ß-Glucan fragmentation by microfluidization and TNF-α-immunostimulating activity of fragmented ß-glucans.

Fragmentation of ß-glucans secreted by the fungus Ophiocordyceps dipterigena BCC 2073 achieved by microfluidization was investigated. The degree of ß-glucan fragmentation was evaluated based on the average number of chain scissions (α). The effects on the α value of experimental variables like solid concentration of the ß-glucan suspension, interaction chamber pressure, and number of passes through the microfluidizer were examined. Kinetic studies were conducted using the relationships of the α and suspension viscosity values with the number of passes. Evidence indicated that α increases with the interaction chamber pressure and the number of passes, whereas the solid concentration shows the inverted effect. Kinetic data indicated that the fragmentation rate increases with ß-glucan solid concentration and interaction chamber pressure. Furthermore, since ß-glucan molecular weight is a key factor determining its biological activity, the effect of ß-glucans of different molecular weights produced by fragmentation on tumor necrosis factor (TNF)-α-stimulating activity in THP-1 human macrophage cells was investigated. Evidence suggested that ß-glucans have an immunostimulating effect on macrophage function, in the absence of cytotoxic effects. Indeed, ß-glucans characterized by a range of molecular weights produced via microfluidization exhibited promise as immunostimulatory agents.

Mass spectral analysis of secondary metabolites from Zingiber montanum rhizome extract using UHPLC-HR-ESI-QTOF-MS/MS.

INTRODUCTION: Zingiber montanum (J.Koenig) Link ex A.Dietr. is a popular medicinal plant in Thailand. Its rhizomes have been used as an ingredient in various Thai traditional medicine formulas. While many reports have focused on the chemical constituents and biological activities of this plant, a comprehensive study on secondary metabolite profiling using tandem mass spectrometry has, to this point, never been documented. OBJECTIVE: To analyze the chemical constituents in Z. montanum rhizomes using ultra-high performance liquid chromatography coupled with ultra-high-resolution electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UHPLC-HR-ESI-QTOF-MS/MS) analyses and to utilize the characteristic fragmentation patterns of these compounds to facilitate their identification. METHODOLOGY: UHPLC-HR-ESI-QTOF-MS/MS in positive ion mode was used for chemical identification of secondary metabolites from the ethanolic extract of the plant material. MS/MS data of some known reference compounds, together with detailed fragmentation pattern information of several compounds obtained from the crude extract, were used to elucidate their chemical structures. RESULTS: In this work, one benzaldehyde, ten phenylbutenoid monomers, six curcuminoids, and nine phenylbutenoid dimers were assigned based on their characteristic fragment ions. Among these compounds, 2-(3,4-dimethoxystyryl)oxirane was tentatively suggested as a potential new compound. Several characteristic fragment ions from these compounds were assigned and the relative ion abundance of these was also used to differentiate the chemical structures of compounds having the same molecular mass. CONCLUSIONS: The results will benefit future high-throughput screening of bioactive compounds and method development for the quality control of raw materials and herbal drugs derived from Z. montanum rhizome extracts.

Inhibitory effects of Gymnema inodorum (Lour.) Decne leaf extracts and its triterpene saponin on carbohydrate digestion and intestinal glucose absorption.

ETHNOPHARMACOLOGICAL RELEVANCE: Chiang-Da, Gymnema inodorum (Lour.) Decne. (GI), is an ethnomedicinal plant that has been used for diabetic treatment since ancient times. One of the anti-diabetic mechanisms is possibly related to the actions of triterpene glycoside, (3ß, 16ß)-16,28-dihydroxyolean-12-en-3-yl-O-ß-D-glucopyranosyl-ß-D-glucopyranosiduronic acid (GIA1) in decreasing carbohydrate digestive enzymes and intestinal glucose absorption in the gut system. AIMS OF THE STUDY: To observe the amount of GIA1 in GI leaf extracts obtained from different ethanol concentrations and to investigate the anti-hyperglycemic mechanisms of the extracts and GIA1. MATERIALS AND METHODS: The crude extracts were prepared using 50%v/v to 95%v/v ethanol solutions and used for GIA1 isolation. The anti-hyperglycemic models included in our study examined the inhibitory activities of α-amylase/α-glucosidase and intestinal glucose absorption related to sodium glucose cotransporter type 1 (SGLT1) using Caco-2 cells. RESULTS: GIA1 was found about 8%w/w to 18%w/w in the GI extract depending on ethanol concentrations. The GI extracts and GIA1 showed less inhibitory activities on α-amylase. The extracts from 75%v/v and 95%v/v ethanol and GIA1 significantly delayed the glycemic absorption by lowering α-glucosidase activity and glucose transportation of SGLT1. However, the 50%v/v ethanolic extract markedly decreased the α-glucosidase activity than the SGLT1 function. CONCLUSION: Differences in the GIA1 contents and anti-glycemic properties of the GI leaf extract was dependent on ethanol concentrations. Furthermore, the inhibitory effects of the 75%v/v and 95%v/v ethanolic extracts on α-glucosidase and SGLT1 were relevant to GIA1 content.

