Comparative transcriptomic analysis primarily explores the molecular mechanism of compound eye formation in Neocaridina denticulata sinensis.

Compound eyes formation in decapod crustaceans occurs after the nauplius stage. However, the key genes and regulatory mechanisms of compound eye development during crustacean embryonic development have not yet been clarified. In this study, RNA-seq was used to investigate the gene expression profiles of Neocaridina denticulata sinensis from nauplius to zoea stage. Based on RNA-seq data analysis, the phototransduction and insect hormone biosynthesis pathways were enriched, and molting-related neuropeptides were highly expressed. There was strong cell proliferation in the embryo prior to compound eye development. The formation of the visual system and the hormonal regulation of hatching were the dominant biological events during compound eye development. The functional analysis of DEGs across all four developmental stages showed that cuticle formation, muscle growth and the establishment of immune system occurred from nauplius to zoea stage. Key genes related to eye development were discovered, including those involved in the determination and differentiation of the eye field, eye-color formation, and visual signal transduction. In conclusion, the results increase the understanding of the molecular mechanism of eye formation in crustacean embryonic stage.

Elucidating the salt-tolerant mechanism of Halomonas cupida J9 and unsterile ectoine production from lignocellulosic biomass.

BACKGROUND: Ectoine as an amino acid derivative is widely applied in many fields, such as the food industry, cosmetic manufacturing, biologics, and therapeutic agent. Large-scale production of ectoine is mainly restricted by the cost of fermentation substrates (e.g., carbon sources) and sterilization. RESULTS: In this study, Halomonas cupida J9 was shown to be capable of synthesizing ectoine using xylose as the sole carbon source. A pathway was proposed in H. cupida J9 that synergistically utilizes both WBG xylose metabolism and EMP glucose metabolism for the synthesis of ectoine. Transcriptome analysis indicated that expression of ectoine biosynthesis module was enhanced under salt stress. Ectoine production by H. cupida J9 was enhanced by improving the expression of ectoine biosynthesis module, increasing the intracellular supply of the precursor oxaloacetate, and utilizing urea as the nitrogen source. The constructed J9U-P8EC achieved a record ectoine production of 4.12 g/L after 60 h of xylose fermentation. Finally, unsterile production of ectoine by J9U-P8EC from either a glucose-xylose mixture or corn straw hydrolysate was demonstrated, with an output of 8.55 g/L and 1.30 g/L of ectoine, respectively. CONCLUSIONS: This study created a promising H. cupida J9-based cell factory for low-cost production of ectoine. Our results highlight the potential of J9U-P8EC to utilize lignocellulose-rich agriculture waste for open production of ectoine.

A Rationally Designed CRISPR/Cas12a Assay Using a Multimodal Reporter for Various Readouts.

The CRISPR/Cas systems offer a programmable platform for nucleic acid detection, and CRISPR/Cas-based diagnostics (CRISPR-Dx) have demonstrated the ability to target nucleic acids with greater accuracy and flexibility. However, due to the configuration of the reporter and the underlying labeling mechanism, almost all reported CRISPR-Dx rely on a single-option readout, resulting in limitations in end-point result readouts. This is also associated with high reagent consumption and delays in diagnostic reports due to protocol differences. Herein, we report for the first time a rationally designed Cas12a-based multimodal universal reporter (CAMURE) with improved sensitivity that harnesses a dual-mode reporting system, facilitating options in end-point readouts. Through systematic configurations and optimizations, our novel universal reporter achieved a 10-fold sensitivity enhancement compared to the DETECTR reporter. Our unique and versatile reporter could be paired with various readouts, conveying the same diagnostic results. We applied our novel reporter for the detection of staphylococcal enterotoxin A due to its high implication in staphylococcal food poisoning. Integrated with loop-mediated isothermal amplification, our multimodal reporter achieved 10 CFU/mL sensitivity and excellent specificity using a real-time fluorimeter, in-tube fluorescence, and lateral flow strip readouts. We also propose, using artificially contaminated milk samples, a fast (2-5 min) Triton X-100 DNA extraction approach with a comparable yield to the commercial extraction kit. Our CAMURE could be leveraged to detect all gene-encoding SEs by simply reprogramming the guide RNA and could also be applied to the detection of other infections and disease biomarkers.

High-efficiency fluorescent coordination polymer nanoparticles co-doped with Ce3+/Tb3+ ions for curcumin detection.

