extract was potentiated after fermentation to suppress the release of inflammatory mediators, such as nitric oxide (NO), reactive oxygen species (ROS), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α), as well as downregulate inflammatory genes in lipopolysaccharides (LPS)-stimulated RAW 264.7 cells. This increased antioxidant and anti-inflammatory activity was mediated through bioconversion of the iridoid glycoside geniposide to its aglycone genipin via the supposed hydrolytic action of β-glucosidases harbored by SN13T. In the complete genome of SN13T, ten putative genes encoding β-glucosidases of glycosyl hydrolase (GH) family 1 organized among eight gene operons were identified. Transcriptional profiling revealed that two 6-phospho-β-glucosidase genes, pbg9 and SN13T_1925, located adjacently in the gene operon SN13T_1923, were transcribed significantly more than the remaining genes during fermentation of the gardenia extract. This suggests the role of these β-glucosidases in bioconversion of geniposide to genipin and the subsequent enhanced bioactivity of the gardenia fructus extract after fermentation with SN13T. [ABSTRACT FROM AUTHOR]

Cyanophycin (CGP) is a polypeptide consisting of amino acids—aspartic acid in the backbone and arginine in the side chain. Owing to its resemblance to cell adhesive motifs in the body, it can be considered suitable for use in biomedical applications as a novel component to facilitate cell attachment and tissue regeneration. Although it has vast potential applications, starting with nutrition, through drug delivery and tissue engineering to the production of value-added chemicals and biomaterials, CGP has not been brought to the industry yet. To develop scaffolds using CGP powder produced by bacteria, its properties (e.g., biocompatibility, morphology, biodegradability, and mechanical strength) should be tailored in terms of the requirements of the targeted tissue. Crosslinking commonly stands for a primary modification method for renovating biomaterial features to these extents. Herein, we aimed to crosslink CGP for the first time and present a comparative study of different methods of CGP crosslinking including chemical, physical, and enzymatic methods by utilizing glutaraldehyde (GTA), UV exposure, genipin, 1-ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride/N-hydroxysuccinimide (EDC/NHS), and monoamine oxidase (MAO). Crosslinking efficacy varied among the samples crosslinked via the different crosslinking methods. All crosslinked CGP were non-cytotoxic to L929 cells, except for the groups with higher GTA concentrations. We conclude that CGP is a promising candidate for scaffolding purposes to be used as part of a composite with other biomaterials to maintain the integrity of scaffolds. The initiative study demonstrated the unknown characteristics of crosslinked CGP, even though its feasibility for biomedical applications should be confirmed by further examinations. Key points: • Cyanophycin was crosslinked by 5 different methods • Crosslinked cyanophycin is non-cytotoxic to L929 cells • Crosslinked cyanophycin is a promising new material for scaffolding purposes [ABSTRACT FROM AUTHOR]

Osteochondral defects present significant challenges for effective tissue regeneration due to the complex composition of bone and cartilage. To address this challenge, this study presents the fabrication of hierarchical scaffolds combining chitosan/β-tricalcium phosphate (β-TCP) to simulate a bone-like layer, interconnected with a silk fibroin layer to mimic cartilage, thus replicating the cartilage-like layer to mimic the native osteochondral tissue architecture. The scaffolds were produced by freeze-drying and then crosslinking with genipin. They have a crosslinking degree of up to 24%, which promotes a structural rearrangement and improved connection between the different layers. Micro-CT analysis demonstrated that the structures have distinct porosity values on their top layer (up to 84%), interface (up to 65%), and bottom layer (up to 77%) and are dependent on the concentration of β-tricalcium phosphate used. Both layers were confirmed to be clearly defined by the distribution of the components throughout the constructs, showing adequate mechanical properties for biomedical use. The scaffolds exhibited lower weight loss (up to 7%, 15 days) after enzymatic degradation due to the combined effects of genipin crosslinking and β-TCP incorporation. In vitro studies showed that the constructs supported ATDC5 chondrocyte-like cells and MC3T3 osteoblast-like cells in duo culture conditions, providing a suitable environment for cell adhesion and proliferation for up to 14 days. Overall, the physicochemical properties and biological results of the developed chitosan/β-tricalcium phosphate/silk fibroin bilayered scaffolds suggest that they may be potential candidates for osteochondral tissue strategies. [ABSTRACT FROM AUTHOR]

Ferroptosis has been demonstrated to suppress cancer development and is targeted for cancer therapy. Genipin, an iridoid constituent in Gardeniae Fructus, has been reported to exert anti-cancer abilities. However, whether genipin could induce ferroptosis remains unclear. The purpose of this study is to explore the anti-gastric cancer (GC) effects of genipin by inducing ferroptosis and to identify the potential targets. CCK-8 and colony formation assays were performed to evaluate the anti-GC effects of genipin. Flowcytometry and western blot were used to indicate ferroptosis-inducing ability of genipin. The potential targets of genipin were analyzed by network pharmacology, screened using UALCAN and KM-plotter database and evaluated by molecular docking. The results showed that genipin inhibited cell viability and proliferation of GC cells. Genipin treatment decreased levels of GPX4 and SLC7A11, induced accumulation of lipid peroxidation intracellularly and led to ferroptosis in GC cells. Network pharmacology analysis identified that lipid- and ROS-related pathways involved in ferroptosis ranked high among genipin-GC common targets. Data from UALCAN and KM-plotter database demonstrated that expression levels of ferroptosis-related targets, including AURKA, BCAT2, DHODH, and GPI, increased in GC tissues and the higher levels of the above four targets were related to tumor stage, tumor grade, and poor prognosis. Among these four targets, AURKA, BCAT2, and DHODH were confirmed by molecular docking with binding energies less than − 5. Taken together, our study demonstrates that genipin could exert anti-GC ability by inducing ferroptosis and provides evidence for the potential application of genipin in GC treatment. [ABSTRACT FROM AUTHOR]

