A Low Transition Temperature Mixture-based viscosupplementation complemented with celecoxib for osteoarthritis treatment.

Viscosupplementation consists of hyaluronic acid (HA) intra-articular injections, commonly applied for osteoarthritis treatment while non-steroidal anti-inflammatory drugs (NSAIDs) are widely administered for pain relief. Here, HA and a NSAID (celecoxib) were combined in a formulation based on a low transition temperature mixture (LTTM) of glycerol:sorbitol, reported to increase celecoxib's solubility, thus rendering a potential alternative viscosupplement envisioning enhanced therapeutic efficiency. The inclusion of glucosamine, a cartilage precursor, was also studied. The developed formulations were assessed in terms of rheological properties, crucial for viscosupplementation: the parameters of crossover frequency, storage (G') and loss (G'') moduli, zero-shear-rate viscosity, stable viscosity across temperatures, and shear thinning behaviour, support viscoelastic properties suitable for viscosupplementation. Additionally, the gels biocompatibility was confirmed in chondrogenic cells (ATDC5). Regarding drug release studies, high and low clearance scenarios demonstrated an increased celecoxib (CEX) release from the gel (6 to 73-fold), compared to dissolution in PBS. The low clearance setup presented the highest and most sustained CEX release, highlighting the importance of the gel structure in CEX delivery. NMR stability studies over time demonstrated the LTTM+HA+CEX (GHA+CEX) gel as viable candidate for further in vivo evaluation. In sum, the features of GHA+CEX support its potential use as alternative viscosupplement.

Quantitative enzymatic-mass spectrometric analysis of the chitinous polymers in fungal cell walls.

Chitin is an essential structural component of complex and dynamic fungal cell walls. It may be converted by partial or full deacetylation to yield chitosan. Here, we describe a method to quantify N-acetyl d-glucosamine (GlcNAc, A) and d-glucosamine (GlcN, D) units and, thus, total amount and average fraction of acetylation (x̅ FA) of the chitinous polymers by complete enzyme hydrolysis of the polymers followed by mass spectrometric analyses of the monomers. First, the native polymers were isotopically N-acetylated, then enzymatically hydrolyzed to A and R (2H3N-acetyl-d-glucosamine - former D) monomers. Relative abundances of A and R units were used to calculate x̅ FA, and a double-isotopically labeled internal standard R* ([13C2,2H3] N-acetyl-d-glucosamine) monomer was used to calculate the absolute amounts of GlcNAc and GlcN units present in the fungal samples. The method was validated using known chitosan polymers and is suitable for both purified cell walls and whole mycelia.

Optimization of enzymatic soy protein isolate-glucosamine conjugates to improve the freeze-thaw stability of emulsion.

BACKGROUND: Using transglutaminase (TGase) is a new method to improve protein properties in order to promote protein glycosylation. This article mainly studies soy protein isolate (SPI) and glucosamine to improve the freeze-thaw stability of emulsion under the action of TGase. The degree of glycosylation was studied by the content of free amino groups and the degree of conjugation. The optimal conditions for preparing soy protein isolate-glucosamine (SPI-G) conjugate were determined by a response surface optimization model based on single-factor experiments using the creaming index of the emulsion after the first freeze-thaw cycle as the response value. RESULTS: The results showed that the emulsion had the lowest creaming index when the conditions of protein concentration was 20 g L-1 , mass ratio of SPI-G was 5:3 (w/w), enzyme addition amount was 10 U g-1 , and reaction time was 2 h. The optimized modified product was measured for the creaming index after the first freeze-thaw cycle. It was found that the creaming index of the modified product SPI-G after the first freeze-thaw cycle was 9.02%, which was less than and close to the optimized model predicted value. The creaming index and optical microscopy results after three freeze-thaw cycles confirmed that the freeze-thaw stability of the SPI-G samples was significantly enhanced after optimization of the response surface model. CONCLUSION: It showed that glycosylation promoted by TGase could improve the freeze-thaw stability of SPI emulsion, thereby broadening the application of SPI in food. © 2022 Society of Chemical Industry.

Preparation, Anticoagulant and Antioxidant Properties of Glucosamine-Heparin Salt.