Coumarin-Caged Compounds of 1-Naphthaleneacetic Acid as Light-Responsive Controlled-Release Plant Root Stimulators.

Six coumarin-caged compounds of 1-naphthaleneacetic acid (NAA) comprising different substituents on the coumarin moiety were synthesized and evaluated for their photophysical and chemical properties as light-responsive controlled-release plant root stimulators. The 1H NMR and HPLC techniques were used to verify the release of NAA from the caged compounds. After irradiation at 365 nm, the caged compounds exhibited the fastest release rate at t1/2 of 6.7 days and the slowest release rate at t1/2 of 73.7 days. Caged compounds at high concentrations (10-5 and 10-6 M) significantly stimulate secondary root germination while free NAA at the same level is toxic and leads to inhibition of secondary root germination. The cytotoxicity of the caged compounds against fibroblasts and vero cells were evaluated, and the results suggested that, at 10-5-10-6 M, caged compounds exhibited no significant cytotoxicity to the cells. Thus, the caged compounds of NAA in this study could be of great benefit as efficient agrochemicals.

Anti-EpCAM scFv gadolinium chelate: a novel targeted MRI contrast agent for imaging of colorectal cancer.

OBJECTIVES: The development of targeted contrast agents for magnetic resonance imaging (MRI) facilitates enhanced cancer imaging and more accurate diagnosis. In the present study, a novel contrast agent was developed by conjugating anti-EpCAM humanized scFv with gadolinium chelate to achieve target specificity. MATERIALS AND METHODS: The material design strategy involved site-specific conjugation of the chelating agent to scFv. The scFv monomer was linked to maleimide-DTPA via unpaired cysteine at the scFv C-terminus, followed by chelation with gadolinium (Gd). Successful scFv-DTPA conjugation was achieved at 1:10 molar ratio of scFv to maleimide-DTPA at pH 6.5. The developed anti-EpCAM-Gd-DTPA MRI contrast agent was evaluated for cell targeting ability, in vitro serum stability, cell cytotoxicity, relaxivity, and MR contrast enhancement. RESULTS: A high level of targeting efficacy of anti-EpCAM-Gd-DTPA to an EpCAM-overexpressing HT29 colorectal cell was demonstrated by confocal microscopy. Good stability of the contrast agent was obtained and no cytotoxicity was observed in HT29 cells after 48 h incubation with 25-100 µM of Gd. Favorable imaging was obtained using anti-EpCAM-Gd-DTPA, including 1.8-fold enhanced relaxivity compared with Gd-DTPA, and MR contrast enhancement observed after binding to HT29. CONCLUSION: The potential benefit of this contrast agent for in vivo MR imaging of colorectal cancer, as well as other EpCAM positive cancers, is suggested and warrants further investigation.

Controllable encapsulation of α-mangostin with quaternized ß-cyclodextrin grafted chitosan using high shear mixing.

In this study, the inclusion complex formation between α-mangostin and water-soluble quaternized ß-CD grafted-chitosan (QCD-g-CS) was investigated. Inclusion complex formation with encapsulation efficiency (%EE) of 5, 15 and 75% can be varied using high speed homogenizer. Tuning %EE plays a role on physicochemical and biological properties of α-mangostin/QCD-g-CS complex. Molecular dynamics simulations indicate that α-mangostin is included within the hydrophobic ß-CD cavity and being absorbed on the QCD-g-CS surface, with these results being confirmed by Fourier transform infrared (FTIR) spectroscopy. Probing the release characteristics of the inclusion complex at various %EE (5%, 15% and 75%) in simulated saliva (pH 6.8) demonstrated that α-mangostin release rates were dependent on % EE (order 5% > 15% > 75%). Additionally, higher antimicrobial and anti-inflammation activities were observed for the inclusion complex than those of free α-mangostin due to enhance the solubility of α-mangostin through the inclusion complex with QCD-g-CS.

Development of an in Vitro System to Simulate the Adsorption of Self-Emulsifying Tea (Camellia oleifera) Seed Oil.

In this study, tea (Camellia oleifera) seed oil was formulated into self-emulsifying oil formulations (SEOF) to enhance the aqueous dispersibility and intestinal retention to achieve higher bioavailability. Self-emulsifying tea seed oils were developed by using different concentrations of lecithin in combination with surfactant blends (Span(®)80 and Tween(®)80). The lecithin/surfactant systems were able to provide clear and stable liquid formulations. The SEOF were investigated for physicochemical properties including appearance, emulsion droplets size, PDI and zeta potential. The chemical compositions of tea seed oil and SEOF were compared using GC-MS techniques. In addition, the oil adsorption measurement on artificial membranes was performed using a Franz cell apparatus and colorimetric analysis. The microscopic structure of membranes was observed with scanning electron microscopy (SEM). After aqueous dilution with fed-state simulated gastric fluid (FeSSGF), the droplet size of all SEOF was close to 200 nm with low PDI values and the zeta potential was negative. GC-MS chromatograms revealed that the chemical compositions of SEOF were not significantly different from that of the original tea seed oil. The morphological study showed that only the SEOF could form film layers. The oil droplets were extracted both from membrane treated with tea seed oil and the SEOF in order to evaluate the chemical compositions by GC-MS.