Further progress in regenerative medicine and bioengineering highly depends on the development of 3D polymeric scaffolds with active biological properties. The most attention is paid to natural extracellular matrix components, primarily collagen. Herein, nonwoven nanofiber materials with various degrees of collagen denaturation and fiber diameters 250–500 nm were produced by electrospinning, stabilized by genipin, and characterized in detail. Collagen denaturation has been confirmed using DSC and FTIR analysis. The comparative study of collagen and gelatin nonwoven materials (NWM) revealed only minor differences in their biocompatibility with skin fibroblasts and keratinocytes in vitro. In long‐term subcutaneous implantation study, the inflammation was less evident on collagen than on gelatin NWM. Remarkably, the pronounced calcification was revealed in the collagen NWM only. The results obtained can be useful in terms of improving the electrospinning technology of collagen from aqueous solutions, as well as emphasize the importance of long‐term study to ensure proper implementation of the material, taking into account the ability of collagen to provoke calcification. [ABSTRACT FROM AUTHOR]

Chitosan nanoparticles (NPs) possess great potential in biomedical fields. Orifice-induced hydrodynamic cavitation (HC) has been used for the enhancement of fabrication of size-controllable genipin-crosslinked chitosan (chitosan-genipin) NPs based on the emulsion cross-linking (ECLK). Experiments have been performed using various plate geometries, chitosan molecular weight and under different operational parameters such as inlet pressure (1–3.5 bar), outlet pressure (0–1.5 bar) and cross-linking temperature (40–70 °C). Orifice plate geometry was a crucial factor affecting the properties of NPs, and the optimized geometry of orifice plate was with single hole of 3.0 mm diameter. The size of NPs with polydispersity index of 0.359 was 312.6 nm at an optimized inlet pressure of 3.0 bar, and the maximum production yield reached 84.82 %. Chitosan with too high or too low initial molecular weight (e.g., chitosan oligosaccharide) was not applicable for producing ultra-fine and narrow-distributed NPs. There existed a non-linear monotonically-increasing relationship between cavitation number (Cv) and chitosan NP size. Scanning electron microscopy (SEM) test indicated that the prepared NPs were discrete with spherical shape. The study demonstrated the superiority of HC in reducing particle size and size distribution of NPs, and the energy efficiency of orifice type HC-processed ECLK was two orders of magnitude than that of ultrasonic horn or high shear homogenization-processed ECLK. In vitro drug-release studies showed that the fabricated NPs had great potential as a drug delivery system. The observations of this study can offer strong support for HC to enhance the fabrication of size-controllable chitosan-genipin NPs.

Traumatic nerve injuries are nowadays a significant clinical challenge and new substitutes with adequate biological and mechanical properties are in need. In this context, fibrin-agarose hydrogels (FA) have shown the possibility to generate tubular scaffolds with promising results for nerve repair. However, to be clinically viable, these scaffolds need to possess enhanced mechanical properties. In this line, genipin (GP) crosslinking has demonstrated to improve biomechanical properties with good biological properties compared to other crosslinkers. In this study, we evaluated the impact of different GP concentrations (0.05, 0.1 and 0.2% (m/v)) and reaction times (6, 12, 24, 72 h) on bioartificial nerve substitutes (BNS) consisting of nanostructured FA scaffolds. First, crosslinked BNS were studied histologically, ultrastructurally and biomechanically and then, its biocompatibility and immunomodulatory effects were ex vivo assessed with a macrophage cell line. Results showed that GP was able to improve the biomechanical resistance of BNS, which were dependent on both the GP treatment time and concentration without altering the structure. Moreover, biocompatibility analyses on macrophages confirmed high cell viability and a minimal reduction of their metabolic activity by WST-1. In addition, GP-crosslinked BNS effectively directed macrophage polarization from a pro-inflammatory (M1) towards a pro-regenerative (M2) phenotype, which was in line with the cytokines release profile. In conclusion, this study considers time and dose-dependent effects of GP in FA substitutes which exhibited increased biomechanical properties while reducing immunogenicity and promoting pro-regenerative macrophage shift. These tubular substitutes could be useful for nerve application or even other tissue engineering applications such as urethra.

In 2004, it was the first time that Wollensak and Spoerl had applied physical and chemical cross-linking methods to scleral tissue. They found that the biomechanical strength of cross-linked sclera, induced by riboflavin/ultraviolet A, glyceraldehyde and glutaraldehyde, could be improved and proposed that scleral collagen cross-linking is expected to be a new method for the treatment of pathologic myopia. In recent years, a series of explorations have been made on the effectiveness and adverse reactions of physical and chemical cross-linking in the prevention and treatment of pathologic myopia, including the establishment of various animal models and different myopia modeling methods, the improvement of cross-linking methods, the amelioration of the measurement of biomechanical strength of scleral tissue and the attention of biological parameters such as the thickness of retinal nerve fiber layer and the amplitude of electroretinogram in vivo. Genipin-crosslinking of the scleral collagen combined with posterior scleral contraction/reinforcement has been applied to clinical research. This review summarizes physical cross-linking and the genipin-crosslinking of scleral collagen to explore the effectiveness and safety of the methods in the prevention and treatment of the pathologic myopia.