Excessive inorganic ions in vivo may lead to electrolyte disorders and induce damage to the human body. Therefore, preparation of enhanced bioactivity compounds, composed of activated organic cations and organic anions, is of great interest among researchers. In this work, glucosamine-heparin salt (GHS) was primarily synthesized with positively charged glucosamine hydrochloride (GAH) and negatively charged heparin sodium (Heps) by ion exchange method. Then, the detailed structural information of the GHS was characterized by FTIR, 1H NMR spectroscopy and ICP-MS. In addition, its anticoagulant potency and antioxidant properties were evaluated, respectively. The results demonstrated that GHS salt achieved enhanced antioxidant activities, including 98.78% of O2•- radical scavenging activity, 91.23% of •OH radical scavenging rate and 66.49% of DPPH radical scavenging capacity at 1.6 mg/mL, severally. Meanwhile, anticoagulant potency (ATTP) of GHS strengthened from 153.10 ± 17.14 to 180.03 ± 6.02 at 0.75 µmol/L. Thus, introducing cationic glucosamine residues into GHS could improve its anticoagulant activity. The findings suggest that GHS product with a small amount of inorganic ions can greatly abate the prime cost of antioxidants and anticoagulants, and has significant economic benefits and practical significance.

Phosphate buffer-catalyzed kinetics of mutarotation of glucosamine investigated by NMR spectroscopy.

Glucosamine (2-amino-2-deoxy-d-glucose, GlcN) is a naturally occurring amino monosaccharide that is essential for a variety of biological functions, it is mainly involved in the formation of polysaccharide structures. It was recently reported to enable the imaging of cancerous tumors as an exogenous contrast agent using the MRI technique of chemical exchange saturation transfer (CEST). In preparation for the clinical use of GlcN, its anomeric equilibrium and mutarotation rate constants were directly investigated in this study utilizing high resolution 1H and 13C NMR spectroscopy. The effects of GlcN concentration, temperature, pH and buffer on the mutarotation rate constant and mutarotation equilibrium were measured. The mutarotation rate constant increased markedly with increasing GlcN concentrations. The rate constant of mutarotation of GlcN at room temperature was 2.2 × 10-4 - 5.0 × 10-4 s-1 at concentrations of 0.02-0.5 M, corresponding to a time of 3.8-1.7 h to reach 95% equilibrium. The anomeric ratio was strongly pH-dependent. The influence of phosphate buffer on the apparent rate constant of GlcN mutarotation was investigated. For phosphate buffer saline values between 0 and 50 mM, there was a six-fold increase in rate at pH 7.0. The mutarotation rate constant rose rapidly with pH at a phosphate concentration of 50 mM: from 0.4 × 10-3 s-1 at pH 5.0 to 7.8 × 10-3 s-1 at pH 9.4, suggesting that the catalysis is due to the HPO42- and PO43- ions. These findings might help researchers design the experimental setting for employing GlcN for cancer detection using GlcN-CEST MRI.

Preparation of the porous carbon-based solid acid from starch for efficient degradation of chitosan to D-glucosamine.

Effective degradation of chitosan to D-glucosamine is considered to make a great contribution for the development of the medical industry. To address this issue, a porous carbon-based solid acid catalyst (PCSA) functionalized with -OH, -COOH and -SO3H groups was successfully prepared. Typically, the physicochemical properties of PCSA were deeply determined by a series of characterization technique including FT-IR, TGA, RM, NH3-TPD, SEM and Element Analysis. Moreover, the catalytic performances of PCSA towards to D-glucosamine production from chitosan were evaluated. In particular, the effects of catalyst acid density, ratio of acidic groups, chitosan concentration, reaction temperature, reaction time and catalyst dosage on the yield of D-glucosamine were investigated in detail. Interestingly, the experimental results indicated that a yield of D-glucosamine as high as 90.5% was achieved, and no obvious deactivation occurred even after six consecutive cycles. In light of the advantages of superior activity/recyclability and low cost, the starch-derived solid acid developed in this work might possess the broad industrial application prospects.

Lignin/Carbohydrate Complex Isolated from Posidonia oceanica Sea Balls (Egagropili): Characterization and Antioxidant Reinforcement of Protein-Based Films.

