The protective effect of vitexin on hypertensive nephropathy rats.

INTRODUCTION: Vitexin is a natural flavonoid compound extracted from Vitex leaves or seeds, exhibiting various pharmacological activities including anticancer, antihypertensive, anti-inflammatory, and spasmolytic effects. However, its protective effects on hypertensive nephropathy (HN) and the underlying mechanisms remain unclear. METHODS: Spontaneous hypertension rats were fed a high-sugar and high-fat diet for 8 weeks to induce the disease HN model. From the 5th week, the rats were administered vitexin via gavage. Blood pressure was measured bi-weekly using the tail-cuff method. Histopathological changes were assessed using HE staining, and biochemical analyses were performed to evaluate the effects of vitexin on HN rats. The underlying mechanisms of vitexin treatment were investigated through western blotting. RESULTS: The data demonstrated that vitexin significantly lowered systolic, diastolic, and mean arterial pressures, and ameliorated histopathological changes in HN rats. Biochemical analyses revealed that vitexin reduced the levels of creatinine (Cr), blood urea nitrogen (BUN), total cholesterol (TC), triglycerides (TG), total protein (TP), low-density lipoprotein cholesterol (LDL-C), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), malondialdehyde (MDA), and advanced glycation end products (AGEs), while increasing the levels of albumin (ALB) and superoxide dismutase (SOD). Western blotting results indicated that vitexin treatment decreased the expression of TNF-α, IL-6, and nuclear factor kappa-B (NF-κB), while increasing the expression of SOD. CONCLUSION: The findings of this study suggest that vitexin exerts protective effects against HN, providing pharmacological evidence for its potential use in HN treatment.

A chemo-selective enrichment strategy to achieve in-depth coverage of methyllysine proteome.

Proteomics is a powerful method to comprehensively understand cellular posttranslational modifications (PTMs). Due to low abundance, tryptic peptides with PTMs are usually enriched for enhanced coverage by LC-MS/MS. Affinity chromatography for phosphoproteomes by metal-oxide and pan-specific antibodies for lysine acetylome allow identification of tens of thousands of modification sites. Lysine methylation is a significant PTM, however, only hundreds of methylation sites were identified from available approaches. Here we report an aryl diazonium-based chemoselective strategy that enables enrichment of monomethyllysine (Kme1) peptides via covalent bond with extraordinary sensitivity. We identified more than ten thousand Kme1 peptides from diverse cell lines and mouse tissues, that implied wide lysine methylation impact on cellular processes. In addition, we found a significant amount of methyl marks that were not S-adenosyl methionine (SAM)-dependent by isotope labeling experiments. And therefore, this method paves a way to broad application in lysine methylation research and new biology discovery.

Chemical O-sulfation of N-sulfoheparosan: a route to rare N-sulfo-3-O-sulfoglucosamine and 2-O-sulfoglucuronic acid.

Heparosan, the capsular polysaccharide of E. coli K5 is currently used as the starting material in the chemoenzymatic synthesis of heparan sulfate and the structurally related anticoagulant drug heparin. Base hydrolysis of N-acetyl groups and their subsequent N-sulfonation, are used to prepare N-sulfoheparosan an intermediate of biosynthesis. In the present study, when excess sulfonation reagent was used during N-sulfonation, some O-sulfation also took place in the N-sulfoheparosan product. After a nearly full digestion, a hexasaccharide fraction exhibited resistance to heparin lyase II. Excessive digestion by heparin lyase II and structural identification by NMR and mass spectroscopy indicated that the resistant hexasaccharide fraction has two structures, ΔUA-GlcNS-GlcA2S-GlcNS-GlcA-GlcNS and ΔUA-GlcNS-GlcA- GlcNS3S-GlcA-GlcNS in similar amounts. The 2-sulfated structure exhibited partial resistance to heparin lyase II; however the structure of ΔUA-GlcNS-GlcA-GlcNS3S was completely resistant to heparin lyase II.