The present work aims to design jackfruit gum-based curcumin-loaded nanoparticles (CUR-NPs) for improved drug entrapment and modified release of CUR using ionotropic gelation. Briefly, the optimization of CUR-NPs was confirmed using a 3² response surface methodology. The diffractogram and thermogram of CUR-NPs confirmed reduction of crystallinity of CUR (optimized batch: F5) due to jackfruit gum and genipin cross-linked polymeric network. The particle size and zeta potential analysis confirmed formation of nanosized and stable CUR-NPs, respectively. Also, the nanoparticles demonstrated 83.99 ± 1.23% entrapment efficiency, whereas they showed 98.36 ± 0.96% of CUR release within 12 h at pH 7.4. The CUR-NPs exhibited good mucoadhesive properties due to the presence of jackfruit gum. Finally, the MTT assay showed a decrease in colorectal cancer cell viability due to tailored CUR release from CUR-NPs. In conclusion, jackfruit gum-genipin-based CUR-NPs offered high entrapment efficiency, tailored releases of CUR, good mucoadhesive property and improved anticancer activity. [ABSTRACT FROM AUTHOR]

Replacing conventional plastics with other products obtained from biobased and biodegradable raw materials is an increasingly studied solution. In this line, the development of protein-based bioplastics is a promising alternative. However, for some applications, such as packaging it would be necessary to improve their properties by including an additional stage to crosslink the protein chains during the production of bioplastics. Therefore, pea protein-based bioplastics crosslinked with genipin, a natural non-toxic chemical crosslinking agent, are evaluated in this study. The bioplastics are obtained via injection moulding and genipin, is included in the initial formulation. Specifically, the concentration of genipin and the effects on the protein blends with genipin over days are evaluated. The evolution of the reaction is followed by the colour change of the blend, together with thermal analyses and infrared spectroscopy. Results showed the evolution of the crosslinking in the blends resulted in conformational changes that resulted in the modification of the initial yellowish colour to a blueish system. Respect their bioplastics, more deformable systems with a lower water absorption capability are obtained by using genipin as crosslinking agent. [ABSTRACT FROM AUTHOR]

The pharmacopoeial limit test for the impurity 2-aminobutanol (AB) in ethambutol hydrochloride (ETB) relies on florescence measurement using a fluorometer. However, this instrument is often unavailable in many laboratories due to its cost. A novel colorimetric method was therefore developed using genipin, an amine-reactive reagent, to react with AB, producing a blue color. The color intensity was subsequently measured using a more affordable UV-vis spectrophotometer. To overcome matrix effects, the standard addition technique was incorporated. The test effectively detected AB at concentrations as low as 0.125% in ETB, below the compendial limit of 1.0%. The analysis of ETB bulk and tablets showed consistent semi-quantitative results with the USP method. Furthermore, it aligned with green chemistry by eliminating the use of organic solvents and employing safe, naturally derived reagent. In summary, the proposed method provides a reliable, green, and cost-effective alternative for analyzing AB in ETB, ensuring compliance with pharmacopeial standards. [ABSTRACT FROM AUTHOR]

Bio-based adsorbents for removing pollutants were prepared using composite sponges with porosities greater than 80%. Herein, we report for the first time the preparation of macroporous sponges via freeze-drying of a chitosan solution and water hyacinth fiber (WHF). Genipin, a chemical crosslinker, was utilized in the process. The results showed that genipin increased the resistance to compression of the sponges while also improving their water and thermal stability. The composite sponges effectively removed C.I. reactive orange 16 (RO16) from water, with adsorption capacities ranging from 37.86 to 95.77 mg/g at pH 7. After 24 h, nearly complete dye removal was achieved with an initial dye concentration of 20 mg/L and an adsorbent dose of 2.5 g/L. The adsorption isotherm followed the Freundlich model, and the adsorption kinetics followed the pseudo-second-order model. The WHF/chitosan sponge could be reused for up to four cycles. These findings demonstrate the potential of the prepared sponges as bio-based adsorbents for the treatment of dye-contaminated wastewater. [ABSTRACT FROM AUTHOR]

Nerve conduits may represent a valuable alternative to autograft for the regeneration of long-gap damages. However, no NCs have currently reached market approval for the regeneration of limiting gap lesions, which still represents the very bottleneck of this technology. In recent years, a strong effort has been made to envision an engineered graft to tackle this issue. In our recent work, we presented a novel design of porous/3D-printed chitosan/poly-ε-caprolactone conduits, coupling freeze drying and additive manufacturing technologies to yield conduits with good structural properties. In this work, we studied genipin crosslinking as strategy to improve the physiochemical properties of our conduit. Genipin is a natural molecule with very low toxicity that has been used to crosslink chitosan porous matrix by binding the primary amino group of chitosan chains. Our characterization evidenced a stabilizing effect of genipin crosslinking towards the chitosan matrix, with reported modified porosity and ameliorated mechanical properties. Given the reported results, this method has the potential to improve the performance of our conduits for the regeneration of long-gap nerve injuries. [ABSTRACT FROM AUTHOR]

Genipin, a natural compound derived from the fruit of Gardenia jasminoides Ellis, was reported to have activity against various cancer types. In this study, we determined the underlying mechanism for genipin‐induced cell death in human oral squamous cell carcinoma (OSCC). The growth‐inhibitory effects of genipin in human OSCC cells was examined by the Cell Counting Kit‐8 and soft agar assays. The effects of genipin on apoptosis were assessed by nuclear morphological changes by 4′,6‐diamidino‐2‐phenylindole staining, measurement of the sub‐G1 population, and Annexin V‐fluorescein isothiocyanate/propidium iodide double staining. The underlying mechanism of genipin activity was analyzed by western blot analysis, subcellular fractionation of the nucleus and cytoplasm, immunocytochemistry, and quantitative real‐time polymerase chain reaction. Genipin inhibited the growth of OSCC cells and induced apoptosis, which was mediated by a caspase‐dependent pathway. Genipin reduced the phosphorylation of signal transducer and activator of transcription 3 (STAT3) at Tyr705 and its nuclear localization. Furthermore, inhibition of p‐STAT3Tyr705 levels following genipin treatment was required for the reduction of survivin and myeloid cell leukemia‐1 (Mcl‐1) expression, leading to apoptotic cell death. The genipin‐mediated reduction in survivin and Mcl‐1 expression was caused by transcriptional and/or posttranslational regulatory mechanisms. The results provide insight into the regulatory mechanism by which genipin induces apoptotic cell death through the abrogation of nuclear STAT3 phosphorylation and suggest that genipin may represent a potential therapeutic option for the treatment of human OSCC. Significance statement: STAT3 is frequently overexpressed in various cancers, including OSCC, indicating that it can be an oncogenic factor and cancer therapy targeting STAT3 is thought to be a promising therapeutic option.In the present study, we investigated the possibility of genipin as an anticancer drug candidate by targeting STAT3 kinase in oral cancer cell lines.The importance of our findings is genipin can mediate STAT3 signaling to induce apoptosis for treating human oral cancer and, to the best our knowledge, it is the first study showing the deactivation of STAT3 by genipin for apoptosis in oral cancer. [ABSTRACT FROM AUTHOR]