A lignin fraction (LF) was extracted from the sea balls of Posidonia oceanica (egagropili) and extensively dialyzed and characterized by FT-IR and NMR analyses. LF resulted water soluble and exhibited a brownish-to-black color with the highest absorbance in the range of 250-400 nm, attributed to the chromophore functional groups present in the phenylpropane-based polymer. LF high-performance size exclusion chromatography analysis showed a highly represented (98.77%) species of 34.75 kDa molecular weight with a polydispersity index of 1.10 and an intrinsic viscosity of 0.15. Quantitative analysis of carbohydrates indicated that they represented 28.3% of the dry weight of the untreated egagropili fibers and 72.5% of that of LF. In particular, eight different monosaccharides were detected (fucose, arabinose, rhamnose, galactose, glucose, xylose, glucosamine and glucuronic acid), glucuronic acid (46.6%) and rhamnose (29.6%) being the most present monosaccharides in the LF. Almost all the phenol content of LF (113.85 ± 5.87 mg gallic acid eq/g of extract) was water soluble, whereas around 22% of it consisted of flavonoids and only 10% of the flavonoids consisted of anthocyanins. Therefore, LF isolated from egagropili lignocellulosic material could be defined as a water-soluble lignin/carbohydrate complex (LCC) formed by a phenol polymeric chain covalently bound to hemicellulose fragments. LCC exhibited a remarkable antioxidant activity that remained quite stable during 6 months and could be easily incorporated into a protein-based film and released from the latter overtime. These findings suggest egagropili LCC as a suitable candidate as an antioxidant additive for the reinforcement of packaging of foods with high susceptibility to be deteriorated in aerobic conditions.

Synthesis and Bioinformatic Characterization of New Schiff Bases with Possible Applicability in Brain Disorders.

(1) Background: The research aims to find new treatments for neurodegenerative diseases, in particular, Alzheimer's disease. (2) Methods: This article presents a bioinformatics and pathology study of new Schiff bases, (EZ)-N'-benzylidene-(2RS)-2-(6-chloro-9H-carbazol-2-yl)propanehydrazide derivatives, and aims to evaluate the drug-like, pharmacokinetic, pharmacodynamic and pharmacogenomic properties, as well as to predict the binding to therapeutic targets by applying bioinformatics, cheminformatics and computational pharmacological methods. (3) Results: We obtained these Schiff bases by condensing (2RS)-2-(6-chloro-9H-carbazol-2-yl)propanehydrazide with aromatic aldehydes, using the advantages of microwave irradiation. The newly synthesized compounds were characterized spectrally, using FT-IR and NMR spectroscopy, which confirmed their structure. Using bioinformatics tools, we noticed that all new compounds are drug-likeness features and may be proposed as potentially neuropsychiatric drugs (4) Conclusions: Using bioinformatics tools, we determined that the new compound 1e had a high potential to be used as a good candidate in neurodegenerative disorders treatment.

Substrate Substitution in Kanosamine Biosynthesis Using Phosphonates and Phosphite Rescue.

Kanosamine is an antibiotic and antifungal compound synthesized from glucose 6-phosphate (G6P) in Bacillus subtilis by the action of three enzymes: NtdC, which catalyzes NAD-dependent oxidation of the C3-hydroxyl; NtdA, a PLP-dependent aminotransferase; and NtdB, a phosphatase. We previously demonstrated that NtdC can also oxidize substrates such as glucose and xylose, though at much lower rates, suggesting that the phosphoryloxymethylene moiety of the substrate is critical for effective catalysis. To probe this, we synthesized two phosphonate analogues of G6P in which the bridging oxygen is replaced by methylene and difluoromethylene groups. These analogues are substrates for NtdC, with second-order rate constants an order of magnitude lower than those for G6P. NtdA converts the resulting 3-keto products to the corresponding kanosamine 6-phosphonate analogues. We compared the rates to the rate of NtdC oxidation of glucose and xylose and showed that the low reactivity of xylose could be rescued 4-fold by the presence of phosphite, mimicking G6P in two pieces. These results allow the evaluation of the individual energetic contributions to catalysis of the bridging oxygen, the bridging C6 methylene, the phosphodianion, and the entropic gain of one substrate versus two substrate pieces. Phosphite also rescued the reversible formation 3-amino-3-deoxy-d-xylose by NtdA, demonstrating that truncated and nonhydrolyzable analogues of kanosamine 6-phosphate can be generated enzymatically.

Molecular docking-guided synthesis of NSAID-glucosamine bioconjugates and their evaluation as COX-1/COX-2 inhibitors with potentially reduced gastric toxicity.