Bottom-up analysis using liquid chromatography-Fourier transform mass spectrometry to characterize fucosylated chondroitin sulfates from sea cucumbers.

Fucosylated chondroitin sulfates (FCSs) from sea cucumbers have repetitive structures that exhibit minor structural differences based on the organism from which they are recovered. A detailed characterization of FCSs and their derivatives is important to establish their structure-activity relationship in the development of new anticoagulant drugs. In the current study, online hydrophilic interaction chromatography-Fourier transform mass spectrometry (FTMS) was applied to analyze the FCS oligosaccharides generated by selective degradation from four species of sea cucumbers, Isostichopus badionotus, Pearsonothuria graeffei, Holothuria mexicana and Acaudina molpadioides. These depolymerized FCS fragments were quantified and compared using the glycomics software package, GlycReSoft. The quantified fragments mainly had trisaccharide-repeating compositions and showed significant differences in fucosylation (including its sulfation) among different species of sea cucumbers. Detailed analysis of FTMS ion peaks and top-down nuclear magnetic resonance spectroscopy of native FCS polysaccharides verified the accuracy of this method. Thus, a new structural model for FCS chains from these different sea cucumbers was defined. This bottom-up approach provides rich detailed structural analysis and provides quantitative information with high accuracy and reproducibility and should be suitable for the quality control in FCSs as well as their oligosaccharides.

Structural elucidation of fucosylated chondroitin sulfates from sea cucumber using FTICR-MS/MS.

Fucosylated chondroitin sulfates are complex polysaccharides extracted from sea cucumber. They have been extensively studied for their anticoagulant properties and have been implicated in other biological activities. While nuclear magnetic resonance spectroscopy has been used to extensively characterize fucosylated chondroitin sulfate oligomers, we herein report the first detailed mass characterization of fucosylated chondroitin sulfate using high-resolution Fourier transform ion cyclotron resonance mass spectrometry. The two species of fucosylated chondroitin sulfates considered for this work include Pearsonothuria graeffei (FCS-Pg) and Isostichopus badionotus (FCS-Ib). Fucosylated chondroitin sulfate oligosaccharides were prepared by N-deacetylation-deaminative cleavage of the two fucosylated chondroitin sulfates and purified by repeated gel filtration. Accurate mass measurements obtained from electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry measurements confirmed the oligomeric nature of these two fucosylated chondroitin sulfate oligosaccharides with each trisaccharide repeating unit averaging four sulfates per trisaccharide. Collision-induced dissociation of efficiently deprotonated molecular ions through Na/H+ exchange proved useful in providing structurally relevant glycosidic and cross-ring product ions, capable of assigning the sulfate modifications on the fucosylated chondroitin sulfate oligomers. Careful examination of the tandem mass spectrometry of both species deferring in the positions of sulfate groups on the fucose residue (FCS-Pg-3,4- OS) and (FCS-Ib-2,4- OS) revealed cross-ring products 0,2Aαf and 2,4X2αf which were diagnostic for (FCS-Pg-3,4- OS) and 0,2X2αf diagnostic for (FCS-Ib-2,4- OS). Mass spectrometry and tandem mass spectrometry data acquired for both species varying in oligomer length (dp3-dp15) are presented.

Depolymerization of Fucosylated Chondroitin Sulfate with a Modified Fenton-System and Anticoagulant Activity of the Resulting Fragments.