Chitosan (CH) is a very well‐known biopolymer that has been widely used for the development of biomaterials with a wide range of applications in the biomedical field, such as the preparation of hydrogels, owing to its outstanding anti‐inflammatory, antibacterial and antifungal properties, biocompatibility and biodegradability, although they present limited mechanical properties. Chemical crosslinking is one of the most recurrent strategies for the reinforcement of these structures and, above all, crosslinking with natural‐origin compounds that do not compromise their biocompatibility is considered a hot topic in this research field. D‐fructose (F), obtained from the hydrolyzation and further isomerization of starch, an abundant raw material and genipin (G), which is extracted from the fruits of Gardenia jasminoides Ellis are used as natural crosslinkers. Chitosan‐based hydrogels crosslinked with each crosslinking agent are prepared and characterized through Fourier transform infrared (FTIR) spectroscopy, crosslinking and swelling degree determination, rheological, microstructural, and biological studies. The results demonstrate that crosslinking with G is more beneficial for chitosan‐based hydrogels since these samples showed more compact structures and better rheological performance. Additionally, excellent biological in vitro behavior due to the crosslinking with G, unlike that of F. [ABSTRACT FROM AUTHOR]

Natural dyes, obtained without the use of chemical treatment, are derived from naturally occurring sources, such as plants, animals, insects, and minerals. The usage of natural substances and their medicinal properties dates back to the origins of human civilization. The purpose of this review is to highlight the medicinal importance of selected natural colors, which sheds light on the critical role played by these dyes in the pharmaceutical industry. The objective is to showcase the health benefits of each color that can be obtained from nature for medicinal purposes based on their chemical structure. The review presents the reasons for utilizing natural resources in addressing various health issues, with a focus on three specific problems: microbial infections, cancer, and oxidative stress. Our review highlights the potential of natural resource structures, particularly anthocyanins, genipin, carotenoids, phycocyanin, and chlorophylls, in combating these ailments, emphasizing the need to explore their resources further for medicinal purposes. While most reviews provide a survey about colorful crude plant extracts in relation to one or a few categories of human health, our review focuses on the specific chromophore extracted not only from plants but also from any natural resource to provide a specific chromophore effect in a whole resource. The review highlights the significant role performed by organic pigments in the medicinal domain, with organic colorants acting as an essential element of the pharmaceutical sector's weaponry. Hence, it is of paramount significance to actively promote and stress the adoptions of naturally existing chromophores in diverse everyday commodities, while simultaneously acknowledging and valuing their substantial importance and worth in the vast realm of the pharmaceutical industry. [ABSTRACT FROM AUTHOR]

This study investigated the impact of a support matrix and active group on the support to the nutritional properties of orange juice after juice clarification. Pectinase was immobilized on chitosan and aminated silica supports, activated with genipin or glutaraldehyde, and applied for juice clarification. The effects on various juice properties, including reducing sugars, total soluble solids, vitamin C, and phenolic compounds, juice color, and pH, were evaluated. The results revealed that the immobilization on chitosan activated using genipin resulted in the highest biocatalyst activity (1211.21 U·g−1). The juice treatments using the biocatalysts led to turbidity reduction in the juice (up to 90%), with the highest reductions observed in treatments involving immobilized enzyme on chitosan. Importantly, the enzymatic treatments preserved the natural sugar content, total soluble solids, and pH of the juice. Color differences between treated and raw juice samples were especially relevant for those treated using enzymes, with significant differences in L* and b*, showing loss of yellow vivid color. Analysis of phenolic compounds and vitamin C showed no significant alterations after the enzymatic treatment of the raw juice. According to our results, the clarification of orange juice using immobilized enzymes can be a compromise in turbidity reduction and color reduction to maintain juice quality. [ABSTRACT FROM AUTHOR]

The immobilized enzymes' properties can be affected by cross-linkers on the surface of supports. To study how cross-linkers alter enzymes function, chitosan-coated magnetic nanoparticles (CMNPs) with immobilized papain were prepared using glutaraldehyde and or genipin, and then, the properties of the nanoparticles and the immobilized enzymes were assessed. The Scanning Electron Microscope (SEM), Fourier Transform Infrared (FTIR), and X-Ray Diffraction (XRD) results showed that the CMNPs were prepared and papain molecules were immobilized on CMNPs by glutaraldehyde (CMNP-Glu-Papain) or by genipin (CMNP-Gen-Papain). Also, the results associated with enzymes activity indicated that the immobilization by glutaraldehyde and genipin increased the pH optimum of papain from 7 to 7.5 and 9, respectively. The kinetic results indicated that the immobilization by genipin slightly affects the enzyme affinity to the substrate. The stability results showed that CMNP-Gen-Papain has more thermal stability than CMNP-Glu-Papain and papain immobilization on CMNPs by genipin leads to stabilization of the enzyme in the presence of polar solvents, probably due to the more hydroxyl groups on CMNPs activated by genipin. In conclusion, this study suggests that there is a relationship between the types of cross-linker on the surface of supports, and the mechanism of action, kinetic parameters, and the stability of immobilized papain. [ABSTRACT FROM AUTHOR]