Curcumin (Cur) possesses diverse biological and pharmacologic effects. It is widely used as a food additive and therapeutic medicine. A study to determine a sensitive detection method for Cur is necessary and meaningful. In this work, double rare earth ions co-doped fluorescent coordination polymer nanoparticles (CPNPs) were developed for the Cur detection. The CPNPs were synthesized by using adenosine monophosphate (AMP) as bridge ligands via coordination self-assembly with Ce3+ and Tb3+. The AMP-Ce/Tb CPNPs exhibited the characteristic green fluorescence of Tb3+ and had high luminescence efficiency. Under the optimal conditions, the fluorescence intensity of AMP-Ce/Tb CPNPs could be significantly quenched by Cur. The fluorescence quenching extent at λex/λem of 300 nm/544 nm showed a good linear relationship with the Cur concentration in the range of 10 to 1000 nM. The detection limit was as low as 8.0 nM (S/N = 3). This method was successfully applied to the determination of Cur in real samples with satisfactory results. The luminescence mechanism of AMP-Ce/Tb CPNPs and the fluorescence quenching mechanism of the CPNPs by Cur were both examined.

Ovarian transcriptome and metabolic responses of RNAi-mediated farnesyl pyrophosphate synthase knockdown in Neocaridina denticulata sinensis.

Methyl farnesoate (MF) is considered the equivalent of JH in crustaceans and plays an essential role in many crucial physiological processes. It is believed that farnesyl pyrophosphate synthase (FPPS) plays an essential role in the biosynthesis of mevalonate, which is a branch of the JH/MF pathway. The full-length cDNA of FPPS (NdFPPS) from Neocaridina denticulata sinensis was isolated and characterized, and the deduced amino acid of NdFPPS contained a polyprenyl_synt domain. In addition to its ubiquitous tissue expression, NdFPPS was significantly expressed in the ovary. In vivo gene silencing with dsRNA interference was performed to further investigate the function of NdFPPS. An ovarian transcriptomic analysis of dsNdFPPS experimental and control groups was used to compare, annotate, and classify differentially expressed genes (DEGs). A total of 9230 DEGs were identified in the experimental and control groups based on FPKM values, of which 5082 were up-regulated genes and 4148 were down-regulated genes. 761 GO terms and 102 KEGG pathways were enriched for the DEGs. Our results suggest that NdFPPS might play an important role in ovarian development, however, further functional study is needed to elucidate physiological role of NdFPPS in reproduction.

Spectroelectrochemistry with Ultrathin Ion-Selective Membranes: Three Distinct Ranges for Analytical Sensing.

We present spectroelectrochemical sensing of the potassium ion (K+) at three very distinct analytical ranges─nanomolar, micromolar, and millimolar─when using the same ion-selective electrode (ISE) but interrogated under various regimes. The ISE is conceived in the all-solid-state format: an ITO glass modified with the conducting polymer poly(3-octylethiophene) (POT) and an ultrathin potassium-selective membrane. The experimental setup is designed to apply a potential in a three-electrode electrochemical cell with the ISE as the working electrode, while dynamic spectral changes in the POT film are simultaneously registered. The POT film is gradually oxidized to POT+, and this process is ultimately linked to K+ transfer at the membrane-sample interface, attending to electroneutrality requirements. The spectroelectrochemistry experiment provides two signals: a voltammetric peak and a transient absorbance response, with the latter of special interest because of its correspondence with the generated charge in the POT and thus with the ionic charge expelled from the membrane. By modifying how the ion analyte (K+ but also others) is initially accumulated into the membrane, we found three ranges of response for the absorbance: 10-950 nM for an accumulation-stripping protocol, 0.5-10 µM in diffusion-controlled cyclic voltammetry, and 0.5-32 mM with thin-layer cyclic voltammetry. This wide response range is a unique feature, one that is rare to find for a sensor and indeed for any analytical technique. Accordingly, the developed sensor is highly appealing for many analytical applications, especially considering the versatility of samples and ion analytes that may be spotted.

Endotoxin tolerance induced by Porphyromonas gingivalis lipopolysaccharide alters macrophage polarization.