Fucosylated chondroitin sulfate (fCS) from sea cucumber Isostichopus badionotus (fCS-Ib) with a chondroitin sulfate type E (CSE) backbone and 2,4-O-sulfo fucose branches has shown excellent anticoagulant activity although has also show severe adverse effects. Depolymerization represents an effective method to diminish this polysaccharide's side effects. The present study reports a modified controlled Fenton system for degradation of fCS-Ib and the anticoagulant activity of the resulting fragments. Monosaccharides and nuclear magnetic resonance (NMR) analysis of the resulting fragments indicate that no significant chemical changes in the backbone of fCS-Ib and no loss of sulfate groups take place during depolymerization. A reduction in the molecular weight of fCS-Ib should result in a dramatic decrease in prolonging activated partial thromboplastin time and thrombin time. A decrease in the inhibition of thrombin (FIIa) by antithromin III (AT III) and heparin cofactor II (HCII), and the slight decrease of the inhibition of factor X activity, results in a significant increase of anti-factor Xa (FXa)/anti-FIIa activity ratio. The modified free-radical depolymerization method enables preparation of glycosaminoglycan (GAG) oligosaccharides suitable for investigation of clinical anticoagulant application.

Proline-Rich Region II (PRR2) Plays an Important Role in Tau-Glycan Interaction: An NMR Study.

(1) Background: Prion-like transcellular spreading of tau pathology in Alzheimer's disease (AD) is mediated by tau binding to the cell-surface glycan heparan sulfate (HS). However, the structural determinants for tau-HS interaction are not well understood. (2) Methods and Results: Binding-site mapping using NMR showed two major binding regions in full-length tau responsible for heparin interaction. Thus, two tau constructs, tau PRR2* and tau R2*, were designed to investigate the molecular details at the tau-heparin binding interface. The 2D 1H-15N HSQC of tau PRR2* and tau R2* lacked dispersion, which is characteristic for intrinsically disordered proteins. NMR titration of Arixtra into 15N-labeled tau R2* induced large chemical shift perturbations (CSPs) in 275VQIINK280 and downstream residues K281-D283, in which L282 and I278 displayed the largest shifts. NMR titration of Arixtra into 15N-labeled tau PRR2* induced the largest CSPs for residue R209 followed by residues S210 and R211. Residue-based CSP fitting showed that tau PRR2*-Arixtra interaction had a much stronger binding affinity (0.37-0.67 mM) than that of tau R2*-Arixtra (1.90-5.12 mM) interaction. (3) Conclusions: Our results suggested that PRR2 is a crucial domain for tau-heparin and tau-HS interaction.

Rhamnan sulfate reduces atherosclerotic plaque formation and vascular inflammation.

OBJECTIVE: While lipid-lowering drugs have become a mainstay of clinical therapy these treatments only slow the progression of the disease and can have side effects. Thus, new treatment options are needed to supplement the effects of lipid lowering therapy for treating atherosclerosis. We examined the use of an inexpensive and widely available marine polysaccharide rhamnan sulfate as an oral therapeutic for limiting vascular inflammation and atherosclerosis. METHODS AND RESULTS: We found rhamnan sulfate enhanced the barrier function of endothelial cells, preventing the deposition of LDL and maintaining barrier function even in the presence of glycocalyx-degrading enzymes. Rhamnan sulfate was also found to bind directly to FGF-2, PDGF-BB and NF-κB subunits with high affinity. In addition, rhamnan sulfate was a potent inhibitor of NF-κB pathway activation in endothelial cells by TNF-α. We treated ApoE-/- mice with a high fat diet for 4 weeks and then an addition 9 weeks of high fat diet with or without rhamnan sulfate. Rhamnan sulfate reduced vascular inflammation and atherosclerosis in both sexes of ApoE-/- mice but had a stronger therapeutic effect in female mice. Oral consumption of rhamnan sulfate induced a significant decrease in cholesterol plasma levels in female mice but not in male mice. In addition, there was a marked reduction in inflammation for female mice in the liver and aortic root in comparison to male mice. CONCLUSIONS: Rhamnan sulfate has beneficial effects in reducing inflammation, binding growth factors and NF-κB, enhancing endothelial barrier function and reducing atherosclerotic plaque formation in ApoE-/- mice.