Natural dyes, obtained without the use of chemical treatment, are derived from naturally occurring sources, such as plants, animals, insects, and minerals. The usage of natural substances and their medicinal properties dates back to the origins of human civilization. The purpose of this review is to highlight the medicinal importance of selected natural colors, which sheds light on the critical role played by these dyes in the pharmaceutical industry. The objective is to showcase the health benefits of each color that can be obtained from nature for medicinal purposes based on their chemical structure. The review presents the reasons for utilizing natural resources in addressing various health issues, with a focus on three specific problems: microbial infections, cancer, and oxidative stress. Our review highlights the potential of natural resource structures, particularly anthocyanins, genipin, carotenoids, phycocyanin, and chlorophylls, in combating these ailments, emphasizing the need to explore their resources further for medicinal purposes. While most reviews provide a survey about colorful crude plant extracts in relation to one or a few categories of human health, our review focuses on the specific chromophore extracted not only from plants but also from any natural resource to provide a specific chromophore effect in a whole resource. The review highlights the significant role performed by organic pigments in the medicinal domain, with organic colorants acting as an essential element of the pharmaceutical sector’s weaponry. Hence, it is of paramount significance to actively promote and stress the adoptions of naturally existing chromophores in diverse everyday commodities, while simultaneously acknowledging and valuing their substantial importance and worth in the vast realm of the pharmaceutical industry.

Natural and synthetic colorants present in food can modulate hemostasis, which includes the coagulation process and blood platelet activation. Some colorants have cardioprotective activity as well. However, the effect of genipin (a natural blue colorant) and synthetic blue colorants (including patent blue V and brilliant blue FCF) on hemostasis is not clear. In this study, we aimed to investigate the effects of three blue colorants—genipin, patent blue V, and brilliant blue FCF—on selected parameters of hemostasis in vitro. The anti- or pro-coagulant potential was assessed in human plasma by measuring the following coagulation times: thrombin time (TT), prothrombin time (PT), and activated partial thromboplastin time (APTT). Moreover, we used the Total Thrombus formation Analysis System (T-TAS, PL-chip) to evaluate the anti-platelet potential of the colorants in whole blood. We also measured their effect on the adhesion of washed blood platelets to fibrinogen and collagen. Lastly, the cytotoxicity of the colorants against blood platelets was assessed based on the activity of extracellular lactate dehydrogenase (LDH). We observed that genipin (at all concentrations (1–200 µM)) did not have a significant effect on the coagulation times (PT, APTT, and TT). However, genipin at the highest concentration (200 µM) and patent blue V at the concentrations of 1 and 10 µM significantly prolonged the time of occlusion measured using the T-TAS, which demonstrated their anti-platelet activity. We also observed that genipin decreased the adhesion of platelets to fibrinogen and collagen. Only patent blue V and brilliant blue FCF significantly shortened the APTT (at the concentration of 10 µM) and TT (at concentrations of 1 and 10 µM), demonstrating pro-coagulant activity. These synthetic blue colorants also modulated the process of human blood platelet adhesion, stimulating the adhesion to fibrinogen and inhibiting the adhesion to collagen. The results demonstrate that genipin is not toxic. In addition, because of its ability to reduce blood platelet activation, genipin holds promise as a novel and valuable agent that improves the health of the cardiovascular system and reduces the risk of cardiovascular diseases. However, the mechanism of its anti-platelet activity remains unclear and requires further studies. Its in vivo activity and interaction with various anti-coagulant and anti-thrombotic drugs, including aspirin and its derivatives, should be examined as well.

Abstract Chitosan (CH) is a very well‐known biopolymer that has been widely used for the development of biomaterials with a wide range of applications in the biomedical field, such as the preparation of hydrogels, owing to its outstanding anti‐inflammatory, antibacterial and antifungal properties, biocompatibility and biodegradability, although they present limited mechanical properties. Chemical crosslinking is one of the most recurrent strategies for the reinforcement of these structures and, above all, crosslinking with natural‐origin compounds that do not compromise their biocompatibility is considered a hot topic in this research field. D‐fructose (F), obtained from the hydrolyzation and further isomerization of starch, an abundant raw material and genipin (G), which is extracted from the fruits of Gardenia jasminoides Ellis are used as natural crosslinkers. Chitosan‐based hydrogels crosslinked with each crosslinking agent are prepared and characterized through Fourier transform infrared (FTIR) spectroscopy, crosslinking and swelling degree determination, rheological, microstructural, and biological studies. The results demonstrate that crosslinking with G is more beneficial for chitosan‐based hydrogels since these samples showed more compact structures and better rheological performance. Additionally, excellent biological in vitro behavior due to the crosslinking with G, unlike that of F.

Alzheimer's disease (AD) is characterized by the aggregation of disordered proteins, such as amyloid beta (Aβ) and tau, leading to neurotoxicity and disease progression. Despite numerous efforts, effective inhibitors of Aβ and tau aggregates have not been developed. Thus, we aimed to screen natural small molecules from crude extracts that target various pathologies and are prescribed for patients with neurological diseases. In this study, we screened 162 natural small molecules prescribed for neurological diseases and identified genipin and pyrogallol as hit compounds capable of simultaneously regulating the aggregation of Aβ and tau K18. Moreover, we confirmed the dual modulatory effects of these compounds on the reduction of amyloid-mediated neurotoxicity in vitro and the disassembly of preformed Aβ42 and tau K18 fibrils. Furthermore, we observed the alleviatory effects of genipin and pyrogallol against AD-related pathologies in triple transgenic AD mice. Molecular dynamics and docking simulations revealed the molecular interaction dynamics of genipin and pyrogallol with Aβ42 and tau K18, providing insights into their suppression of aggregation. Our findings suggest the therapeutic potential of genipin and pyrogallol as dual modulators for the treatment of AD by inhibiting aggregation or promoting dissociation of Aβ and tau.