Endotoxin tolerance refers to a state refractory to subsequent lipopolysaccharide (LPS) stimulations following a primary LPS exposure. To study the relationship between endotoxin tolerance and macrophage polarization, endotoxin tolerance was induced by 1 µg/mL LPS from the periodontal pathogen, Porphyromonas gingivalis (P. gingivalis), in peritoneal macrophages (PMs) and bone marrow-derived macrophages (BMDMs). Repeated P. gingivalis LPS challenges increased the quantities of CD206+ PMs, while the number of CD86+CD206+ PMs was reduced compared with the non-tolerant group (p < 0.05). However, there were no changes in BMDMs (p > 0.05). Down regulations of TNF-α, IL-12, nitric oxide and MMP-2 production, and upregulated IL-10, MMP-9 levels and arginase-1 activities occurred in tolerant PMs and BMDMs (p < 0.05). P. gingivalis LPS-tolerant PMs and BMDMs also enhanced scrape-wound healing abilities of 15p-1 cells (p < 0.05). Expressions of phospho-signal transducer and activator of transcription 6 (p-STAT6) and protein tyrosine phosphatase 1B (PTP1B) were increased, while p-MEK1/2 levels were downregulated in tolerant PMs and BMDMs (p < 0.05). IL-10 production in tolerant Stat6 knockdown RAW264.7 cells was lower than tolerant control cells (p < 0.05). P. gingivalis LPS-tolerant macrophages represented an intermediate state between M1/M2 polarization, which functioned as M2-like cells, and led to limited inflammatory responses and enhanced wound healing activities. The PTP1B-MEK1/2-STAT6 signaling pathway might be involved in the polarization of tolerant macrophages.

Recombinase polymerase amplification assay for rapid detection of Seneca Valley Virus.

Seneca Valley virus (SVV) is related to vesicular disease in pigs, and its clinical symptoms are indistinguishable from other notifiable clinical symptoms of vesicular disease such as foot-and-mouth disease. The rapid and accurate detection of SVV is essential to confirm the pathogenic factors and initiate the implementation of control measures. The development of a rapid, simple, convenient, and low-cost molecular (nucleic acid amplification) test that can be used at the sample collection point has been identified as a key component for controlling SVV. This study describes the development and demonstration of recombinase polymerase amplification (RPA) test targeting the conserved regions of SVV for detection of SVV. The Primers and probes designed by us have shown good sensitivity and specificity in RPA test, which is helpful for RPA to be an effective tool for rapid diagnosis of SVV.

RNA-seq analysis revealing the immune response of Neocaridina denticulata sinensis gill to Vibrio parahaemolyticus infection.

Vibrio parahaemolyticus causes serious economic losses to the shrimp farming industry. There is still a lack of adequate understanding of the changes in the overall response of N. denticulata sinensis caused by V. parahaemolyticus, particularly with respect to gill tissue, which is severely damaged by the pathogen. In this study, a total of 1358 differentially expressed genes were identified between the PBS control and Vibrio stimulation groups using transcriptome sequencing techniques. After further screening and analysis, many immune-related genes were obtained, involving lysosome pathway, metabolic process, chitin-binding protein, and serine protease family members. In addition, we randomly selected six DEGs in the lysosome pathway for qRT-PCR verification, and the results showed that their expression patterns were consistent with the RNA-seq. The results demonstrate the molecular regulation of the gill tissue response to V. parahaemolyticus infection in N. denticulata sinensis, contributing to the understand of the complex and efficient innate immune system and defense mechanisms in crustaceans.

Transcriptome analysis of Neocaridina denticulate sinensis challenged by Vibrio parahemolyticus.

As a common aquatic pathogen, Vibrio parahaemolyticus can cause a variety of diseases of shrimp, especially acute hepatopancreatic necrosis disease (AHPND), which leads to great losses to the aquaculture industry around the world. However, the molecular mechanism of V. parahaemolyticus infection is still unclear. Neocaridina denticulate sinensis is a kind of small ornamental shrimp that is popular in aquarium trade, and due to its tenacious vitality, rapid growth, high reproductive capacity, it is very suitable to be developed as an animal model for basic research on decapod crustaceans. Thus, in this paper, transcriptomes of N. denticulate sinensis hepatopancreas with or without V. parahaemolyticus injection were explored. The results showed that a total of 23,624 genes with the N50 of 2705 bp were obtained. Comparative transcriptomic analysis revealed 21,464 differentially expressed genes between the V. parahaemolyticus infected and non-infected group, of which, 11,127 genes were up-regulated and 10,337 genes were down-regulated. Functional enrichment analysis suggested that many DEGs enriched in immune related pathways, including MAPK signaling pathway, Phosphatidylinositol signaling system, Chemokine signaling pathway, Phagosome and Jak-STAT signaling pathway and so on. Eight genes were selected randomly for qRT-PCR to verify the transcriptome sequencing results and the results showed the expression of these genes were consistent with the transcriptome results. Our work provides a unique and important dataset that contributes to the understanding of the molecular mechanisms of the immune response to V. parahaemolyticus infection and may further provide the basis for the prevention and resolution of bacterial diseases.