Heparosan Chain Characterization: Sequential Depolymerization of E. Coli K5 Heparosan by a Bacterial Eliminase Heparin Lyase III and a Bacterial Hydrolase Heparanase Bp to Prepare Defined Oligomers.

Heparosan is a non-sulfated polysaccharide and potential applications include, chemoenzymatic synthesis of heparin and heparan sulfates. Heparosan is produced using microbial cells (natural producers or engineered cells). The characterization of heparosan isolated from both natural producers and engineered-cells are critical steps towards the potential applications of heparosan. Heparosan is characterized using 1) analysis of intact chain size and polydispersity, and 2) disaccharide composition. The current paper describes a novel method for heparosan chain characterization, using heparin lyase III (Hep-3, an eliminase from Flavobacterium heparinum) and heparanase Bp (Hep-Bp, a hydrolase from Burkholderia pseudomallei). The partial digestion of E. coli K5 heparosan with purified His-tagged Hep-3 results in oligomers of defined sizes. The oligomers (degree of polymerization from 2 to 8, DP2-DP8) are completely digested with purified GST-tagged Hep-Bp and analyzed using gel permeation chromatography. Hep-Bp specifically cleaves the linkage between d-glucuronic acid (GlcA) and N-acetyl-d-glucosamine (GlcNAc) but not the linkage between 4-deoxy-α-L-threo-hex-4-enopyranosyluronic acid (deltaUA) and GlcNAc, and results in the presence of a minor resistant trisaccharide (GlcNAc-GlcA-GlcNAc). This method successfully demonstrated the substrate selectivity of Hep-BP on heparosan oligomers. This analytical tool could be applied towards heparosan chain mapping and analysis of unnatural sugar moieties in the heparosan chain.

Oral Administration of Fucosylated Chondroitin Sulfate Oligomers in Gastro-Resistant Microcapsules Exhibits a Safe Antithrombotic Activity.

Fucosylated chondroitin sulfate (FCS) polysaccharide isolated from sea cucumber has potent anticoagulant activity. Based on its resistance to the enzymes present in vertebrates, it may serve as an anticoagulant and shows antithrombotic effects when delivered through gastro-resistant (GR) tablets. However, due to the multiple plasma targets of FCS polysaccharide in the coagulation pathway, bleeding can occur after its oral administration. In the current study, we used FCS oligomers, in particular a mixture of oligosaccharides having 6 to 18 saccharide units, as the active ingredient in GR microcapsules for oral anticoagulation. In a Caco-2 model, the FCS oligomers showed higher absorption than native FCS polysaccharides. Oral administration of FCS oligomer-GR microcapsules provided a dose-dependent, prolonged anticoagulant effect with a selective inhibition of the intrinsic coagulation pathway when compared with subcutaneous administration of FCS oligomers or oral administration of unformulated FCS oligomers or native FCS-GR microspheres. Continued oral administration of FCS oligomer-GR microcapsules did not result in the accumulation of oligosaccharides in the plasma. Venous thrombosis animal models demonstrated that FCS oligomers delivered via GR microcapsules produced a potent antithrombotic effect dependent on their anticoagulant properties in the plasma, while oral administration of unformulated FCS oligomers at the same dose exhibited a weaker antithrombotic effect than the formulated version. Oral administration of FCS oligomer-GR microcapsules resulted in no bleeding, while oral administration of native FCS-GR microcapsules resulted in bleeding (p < 0.05). Our present results suggest that a FCS oligomer-GR microcapsule formulation represents an effective and safe oral anticoagulant for potential clinical applications.

Heparan sulfates from bat and human lung and their binding to the spike protein of SARS-CoV-2 virus.