Non-steroidal anti-inflammatory drugs (NSAIDs) are a powerful class of inhibitors targeting two isoforms of the family of cyclooxygenase enzymes (COX-1 and COX-2). While NSAIDs are widely used in the management of pain, in particular as a treatment for osteo- and rheumatoid arthritis, their long-term use has been associated with numerous on- and off-target effects. As the carboxylic acid moiety present in common NSAIDs is responsible for some of their adverse effects, but is not required for their anti-inflammatory activity, we sought to mask this group through direct coupling to glucosamine, which is thought to prevent cartilage degradation. We report herein the conjugation of commonly prescribed NSAIDs to glucosamine hydrochloride and the use of molecular docking to show that addition of the carbohydrate moiety to the parent NSAID can enhance binding in the active site of COX-2. In a preliminary, in vitro screening assay, the diclofenac-glucosamine bioconjugate exhibited 10-fold greater activity toward COX-2, making it an ideal candidate for future in vivo studies. Furthermore, in an intriguing result, we observed that the mefenamic acid-glucosamine bioconjugate displayed enhanced activity toward COX-1 rather than COX-2.

Positional Isomeric Effects on the Optical Properties, Multivalent Glycosidase Inhibition Effect, and Hypoglycemic Effect of Perylene Bisimide-deoxynojirimycin Conjugates.

Although multivalent glycosidase inhibitors have shown enhanced glycosidase inhibition activities, further applications and research directions need to be developed in the future. In this paper, two positional isomeric perylene bisimide derivatives (PBI-4DNJ-1 and PBI-4DNJ-2) with 1-deoxynojirimycin conjugated were synthesized. Furthermore, PBI-4DNJ-1 and PBI-4DNJ-2 showed positional isomeric effects on the optical properties, self-assembly behaviors, glycosidase inhibition activities, and hypoglycemic effects. Importantly, PBI-4DNJ-1 exhibited potent hypoglycemic effects in mice with 41.33 ± 2.84 and 37.45 ± 3.94% decreases in blood glucose at 15 and 30 min, respectively. The molecular docking results showed that the active fragment of PBI-4DNJ-1 has the highest binding energy (9.649 kcal/mol) and the highest total hydrogen bond energy (62.83 kJ/mol), which were related to the positional isomeric effect on the hypoglycemic effect in mice. This work introduced a new means to develop antihyperglycemic agents in the field of multivalent glycomimetics.

Genomic-Led Discovery of a Novel Glycopeptide Antibiotic by Nonomuraea coxensis DSM 45129.

Glycopeptide antibiotics (GPAs) are last defense line drugs against multidrug-resistant Gram-positive pathogens. Natural GPAs teicoplanin and vancomycin, as well as semisynthetic oritavancin, telavancin, and dalbavancin, are currently approved for clinical use. Although these antibiotics remain efficient, emergence of novel GPA-resistant pathogens is a question of time. Therefore, it is important to investigate the natural variety of GPAs coming from so-called "rare" actinobacteria. Herein we describe a novel GPA producer-Nonomuraea coxensis DSM 45129. Its de novo sequenced and completely assembled genome harbors a biosynthetic gene cluster (BGC) similar to the dbv BGC of A40926, the natural precursor to dalbavancin. The strain produces a novel GPA, which we propose is an A40926 analogue lacking the carboxyl group on the N-acylglucosamine moiety. This structural difference correlates with the absence of dbv29-coding for an enzyme responsible for the oxidation of the N-acylglucosamine moiety. Introduction of dbv29 into N. coxensis led to A40926 production in this strain. Finally, we successfully applied dbv3 and dbv4 heterologous transcriptional regulators to trigger and improve A50926 production in N. coxensis, making them prospective tools for screening other Nonomuraea spp. for GPA production. Our work highlights genus Nonomuraea as a still untapped source of novel GPAs.

Reversible derivatization of sugars with carbobenzyloxy groups and use of the derivatives in solution-phase enzymatic oligosaccharide synthesis.

Simple protocols for attaching and detaching carbobenzyloxy (Cbz) groups at the reducing end of sugars was developed. Briefly, lactose was converted into its glycosylamine, which was then acylated with carbobenzyloxy chloride in high overall yield. The obtained lactose Cbz derivative was used in sequential glycosylations using glycosyltransferases and nucleotide sugars in aqueous buffers. Isolation of the reaction products after each step was by simple C-18 solid-phase extraction. The Cbz group was removed by catalytic hydrogenolysis or catalytic transfer hydrogenation followed by in situ glycosylamine hydrolysis. In this way, a trisaccharide (GlcNAc-lactose), a human milk tetrasaccharide (LNnT), and a human milk pentasaccharide (LNFPIII) were prepared in a simple and efficient way.