Transcriptomic analysis of Neocaridina denticulate sinensis hepatopancreas indicates immune changes after copper exposure.

Neocaridina denticulate sinensis is a promising crustacean model species due to its merits in raising and breeding. However, its molecular responses to copper remains largely unknown. In the present research, RNA-seq was used to mine the alteration in transcriptome of N. denticulate sinensis hepatopancreas under copper exposure. A total of 16,423 DEGs was identified between control and Cu2+ treatment groups. GO enrichment analysis of all DEGs suggested down-regulated genes exceeded up-regulated genes in all the significantly enriched terms, except for RNA polymerase III complex (GO:0005666). KEGG analysis showed Cu exposure only induced two significantly enriched pathways, including Phagosome (ko04145) and Pathogenic Escherichia coli infection (ko05130). Besides, pattern recognition receptors as Toll, lectin B, CTL1 and SRB, AMPs as crustin type I, lysozyme, and NOS were down-regulated after Cu2+ exposure, while hemocyanin, MT, HSP70 and HSP90 were significantly up-regulated, implying these molecules may play vital role in Cu2+ detoxification of N. denticulate sinensis. Our results here provide research direction of heavy metal detoxification of N. denticulate sinensis, simultaneously enriched its genomic information.

An extracellular Cu/Zn superoxide dismutase from Neocaridina denticulata sinensis: cDNA cloning, mRNA expression and characterizations of recombinant protein.

Neocaridina denticulata sinensis possesses characters of rapid growth, tenacious vitality, short growth cycle, transparent, and easy feeding. Therefore, it is gradually being developed into an animal model for basic research on decapod crustaceans. Herein, a Cu/Zn superoxide dismutase (Cu/Zn-SOD), named as Nd-ecCu/Zn-SOD, was identified and characterized from N. denticulata sinensis. The full-length cDNA sequence of Nd-ecCu/Zn-SOD is 829 bp containing a 684 bp open reading frame, which encodes a protein of 227 amino acid residues with a typical Sod_Cu domain. The quantitative real-time PCR analysis showed that Nd-ecCu/Zn-SOD mRNA was expressed in all the tested tissues. Under challenge with copper, the mRNA expression of Nd-ecCu/Zn-SOD reached the maximum at 6 h, and decreased until 24 h. After 24 h of exposure, its expression was up-regulated significantly at 36 h. After then its expression sharply decreased with a comeback at 48 h. The result indicated that Nd-ecCu/Zn-SOD might play an important role in the stress response of N. denticulata sinensis. The expression of Nd-ecCu/Zn-SOD in gills challenged with Vibrio parahaemolyticus changed in a time-dependent manner. Nd-ecCu/Zn-SOD was lowly expressed in early developmental stages by RNA-Seq technology, yet it showed that a cyclical rise and fall occurred between middle stages and late stages. In addition, Nd-ecCu/Zn-SOD was recombinantly expressed using E. coli and the recombinant protein was purified as a single band on SDS-PAGE. The recombinant Nd-ecCu/Zn-SOD (rNd-ecCu/Zn-SOD) existed enzymatic activity under a wide range of temperature and pH. The exposure of metal ions was found that Zn2+, Mg2+, Ca2+, Ba2+, and Cu2+ could inhibit the enzymatic activity of rNd-ecCu/Zn-SOD, and Mn2+ increased the enzymatic activity of rNd-ecCu/Zn-SOD. These results indicate that Nd-ecCu/Zn-SOD may play a pivotal role in resistant against oxidative damage and act as a biomarker under stressful environment.

Anti-Infective and Pro-Coagulant Chitosan-Based Hydrogel Tissue Adhesive for Sutureless Wound Closure.

Multifunctional tissue adhesives with excellent adhesion, antibleeding, anti-infection, and wound healing properties are desperately needed in clinical surgery. However, the successful development of multifunctional tissue adhesives that simultaneously possess all these properties remains a challenge. We have prepared a novel chitosan-based hydrogel adhesive by integration of hydrocaffeic acid-modified chitosan (CS-HA) with hydrophobically modified chitosan lactate (hmCS lactate) and characterized its gelation time, mechanical properties, and microstructure. Tissue adhesion properties were evaluated using both pigskin and intestine models. In situ antibleeding efficacy was demonstrated via the rat hemorrhaging liver and full-thickness wound closure models. Good antibacterial activity and anti-infection capability toward S. aureus and P. aeruginosa were confirmed using in vitro contact-killing assays and an infected pigskin model. The result of coculturing with 3T3 fibroblast cells indicated that the hydrogels have no significant cytotoxicity. Most importantly, the biocompatible and biodegradable CS-HA/hmCS lactate hydrogel was able to close the wound in a sutureless way and promote wound healing. Our results demonstrate that this hydrogel has great promise for sutureless closure of surgical incisions.