The discrepancy between in vitro and in vivo bioefficacies of hydrophilic polyphenols often stems from their low intestinal permeabilities. Chitosan (CS)-based polysaccharide-polypeptide nanocomplexes are promising delivery systems for enhancing the intestinal permeability of hydrophilic polyphenols. However, these nanocomplexes are intrinsically susceptible to pH changes, which limits their applications. In this study, the nanocomplexes self-assembled by CS and caseinophosphopeptides (CPPs) were crosslinked by a natural crosslinker genipin, aiming to improve their pH stabilities. The crosslinking reaction altered not only the size, surface charge, and morphology but also the microstructures of the CS-CPPs nanocomplexes (CCNs). Compared to the non-crosslinked counterparts, genipin-crosslinked CCNs (GCCNs) showed higher stability against pH change as they were more resistant to acid-induced dissociation at gastric pH. GCCNs were further used to encapsulate theaflavin-3,3′-digallate (TF-3), an important polyhydroxylated polyphenol in black tea that has low intestinal permeability. Encapsulation of TF-3 also influenced the physicochemical features of the nanocomplexes. Notably, loading in GCCNs significantly enhanced the in vitro intestinal permeability of TF-3. This study demonstrated that GCCNs not only had high pH stability but also had the capacity to enhance the in vitro intestinal permeability of TF-3.

Gardenia, as a medicinal and edible herb, has the pharmacological activity of protecting the liver and cholagogue, but the hepatotoxicity induced by the chemical component genipin (GP) limits its application. The aim of this study was to evaluate the acute and subacute hepatotoxicity of genipin in normal mice and mice with α-naphthalene isothiocyanate (ANIT)-induced liver injury. The results of the acute study showed that the LD50 of genipin was 510 mg/kg. Genipin exhibited hepatotoxicity in normal and jaundiced mice at doses of 125 mg/kg, 250 mg/kg, and 500 mg/kg, which increased with dose. In a 28-day subacute study, the 50 mg/kg and 100 mg/kg dose groups showed some pharmacodynamic effects at 7 days but exhibited hepatotoxicity that increased with time and improved after drug withdrawal. In addition, based on proteomics, the mechanism of liver injury induced by genipin may be related to the disruption of the UDP-glucuronosyltransferase system and cytochrome P450 enzyme activity. In conclusion, this study showed that genipin hepatotoxicity was time- and dose dependent, but it is worth mentioning that hepatotoxicity was reversible. It is hoped that this study will provide a scientific basis for circumventing the adverse effects of genipin.

The effect of genipin (GP) concentrations on the structural, mechanical, and water barrier properties of chitosan (CS)/rice protein hydrolysate (RH) composite films was investigated for further incorporating of curcumin. GP increased the stability of CS/RH conjugate in solution. The degree of CS/RH crosslinking depended on genipin concentration, as indicated in particle size and UV spectra. At optimal genipin concentration (0.4 mg GP/g total biopolymers), the CS/RH film had the lowest moisture content (10.67%) and water vapor transmission rate (829.35 g/m2·24 h.). Genipin could enhance the tensile strength and thermal stability of the CS/RH films depending on GP addition concentration. With an increased amount of GP, the DPPH scavenging activity decreased due to the cross-linking of antioxidant peptides, and the E* value increased indicating the significant color differences caused by increasing GP. Notably, the addition of curcumin had a positive effect on the DPPH scavenging activity and antimicrobial activity of the CS/RH films. Curcumin-loaded CS/RH films had higher antibacterial activity against S. aureus than E. coli. In general, the suitable addition content of GP was a promising and green strategy to improve the properties of the CS/RH films. [ABSTRACT FROM AUTHOR]

Silk fibroin (SF) holds promise for the preparation of matrices for tissue engineering and regenerative medicine or for the development of drug delivery systems. Regenerated fibroin from Bombyx mori cocoons is water-soluble and can be processed into scaffolds of various forms, such as fibrous matrices, using the electrospinning method. In the current study, we studied the correlation between concentrations of fibroin aqueous solutions and their properties, in order to obtain electrospun mats for tissue engineering. Two methods were used to prevent solubility in fibroin-based matrices: The conversion of fibroin to the β-conformation via treatment with an ethanol solution and chemical cross-linking with genipin (Gp). The interaction of Gp with SF led to the appearance of a characteristic blue color but did not lead to the gelation of solutions. To speed up the cross-linking reaction with Gp, we propose using chitosan-containing systems and modifying fibrous materials via treatment with a solution of Gp in 80% ethanol. It was shown that the composition of fibroin with chitosan contributes to an improved water resistance, reduces defective material, and leads to a decrease in the diameter of the fibers. The electrospun fiber matrices based on regenerated fibroin modified by cross-linking with genipin in water–alcohol solutions were shown to promote cell adhesion, spreading, and growth and, therefore, could hold promise for tissue engineering. [ABSTRACT FROM AUTHOR]

Decellularized extracellular matrix (dECM) hydrogels have emerged as promising materials in tissue engineering. The steps to produce dECM hydrogels containing the bioactive epitopes found in the native matrix are often laborious, including the initial harvesting and decellularization of the animal organ. Furthermore, resulting hydrogels often exhibit weak mechanical properties that require the use of additional crosslinkers such as genipin to truly simulate the mechanical properties of the desired study tissue. In this work, we have developed a protocol to readily obtain tens of thin dECM hydrogel cryosections attached to a glass slide as support, to serve as scaffolds for two-dimensional (2D) or three-dimensional (3D) cell culture. Following extensive atomic force microscopy (AFM)-based mechanical characterization of dECM hydrogels crosslinked with increasing genipin concentrations (5 mM, 10 mM, and 20 mM), we provide detailed protocol recommendations for achieving dECM hydrogels of any biologically relevant stiffness. Given that our protocol requires hydrogel freezing, we also confirm that the approach taken can be further used to increase the mechanical properties of the scaffold in a controlled manner exhibiting twice the stiffness in highly crosslinked arrays. Finally, we explored the effect of ethanol-based short- and long-term sterilization on dECM hydrogels, showing that in some situations it may give rise to significant changes in hydrogel mechanical properties that need to be taken into account in experimental design. The hydrogel cryosections produced were shown to be biocompatible and support cell attachment and spreading for at least 72 h in culture. In brief, our proposed method may provide several advantages for tissue engineering: (1) easy availability and reduction in preparation time, (2) increase in the total hydrogel volume eventually used for experiments being able to obtain 15–22 slides from a 250 µL hydrogel) with a (3) reduction in scaffold variability (only a 17.5 ± 9.5% intraslide variability provided by the method), and (4) compatibility with live-cell imaging techniques or further cell characterization of cells. [ABSTRACT FROM AUTHOR]