Effects of transglutaminase glycosylated soy protein isolate on its structure and interfacial properties.

BACKGROUNDS: The structural and interfacial properties of soybean protein isolate (SPI) after glycosylation by the transglutaminase method were studied. It is hoped that preliminary explorations will find a new food ingredient and broader application of SPI in the food industry. RESULTS: The contents of free amino proves that transglutaminase can insert glucosamine into SPI through its transamination, and realize the enzymatic glycosylated SPI. The results of structure properties showed that a decrease in the content of the α-helical structure indicates that the rigid structure of the protein is opened and the flexibility is increased. The blue shift of the maximum fluorescence intensity of soy protein isolate-glucosamine with transglutaminase (SPI-G) indicates the formation of a new substance; scanning electron microscopy shows that the SPI-G powder can be seen at a magnification of 2000×, and the protein structure becomes soft. The results of interfacial properties found that enzymatic protein glycosylation exposes the internal hydrophobic groups of SPI, resulting in increased surface hydrophobicity, increased emulsification and emulsification stability, and reduced surface tension. CONCLUSION: It shows that SPI-G effectively improves the interfacial properties of SPI, providing a theoretical basis for the application of enzymatic glycosylation of SPI in the food industry. © 2021 Society of Chemical Industry.

ADP-dependent glucose/glucosamine kinase from Thermococcus kodakarensis: cloning and characterization.

The genome sequence of Thermococcus kodakarensis contains an open reading frame, TK1110, annotated as ADP-dependent glucokinase. The encoding gene was expressed in Escherichia coli and the gene product, TK-GLK, was produced in soluble and active form. The recombinant enzyme was extremely thermostable. Thermostability was increased significantly in the presence of ammonium sulfate. ADP was the preferred co-factor for TK-GLK, which could be replaced with CDP but with a 60% activity. TK-GLK was a metal ion-dependent enzyme which exhibited glucokinase, glucosamine kinase and glucose 6-phosphatase activities. It catalyzed the phosphorylation of both glucose and glucosamine with nearly the same rate and affinity. The apparent Km values for glucose and glucosamine were 0.48 ± 0.03 and 0.47 ± 0.09 mM, respectively. The catalytic efficiency (kcat/Km) values against these two substrates were 6.2 × 105 ± 0.25 and 5.8 × 105 ± 0.75 M-1 s-1. The apparent Km value for dephosphorylation of glucose 6-phosphate was ~14-fold higher than that of glucose phosphorylation. Similarly, catalytic efficiency (kcat/Km) for phosphatase reaction was ~19-fold lower than that for the kinase reaction. To the best of our knowledge, this is the first report that describes the reversible nature of a euryarchaeal ADP-dependent glucokinase.

The study of the acid-catalyzed reaction between 2-methyl and 2-(2,2,2-trichloroethoxy) gluco-[2,1-d]-2-oxazolines. Synthesis of macrocyclic pseudo-tetrasaccharide derivative of d-glucosamine.

The formation of macrocyclic pseudo-tetrasaccharide derivative of d-glucosamine as a result of the acid-catalyzed reaction between 2-methyl- and 2-(2,2,2-trichloroethoxy)-substituted oxazoline derivatives of sugars was discovered. The structure of the obtained product was determined using NMR spectroscopy and mass spectrometry. An explanation of the obtained results based on the mechanism of the reaction of electrophilic polymerization of 2-substituted glyco-[2,1-d]-2-oxazolines and the principle of hard and soft acids and bases (HSAB) was proposed.

Treatment of knee osteoarthritis with a new formulation of a fixed-dose combination of glucosamine sulfate and bovine chondroitin: a multicenter, randomized, single-blind, non-inferiority clinical trial.