Optimization and Evaluation of the Thermosensitive In Situ and Adhesive Gel for Rectal Delivery of Budesonide.

Budesonide is a glucocorticoid for the treatment of ulcerative colitis (UC). The current study aims to develop a thermosensitive in situ and adhesive gel for rectal delivery of budesonide. HPMC K4M was selected as the adhesive agent based on the adhesive force and the effect on gel performance. The formulation of gel was optimized by using the central composite design-response surface methodology (CCD-RSM); a mathematical model was successfully developed to predict desired formulations as well as to analyze relationships between the amount of Pluronic F-127, Pluronic F-68, and HPMC K4M and the performances of gel. Based on CCD-RSM, a thermosensitive in situ and adhesive gel consisting of 0.002% budesonide, 0.74% HPMC, 4.87% F-68, and 19.0% F-127 was developed. Furthermore, the in vivo behavior of gel was evaluated in Sprague-Dawley rats. In comparison with budesonide solution, rectal administration of budesonide gel at 0.1 mg/kg in rats showed relative bioavailability of 230% with significant increase in rectum uptake.

Delivering siRNA and Chemotherapeutic Molecules Across BBB and BTB for Intracranial Glioblastoma Therapy.

For aggressive brain glioblastoma, the therapy is significantly impaired by blood-brain barrier (BBB) and blood-tumor barrier (BTB). Choosing more than one target from the pool of tumor-stroma interactions is profoundly beneficial to therapeutic approaches. Thus, a multifunctional liposomal system based on anchoring two receptor-specific and penetrable peptides was designed for the combination delivery of BBB-impermeable siRNA and chemotherapeutic docetaxel to brain glioblastoma. Both macroscopic and microscopic specific distributions and targeting effect of the liposomes in the intracranial glioblastoma were confirmed. Superiority in therapeutic efficacies of the siRNA and DTX combination delivery system was revealed from encouraged VEGF gene silencing, tumor cell apoptosis, prolonged survival time, subdued glioblastoma cells in intracranial glioblastoma, and negligible system toxicities after systemic application. Furthermore, the liposomes made better modulation of glioblastoma microenvironment such as the down-regulation of CD31-positive tumor vessels and HIF-1α expression. The transport mechanism of the liposomes delivering the cargos across BBB via receptor-mediated transcytosis without destroying the integrity of BBB has been evaluated from in vitro and in vivo. Therefore, the dual peptides-modified liposomal system provides a safe and noninvasive approach for the delivery of siRNA and chemotherapeutic molecules across the BBB and BTB to target therapy of brain glioblastoma.

Kapok Fiber: A Natural Biomaterial for Highly Specific and Efficient Enrichment of Sialoglycopeptides.

Cancer development and chronic diseases are associated with the overexpression of sialoglycans terminated to the surface proteins and lipids of cancer cells compared with normal cells. The isolation and detection of sialoglycopeptides from complex peptides mixture still remain challenges due to their low abundance, low ionization, and losses of sialic acid residues and water molecule during analytical processes. In this study, kapok fiber, a natural fiber derived from the kapok tree (Bombax ceiba L.), has shown excellent capability to specifically and efficiently enrich sialoglycopeptides, without losses of sialic acid residues and water molecule from sialoglycans. The main components on the surface of kapok fiber are syringyl and guaiacyl lignins which play an important role in isolating sialoglycopeptides from complex peptide mixtures.

Anti-adhesive and antibacterial chitosan/PEO nanofiber dressings with high breathability for promoting wound healing.