Background: Acute myeloid leukemia (AML), one of the common malignancies of the hematologic system, has progressively increased in incidence. Aging is present in both normal tissues and the tumor microenvironment. However, the relationship between senescence and AML prognosis is still not elucidated. Methods: In this study, RNA sequencing data of AML were obtained from TCGA, and prognostic prediction models were established by LASSO-Cox analysis. Differences in immune infiltration between the different risk groups were calculated using the CIBERSORT and ESTIMATE scoring methods. The KEGG and GO gene enrichment and GSEA enrichment were also used to enrich for differential pathways between the two groups. Subsequently, this study collected bone marrow samples from patients and healthy individuals to verify the differential expression of uncoupling protein 2 (UCP2) in different populations. Genipin, a UCP2 protein inhibitor, was also used to examine its effects on proliferation, cell cycle, and apoptosis in AML cell lines in vitro. Results: It showed that aging-related genes (ARGs) expression was correlated with prognosis. And there was a significant difference in the abundance of immune microenvironment cells between the two groups of patients at high risk and low risk. Subsequently, UCP2 expression was found to be elevated in AML patients. Genipin inhibits UCP2 protein and suppresses the proliferation of AML cell lines in vitro. Conclusion: ARGs can be used as a predictor of prognosis in AML patients. Moreover, suppressing UCP2 can reduce the proliferation of AML cell lines, alter their cell cycle, and promote apoptosis in vitro. [ABSTRACT FROM AUTHOR]

It is essential to further characterize liver injury aimed at developing novel therapeutic approaches. This study investigated the mechanistic basis of genipin against carbon tetrachloride (CCl4)-triggered acute liver injury concerning ferroptosis, a novel discovered modality of regulated cell death. All experiments were performed using hepatotoxic models upon CCl4 exposure in mice and human hepatocytes in vitro. Immunohistochemistry, immunoblotting, molecular docking, RNA-sequencing and ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) were conducted. CCl4 intoxication was manifested with lipid peroxidation-dictated ferroptotic cell death, together with changes in a cascade of ferroptosis-associated events and several regulatory pathways. Both the administration of genipin and ferrostatin-1 (Fer-1) significantly prevented this hepatotoxicity in response to CCl4 intoxication via upregulating GPX4 and xCT (i.e., critical regulators of ferroptosis). RNA-sequencing unraveled that arachidonic acid metabolism was considerably influenced upon genipin treatment. Accordingly, genipin treatment attenuated arachidonate 15-lipoxygenase (ALOX15)-launched lipid peroxidation in terms of UHPLC-MS/MS analysis and inflammation. In vitro, genipin supplementation rescued erastin-induced hepatocellular inviability and lipid ROS accumulation. The siRNA knockdown of GPX4 partially abrogated the protective effects of genipin on erastin-induced cytotoxicity, whereas the cytotoxicity was less severe in the presence of diminished ALOX15 expression in L-O2 cells. In conclusion, our findings uncovered that genipin treatment protects against CCl4-triggered acute liver injury by abrogating hepatocyte ferroptosis, wherein the pharmacological modification of dysregulated GPX4 and ALOX15-launched lipid peroxidation was responsible for underlying medicinal effects as molecular basis. [ABSTRACT FROM AUTHOR]

Genipin, an aglycone of geniposide, is a rich iridoid component in the fruit of Gardenia jasminoides Ellis and has numerous biological activities. However, its metabolic profiles in vivo and vitro remain unclear. In this study, an effective analytical strategy based on ultra-high-performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) in positive and negative ion modes was developed to analyze and identify genipin metabolites in rat urine, blood, feces, and fecal fermentation in combination with many methods including post-collection data mining methods, high-resolution extracted ion chromatography (HREIC), and multiple mass defect filtering (MMDF). Simultaneously, the metabolites of genipin in vivo were verified by fecal fermentation of SD rats at different times. Finally, based on information such as reference substances, chromatographic retention behavior, and accurate mass determination, a total of 50 metabolites (including prototypes) were identified in vivo. Among them, 7, 31 and 28 metabolites in vivo were identified in blood, urine, and feces, respectively. Our results showed that genipin could generate different metabolites that underwent multiple metabolic reactions in vivo including methylation, hydroxylation, dehydroxylation, hydrogenation, sulfonation, glucuronidation, demethylation, and their superimposed reactions. Forty-six metabolites were verified in vitro. Meanwhile, 2 and 19 metabolites identified in blood and urine were also verified in fecal fermentation at different times. These results demonstrated that metabolites were produced in feces and reabsorbed into the body. In conclusion, the newly discovered metabolites of genipin can provide a new perspective for understanding its pharmacological effects and build the foundation for thee toxicity and safety evaluations of genipin. [ABSTRACT FROM AUTHOR]

Summary: Chitosan (CS) nanoparticles were formed by two different methodologies: ionic (with sodium tripolyphosphate ‐TPP) and chemical (with genipin ‐GN) cross‐linking. CS–TPP and CS–GN nanoparticles showed positive zeta potential (+46.24 and +62.86 mV, respectively) and sizes ranging from 83.70 to 835.91 nm, respectively. Ionic cross‐linking (with TPP) resulted in particles smaller than those formed by chemical cross‐linking (with GN). The in vitro antibacterial activity revealed an efficient inhibitory effect, especially regarding CS–TPP. In situ, antibacterial and antioxidant studies were carried out on O/W emulsions. The mesophilic and psychrotrophic counts, peroxide value and quantification of conjugated dienes and trienes during time revealed that nanoparticles tend to improve the microbiological and oxidative stability of emulsions. These findings underline the direct relationship between the reduction of particle size and the increase in biopolymer functional properties and expand the possibilities of CS application as a natural preservative of emulsified systems. [ABSTRACT FROM AUTHOR]