OBJECTIVES: To compare the efficacy and safety of a new formulation of a fixed dose combination of glucosamine sulfate (GS; 1500 mg) and bovine chondroitin sulfate (CS; 1200 mg) versus the reference product (RP) in patients with knee osteoarthritis (OA). METHODS: In this multicenter, randomized, single-blind trial, 627 patients with knee osteoarthritis (OA)-Kellgren-Lawrence grades 2 or 3 and mean score ≥ 40 mm in the WOMAC pain subscale-were randomized to receive GS/CS or the RP for 24 weeks. The primary efficacy endpoint was the absolute change in WOMAC pain subscale score. The secondary endpoints included the following: WOMAC total and subscale scores, overall assessment of the disease by the patient and the investigator, SF-12 score, OMERACT-OARSI response rate to the treatment, and rescue medication use. RESULTS: Mean reductions of WOMAC pain score were - 35.1 (sd = 23.2) mm in the GS/CS group and - 36.5 (sd = 24.9) mm in the RP group. The difference between the adjusted means of both treatments confirmed the non-inferiority of GS/CS versus the RP. Improvement was observed in pain, stiffness, physical function and total WOMAC score, as well as in overall OA assessment by the patient and the investigator for both groups. No improvement was observed in SF-12. The rate of OMERACT-OARSI responders was 89.4% in GS/CS group and 87.9% in the RP group. Headache and changes in glucose tolerance were the most frequent treatment-related adverse events. CONCLUSIONS: The new formulation of a fixed-dose combination of glucosamine sulfate and bovine chondroitin sulfate was non-inferior to the RP in symptomatic treatment of knee OA, with a high responder rate and good tolerability profile. TRIAL REGISTRATION: ClinicalTrials.gov; Registration number NCT02830919 ; Date of registration: July 13, 2016; First randomization date: December 05, 2016).

The effect of glucosamine and glucosamine caramel on quality and consumer acceptability of regular and reduced salt breakfast sausages.

Given the more recent interest in its flavour enhancing potential, the effects of the addition of glucosamine or glucosamine caramel on both technological and consumer acceptability of regular and reduced salt breakfast sausages were studied. A 2 × 3 complete factorial design was used with salt level (regular salt, RS (1.1%) and low salt, LS (0.825%)) and formulation treatment (control, GlcN - glucosamine (1%), CAR - glucosamine caramel (1% GlcN equivalent)) as main effects. Raw or cooked sausages were analyzed for CIE L*, a* and b*, physical and textural properties and consumer acceptance. Different salt levels did not affect the pH of meat batter, while the reduced salt treatment resulted in higher cook loss. On the contrary, addition of GlcN and CAR reduced the pH of sausage with no effect on cook loss. Neither salt levels nor treatment formulation affected the textural attributes of sausages. The inclusion of CAR significantly reduced L* value and increased redness (a*) and yellowness (b*) of cooked sausages. Salt reduction resulted in decreased a* and b* values in raw batter; the effect which disappeared in cooked sausages. Glucosamine caramel increased the overall and flavour acceptability score of low salt breakfast sausages. The present study showed that glucosamine caramel could potentially improve the flavour of low salt breakfast sausage with limited effect on textural attributes.

Profiling protein expression in Klebsiella pneumoniae with a carbohydrate-based covalent probe.

We report the application of a covalent probe based on a d-glucosamine scaffold for the profiling of the bacterial pathogen Klebsiella pneumoniae. Incubation of K. pneumoniae lysates with the probe followed by electrophoretic separation and in-gel fluorescence detection allowed the generation of strain-specific signatures and the differentiation of a carbapenem-resistant strain. The labelling profile of the probe was independent of its anomeric configuration and included several low-abundance proteins not readily detectable by conventional protein staining. Initial target identification experiments by mass spectrometry suggest that target proteins include several carbohydrate-recognising proteins, which indicates that the sugar scaffold may have a role for target recognition.

Development and validation of an improved 3-methyl-2-benzothiazolinone hydrazone method for quantitative determination of reducing sugar ends in chitooligosaccharides.

An accurate and sensitive analytical method for detecting and quantifying reducing sugar ends (RSE) in chitooligosaccharides (COSs) is the key quality parameter for evaluating their structure-function relationship and potential applications. In this work, we develop and validate a novel colorimetric assay with high accuracy and precision for determining RSE content using 3-methyl-2-benzothiazolinone hydrazone (MBTH). Under optimal conditions, the stoichiometry is verified using mono-, di-, and tri- glucosamine hydrochlorides, and the dilution ratio does not interfere with the RSE content measured at 590 nm. The regression equation of glucosamine reveal a good linear relationship (R2 = 0.9999). The detection limit, quantification limit, mean relative standard deviation (RSD), and recovery are 2.28 µM, 9.11 µM, 1.90%, and 98.0%, respectively. The newly developed method is potentially useful for monitoring COS hydrolysis, number average molecular weight, and chitosanase activity.