Wound dressings are crucial for wound healing. Ideal wound dressings should possess many functions such as wettability, antibacterial activity and anti-adherent property to promote wound healing. In the present study solution blown spinning (SBS) technology was applied to prepare chitosan/polyethylene oxide (CS/PEO) nanofiber dressings in high efficiency. The obtained nanofiber dressings were treated with anhydrous ethanol to improve the fiber structure and enhance the functionality of the fiber dressings. The results show that the treated nanofibers had higher crystallinities and higher CS contents. The CS/PEO nanofiber dressings fabricated by using no additives and crosslinking had excellent wettability, water stability and antibacterial activity against Escherichia coli and Staphylococcus aureus reached to over 99.99 %. In addition, the CS/PEO nanofiber dressings exhibited high breathability, antioxidant activity and anti-adhesion function. The in vivo animal experiment confirmed that the nanofiber dressings enhanced cell proliferation and significantly accelerated the wound healing within 10 days. The developed CS/PEO nanofiber dressings have great potential in the clinical field of wound healing.

Membrane-bound trehalase enhances cadmium tolerance by regulating cell apoptosis in Neocaridina denticulata sinensis.

Trehalase gene is mainly expressed in the digestive circulatory system for regulating energy metabolism and chitin synthesis in insects, but it is significantly expressed in gill for immunomodulation in shrimp. However, its function in regulating immunity, particularly metal resistance in crustaceans has yet to be elucidated. In this study, one Tre2 gene (NdTre2) was isolated from Neocaridina denticulata sinensis. It could bind to Cd2+ and inhibit its toxicity. Spatiotemporal expression analysis showed that the expression of NdTre2 was highest in the gill and significantly reduced at 12 h after Cd2+ stimulation. The transcriptomic analysis of the gill after NdTre2 knockdown showed that the expression of genes synthetizing 20E was up-regulated and the increased 20E could further induce apoptosis by activating the intrinsic mitochondrial pathway, exogenous death receptor-ligand pathway, and MAPK pathway. In vitro, overexpressing NdTre2 enhanced the tolerance of E. coli in Cd2+ environment. In summary, these results indicate that NdTre2 plays an essential role in regulating immunity and chitin metabolism in N. denticulata sinensis.

Physiological and genetic responses of the benthic dinoflagellate Prorocentrum lima to polystyrene microplastics.

Microplastics are well known as contaminants in marine environments. With the development of biofilms, most microplastics will eventually sink and deposit in benthic environment. However, little research has been done on benthic toxic dinoflagellates, and the effects of microplastics on benthic dinoflagellates are unknown. Prorocentrum lima is a cosmopolitan toxic benthic dinoflagellate, which can produce a range of polyether metabolites, such as diarrhetic shellfish poisoning (DSP) toxins. In order to explore the impact of microplastics on marine benthic dinoflagellates, in this paper, we studied the effects of polystyrene (PS) on the growth and toxin production of P. lima. The molecular response of P. lima to microplastic stress was analyzed by transcriptomics. We selected 100 nm, 10 µm and 100 µm PS, and set three concentrations of 1 mg L-1, 10 mg L-1 and 100 mg L-1. The results showed that PS exposure had limited effects on cell growth, but increased the OA and extracellular polysaccharide content at high concentrations. After exposure to PS MPs, genes associated with DSP toxins synthesis, carbohydrate synthesis and energy metabolism, such as glycolysis, TCA cycle and pyruvate metabolism, were significantly up-regulated. We speculated that after exposure to microplastics, P. lima may increase the synthesis of DSP toxins and extracellular polysaccharides, improve the level of energy metabolism and gene expression of ABC transporter, thereby protecting algal cells from damage. Our findings provide new insights into the effects of microplastics on toxic benthic dinoflagellates.

Enhancement of hepatoprotective activity of limonin from citrus seeds against acetaminophen-induced liver injury by HSCCC purification and liposomal encapsulation.

Limonin is a natural tetracyclic triterpenoid compound in citrus seeds that presents hepatoprotective effects but is often discarded as agricultural waste because of its low content and low solubility. Herein, limonin with high purity (98.11%) from citrus seeds was obtained via purification by high-speed counter-current chromatography (HSCCC) and recrystallization. Limonin-loaded liposomes (Lip-LM) prepared by thin film hydration and high pressure homogenization method to enhance its solubility and hepatoprotective effect on APAP-induced liver injury (AILI). Lip-LM appeared as lipid nanoparticles under a transmission electron microscope, and showed well dispersed nano-scale size (69.04 ± 0.42 nm), high encapsulation efficiency (93.67% ± 2.51%), sustained release, fine stability. Lip-LM also exhibited significantly better hepatoprotective activity on AILI than free limonin in vivo. In summary, Lip-LM might be used as a potential hepatoprotective agent in the form of dietary supplement and provide an effective strategy to improve the potential value of citrus seeds.