Severe acute respiratory syndrome-related coronavirus-2 (SARS-CoV-2) has resulted in a pandemic and continues to spread at an unprecedented rate around the world. Although a vaccine has recently been approved, there are currently few effective therapeutics to fight its associated disease in humans, COVID-19. SARS-CoV-2 and the related severe acute respiratory syndrome (SARS-CoV-1), and Middle East respiratory syndrome (MERS-CoV) result from zoonotic respiratory viruses that have bats as the primary host and an as yet unknown secondary host. While each of these viruses has different protein-based cell-surface receptors, each rely on the glycosaminoglycan, heparan sulfate as a co-receptor. In this study we compare, for the first time, differences and similarities in the structure of heparan sulfate in human and bat lungs. Furthermore, we show that the spike glycoprotein of COVID-19 binds 3.5 times stronger to human lung heparan sulfate than bat lung heparan sulfate.

EGCG binds intrinsically disordered N-terminal domain of p53 and disrupts p53-MDM2 interaction.

Epigallocatechin gallate (EGCG) from green tea can induce apoptosis in cancerous cells, but the underlying molecular mechanisms remain poorly understood. Using SPR and NMR, here we report a direct, µM interaction between EGCG and the tumor suppressor p53 (KD = 1.6 ± 1.4 µM), with the disordered N-terminal domain (NTD) identified as the major binding site (KD = 4 ± 2 µM). Large scale atomistic simulations (>100 µs), SAXS and AUC demonstrate that EGCG-NTD interaction is dynamic and EGCG causes the emergence of a subpopulation of compact bound conformations. The EGCG-p53 interaction disrupts p53 interaction with its regulatory E3 ligase MDM2 and inhibits ubiquitination of p53 by MDM2 in an in vitro ubiquitination assay, likely stabilizing p53 for anti-tumor activity. Our work provides insights into the mechanisms for EGCG's anticancer activity and identifies p53 NTD as a target for cancer drug discovery through dynamic interactions with small molecules.

A Revised Structure for the Glycolipid Terminus of Escherichia coli K5 Heparosan Capsular Polysaccharide.

The structure of heparosan capsular polysaccharide (CPS) has been determined using enzymatic digestion with nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry. Previous errors in the assignment of the glycolipid acceptor structure, from which heparosan is extended, have been corrected. The structure of heparosan CPS is GlcNAc α-1,[4GlcA ß-1,4GlcNAc α-1,]n4GlcA ß-1,[4Kdo ß-2,7Kdo ß-2,]0 or 14Kdo ß-2,7Kdo ß-2,4Kdo ß-2,7Kdo ß-2,4Kdo ß-2,7Kdo ß-2,4Kdo ß-PG-I (C16:0 or C18:0) (where n is ~250 for a CPS of 100 kDa).

Unique Cell Surface Mannan of Yeast Pathogen Candida auris with Selective Binding to IgG.

The emerging, multidrug-resistant yeast pathogen Candida auris is responsible for healthcare-associated outbreaks across the globe with high mortality. The rapid spread of C. auris is linked to its successful colonization of human skin, followed by bloodstream infections. We compared glycomics and proteomics of C. auris to closely and distantly related human pathogenic yeasts, C. haemulonii and C. albicans, with the aim to understand the role of cell surface molecules in skin colonization and immune system interactions. Candida auris mannan is distinct from other pathogenic Candida species, as it is highly enriched in ß-1,2-linkages. The experimental data showed that C. auris surface mannan ß-1,2-linkages were important for the interactions with the immune protein IgG, found in blood and in sweat glands, and with the mannose binding lectin, found in the blood. Candida auris mannan binding to IgG was from 12- to 20-fold stronger than mannan from the more common pathogen C. albicans. The findings suggest unique C. auris mannan could be crucial for the biology and pathogenesis of this emerging pathogen.

Study on the mechanism of ultrasound-accelerated enzymatic hydrolysis of starch: Analysis of ultrasound effect on different objects.