We demonstrated that the combination of poly(dimethylsiloxane) (PDMS) and a hybrid hydrogel based on chitosan/poly(vinyl alcohol)/genipin (CS/PVA/GEN)—with ratios 7:3 (SIPNs1), 5:5 (SIPNs2), and 3:7 (SIPNs3) (w/w)—were useful for obtaining semi-interpenetrated polymeric networks (SIPNs). These SIPNs successfully combined the PDMS's elasticity, the PVA's swelling capability, and the excellent biological response of CS and GEN. Noticing these features are desirable in materials intended for biomedical applications such as wound healing and tissue engineering. Through evaluating the influence of PDMS and CS/PVA/GEN hybrid hydrogel ratios on the physico-chemical, mechanical, and the response of seeded cells, we found contact angles between 55 ∘ and 75 ∘ , while the swelling percentage was enhanced up to three times concerning blank PDMS. Besides, the elastic moduli presented values between 1 and 1.6 MPa. The fibroblasts seeded on the tested semi-interpenetrating polymeric networks were viable, and no cytotoxic effects were found. The cells presented fusiform shapes indicating an excellent attachment to the material's surface. The highest cell densities were found for SIPNs1 and SIPNs2, suggesting that these compositions in a membrane shape could potentially be used in wound healing/dressing or as a scaffold for tissue engineering. [ABSTRACT FROM AUTHOR]

Osteochondral defects present significant challenges for effective tissue regeneration due to the complex composition of bone and cartilage. To address this challenge, this study presents the fabrication of hierarchical scaffolds combining chitosan/β-tricalcium phosphate (β-TCP) to simulate a bone-like layer, interconnected with a silk fibroin layer to mimic cartilage, thus replicating the cartilage-like layer to mimic the native osteochondral tissue architecture. The scaffolds were produced by freeze-drying and then crosslinking with genipin. They have a crosslinking degree of up to 24%, which promotes a structural rearrangement and improved connection between the different layers. Micro-CT analysis demonstrated that the structures have distinct porosity values on their top layer (up to 84%), interface (up to 65%), and bottom layer (up to 77%) and are dependent on the concentration of β-tricalcium phosphate used. Both layers were confirmed to be clearly defined by the distribution of the components throughout the constructs, showing adequate mechanical properties for biomedical use. The scaffolds exhibited lower weight loss (up to 7%, 15 days) after enzymatic degradation due to the combined effects of genipin crosslinking and β-TCP incorporation. In vitro studies showed that the constructs supported ATDC5 chondrocyte-like cells and MC3T3 osteoblast-like cells in duo culture conditions, providing a suitable environment for cell adhesion and proliferation for up to 14 days. Overall, the physicochemical properties and biological results of the developed chitosan/β-tricalcium phosphate/silk fibroin bilayered scaffolds suggest that they may be potential candidates for osteochondral tissue strategies.

Genipin (GP) as a natural crosslinker has gained much attention in the food industry on account of its high biocompatibility and safety. In this paper, chitosan (CS)‐soy protein isolate (SPI) crosslinked films were prepared using GP as a crosslinking agent. Crosslinking reaction with high contents of GP (0.03%, 0.05%) changed the rheological property of the film matrixes. The crosslinking degree was positively correlated with the content of GP, which was from 21.08 ± 3.95% of CS‐SPI‐1 to 39.66 ± 1.62% of CS‐SPI‐5. Fourier transform infrared spectroscopy spectra confirmed the crosslinking reaction. The X‐ray diffraction and scanning electron microscopy analyses indicated that the crosslinking reaction decreased the crystallinity degree and enhanced the surface roughness of CS‐SPI film, respectively. The crosslinking reaction did not significantly affect the thickness, moisture content and water solubility of CS‐SPI film. Crosslinked films presented better light barrier capacity. The tensile strength (TS) of CS‐SPI‐1 was increased by 50%, however, further increasing the contents of GP (0.03%, 0.05%) reduced the TS of the crosslinked films. With the increasing incorporation with GP, the EAB was significantly decreased, while the modulus increased from 95.76 ± 25.78 to 508.78 ± 77.95 MPa. The crosslinking reaction improved the rigidity of CS‐SPI film. Low crosslinking degree could improve thermal stability of CS‐SPI, while higher GP addition (0.03%, 0.05%) decreased the thermal stability and prolonged the degradation time of the film in soil. The results indicated that low crosslinking reaction could improve the property of CS‐SPI film, enhancing its potential application in food packaging. [ABSTRACT FROM AUTHOR]

Research on the use of polysaccharides as hydrophobic bioactive carriers instead of proteins is still scarce. Sugar beet pectin (SBP) contains a small amount of protein and is a potential carrier for loading curcumin. In this work, SBP encapsulation, genipin crosslinking, and laccase-induced gelation were used to develop novel jelly food and improve the stability of curcumin without the incorporation of oil. By mixing the SBP solution (40 mg/mL) with curcumin powder (25 mg/mL SBP solution), an SBP–curcumin complex (SBP–Cur) was fabricated with a loading amount of 32 mg/g SBP, and the solubility of curcumin improved 116,000-fold. Fluorescence spectroscopy revealed that hydrophobic interactions drove the complexation of curcumin and SBP. Crosslinked by genipin (10 mM), SBP–Cur showed a dark blue color, and the gel strength of laccase-catalyzed gels was enhanced. Heating and UV radiation tests suggested that the genipin crosslinking and gelation strategies substantially improved the stability of curcumin. Because of the unique UV-blocking capacity of blue pigment, crosslinked samples retained 20% more curcumin than control samples. With the enhanced stability of curcumin, the crosslinked SBP–curcumin complexes could be a functional food ingredient used in functional drinks, baked food, and jelly food. [ABSTRACT FROM AUTHOR]