Enzymatic hydrolysis of starch is an important process in the food industry. In the present work, ultrasound was introduced in glucoamylase pretreatment, starch pretreatment and mixed reaction system treatment to enhance starch hydrolysis efficiency. These different processes were studied to explore the mechanism of ultrasound in promoting enzymatic reactions. The hydrolysis degree of starch was determined via measuring the reducing sugar yield. Ultrasound caused enzyme inactivation under high temperatures, high ultrasonic power and long-time treatment, especially at high temperatures exceeding 65 °C. Ultrasound pretreatment of starch before enzymolysis led to the furtherance of starch hydrolysis degree. Meanwhile, sonicating the mixed enzymatic reaction system below 65 °C promoted starch hydrolysis significantly, inducing more than five- fold growth in the degree of starch hydrolysis as much as the ultrasound pretreatment caused. Molecular weights analysis conducted by the MALLS system reflected the enormous damage of starch molecules caused by ultrasound. The amylose contents and chain length distributions of samples were separately analyzed by iodine binding method and size exclusion chromatography. The results of the two experiments illustrate that ultrasound could promote the enzymatic hydrolysis of amylopectin, which is harder for glucoamylase to hydrolyze compared to amylose.

Fucosylated Chondroitin Sulfate 9-18 Oligomers Exhibit Molecular Size-Independent Antithrombotic Activity while Circulating in the Blood.

Fucosylated chondroitin sulfate (FCS) oligosaccharides extracted from sea cucumber and depolymerized exhibit potent anticoagulant activity. Knowledge of the antithrombotic activity of different size oligosaccharides and their fucose (Fuc) branch sulfation pattern should promote their development for clinical applications. We prepared highly purified FCS trisaccharide repeating units from hexasaccharide (6-mer) to octadecasaccharide (18-mer), including those with 2,4-disulfated and 3,4-disulfated Fuc branches. All 10 oligosaccharides were identified by their nuclear magnetic resonance structures and ESI-FTMS spectroscopy. In vitro anticoagulant activities and surface plasmon resonance binding tests indicated those of larger molecular sizes and 2,4-disulfated Fuc branches showed stronger anticoagulant effects with respect to anti-FXase activity, as well as stronger binding to FIXa among various clotting proteins. However, both types of FCS 9-mer to 18-mer exhibited molecular size-independent potent antithrombotic activity in vivo at the same dose. In addition, both types of the FCS 6-mer exhibited favorable antithrombotic activity in vivo, although they showed weak anticoagulant activity in vitro. Combining absorption and metabolism studies, we conclude that FCS 9-18 oligomers could remain in the circulation to interact with various clotting proteins to prevent thrombus formation, and appreciable quantities of these oligomers could be excreted through the kidneys. All FCS 9-18 oligomers also resulted in no bleeding, hypotension, or platelet aggregation risk during blood circulation. Thus, FCS 9-18 oligomers with 2,4-disulfated or 3,4-disulfated Fuc branches exhibit potent and safe antithrombotic activity needed for clinical applications.

Highly purified fucosylated chondroitin sulfate oligomers with selective intrinsic factor Xase complex inhibition.

Fucosylated chondroitin sulfate (FCS) oligosaccharides of specific molecular weight have shown potent anticoagulant activities with selectivity towards intrinsic factor Xase complex. However, the preparation of FCS oligosaccharides by traditional methods requires multiple purification steps consuming large amounts of time and significant resources. The current study focuses on developing a method for the rapid preparation of FCS oligomers from sea cucumber Pearsonothuria graeffei having 6-18 saccharide residues. The key steps controlling molecular weight (Mw) and purity of these FCS oligomers were evaluated. Structural analysis showed the resulting FCS oligomers were primarily l-Fuc3,4diS-α1,3-d-GlcA-ß1,3-(d-GalNAc4,6diS-ß1,4-[l-Fuc3,4diS-α1,3-]d-GlcA-ß1,3-)nd-anTal-ol4,6diS (n = 1˜5) accompanied by partial de-fucosylation and/or de-sulfation. In vitro and in vivo experiments demonstrate that these FCS oligomers selectively inhibit intrinsic factor Xase complex and exhibit remarkable antithrombotic activity without hemorrhagic and hypotension side effects. This method is suitable for large-scale preparation of FCS oligosaccharides as clinical anticoagulants.

Development of low molecular weight heparin by H2O2/ascorbic acid with ultrasonic power and its anti-metastasis property.

Low molecular weight heparins (LMWHs) are currently used as an anticoagulant agent since unfractionated heparin (UFH) can cause serious adverse drug reactions. LMWHs are commercially prepared using different methods such as nitrous acid cleavage and ß-elimination under strong reaction conditions or with harsh chemicals, which may cause the saccharide units within the polysaccharide backbone to be decomposed and noticeably modified. This study demonstrates an effective method for depolymerizing heparin via the production of large amounts of free radicals from H2O2/ascorbic acid and ultrasonic power; this results in highly pure products because ascorbic acid can decompose during the reaction, which is different from the previously reported H2O2/Cu2+ method. The reaction conditions-including concentration of ascorbic acid, reaction temperature and intensity of ultrasonic power-were investigated and optimized. We found that the degradation behavior of heparin in this combined physicochemical process conformed to first-order reaction kinetics. The chemical composition and structures of different LMWHs were analyzed. The results showed the primary structure and sulfate esters were well preserved after the depolymerization, the major repeat units are (1-4)-linked glucosamine and iduronic acid. The further in vitro assays indicated that the LMWHs produced by H2O2/ascorbic acid with ultrasonic power have an anti-metastatic effect in A549 cells, which suggested the LMWHs rapidly prepared in this physicochemical way have a potential for anti-tumor metastatic function.

Pectic oligosaccharides hydrolyzed from citrus canning processing water by Fenton reaction and their antiproliferation potentials.

Citrus canning processing water contains a valuable and renewable source of biopolymers and bioactive compounds including pectic polysaccharides. Upgrading these processing wastes can not only alleviate environmental pollution but also add value to the commodity's production. In a previous study we recovered pectic polysaccharides from citrus canning processing water. In the present study, pectic polysaccharides recycled from citrus canning processing water was depolymerized by an optimized Fenton system. The hydrolyzate was fractionated via size-exclusion chromatography into six fractions: 500 Da < LMP1 < 3 kDa; 3 kDa < LMP2 < 5 kDa; 5 kDa < LMP3 < 12 kDa; 12 kDa < LMP4 < 25 kDa; 25 kDa < LMP5 < 100 kDa and LMP6 > 10 wDa. Structure analyses showed that LMP1 were homogalacturonans-enriched non-esterified polysaccharides. While LMP2 contained both HG and rhamnogalacturonan-I (RG-I). Further antitumor assay showed that in comparison with the native pectic polysaccharide with moderate antitumor activity, both LMP1 and LMP2 possessed significant antitumor activity, while the inhibitory effect of LMP1 was higher than that of LMP2, suggesting that the biological properties of LMPs was influenced by structural characteristics, including molecular weight and monosaccharide composition.

Ultrasound promotes enzymatic reactions by acting on different targets: Enzymes, substrates and enzymatic reaction systems.

With the extensive application of enzyme-catalyzed reactions in numerous fields, improving enzymatic efficiency has attracted wide attention for reducing operating costs and increasing output. There are three targets throughout enzymatic reactions: the enzyme, substrate, and mixed reaction system. Ultrasound has been known to accelerate enzymatic reactions by acting on different targets. It can modify both enzyme and substrate macromolecules, which is helpful for enhancing enzyme activity and product yields. The synergistic effect of ultrasound and enzymes is widely reported to increase catalytic rates. The present review discusses the positive effect induced by ultrasound throughout the enzymatic process, including ultrasonic modification of enzymes, ultrasound assisted immobilization, ultrasonic pretreatment of substrates, and ultrasound assisted enzymatic reactions.