A comparison of eugenol and menthol on encapsulation characteristics with water-soluble quaternized ß-cyclodextrin grafted chitosan.

Two guest molecules (eugenol and (-)-menthol) were investigated on inclusion complex formation with water-soluble quaternized ß-CD grafted with chitosan (QCD-g-CS). The inclusion complexes were prepared at varying mole ratios between eugenol or (-)-menthol and ß-CD (substituted on QCD-g-CS) by a conventional shaking method and obtained as solid powder by freeze-drying process. The results showed that encapsulation efficiency %EE decreased with increasing of initial eugenol or (-)-menthol loading whereas %loading increased with increasing of initial eugenol or (-)-menthol loading. The results indicated that inclusion complex formation between eugenol and QCD-g-CS was more favorable than that of (-)-menthol. To clarify this mechanism, molecular dynamics simulations were performed to explore their binding energy, solvation energy and total free energy of those complexes. It was found that the total free energy (ΔG) of eugenol and (-)-menthol against QCD-g-CS (mole ratio of 1) in water-explicit system were -2108.91 kJ/mol and -344.45 kJ/mol, respectively. Moreover, molecular dynamic simulation of eugenol absorbed on surface QCD-g-CS (-205.73 kJ/mol) was shown to have a higher negative value than that of (-)-menthol on QCD-gCS (3182.31 kJ/mol). Furthermore, the release characteristics of the encapsulated powder were also investigated in simulated saliva pH 6.8 at 32 °C. The results suggested that (-)-menthol had higher release rate from the complexes than eugenol. In all cases, the release characteristics for those guest molecules could be characterized by the limited-diffusion kinetics.

A minocycline derivative reduces nerve injury-induced allodynia, LPS-induced prostaglandin E2 microglial production and signaling via toll-like receptors 2 and 4.

Many studies have shown that minocycline, an antibacterial tetracycline, suppresses experimental pain. While minocycline's positive effects on pain resolution suggest that clinical use of such drugs may prove beneficial, minocycline's antibiotic actions and divalent cation (Ca(2+); Mg(2+)) chelating effects detract from its potential utility. Thus, we tested the antiallodynic effect induced by a non-antibacterial, non-chelating minocycline derivative in a model of neuropathic pain and performed an initial investigation of its anti-inflammatory effects in vitro. Intraperitoneal minocycline (100mg/kg) and 12S-hydroxy-1,12-pyrazolinominocycline (PMIN; 23.75 mg/kg, 47.50mg/kg or 95.00 mg/kg) reduce the mechanical allodynia induced by chronic constriction injury of mouse sciatic nerve. PMIN reduces the LPS-induced production of PGE2 by primary microglial cell cultures. Human embryonic kidney cells were transfected to express human toll-like receptors 2 and 4, and the signaling via both receptors stimulated with PAM3CSK4 or LPS (respectively) was affected either by minocycline or PMIN. Importantly, these treatments did not affect the cell viability, as assessed by MTT test. Altogether, these results reinforce the evidence that the anti-inflammatory and experimental pain suppressive effects induced by tetracyclines are neither necessarily linked to antibacterial nor to Ca(2+) chelating activities. This study supports the evaluation of the potential usefulness of PMIN in the management of neuropathic pain, as its lack of antibacterial and Ca(2+) chelating activities might confer greater safety over conventional tetracyclines.

Antifungal activity of alkaloids from the seeds of Chimonanthus praecox.

Two alkaloids, D-calycanthine (1) and L-folicanthine (2), were isolated from the active MeOH extract of the seeds of Chimonanthus praecox LINK. The structures of the two compounds were established by (1)H- and (13)C-NMR, and MS (FAB, ESI) analyses. In the in vitro tests, compounds 1 and 2 showed significant inhibitory activities against five plant pathogenic fungi Exserohilum turcicum, Bipolaris maydis, Alternaria solani, Sclerotinia sderotiorum, and Fusarium oxysportium, among which B. maydis was found to be the most susceptible to 1 with an EC(50) value of 29.3 microg/ml, followed by S. sderotiorum to 2 with an EC(50) value of 61.2 microg/ml. To our knowledge, this is the first report of isolation and LC/MS/MS identification as well as of antifungal properties of these alkaloids from the seeds of this plant.

Automated molecular formula determination by tandem mass spectrometry (MS/MS).

Automated software was developed to analyze the molecular formula of organic molecules and peptides based on high-resolution MS/MS spectroscopic data. The software was validated with 96 compounds including a few small peptides in the mass range of 138-1569 Da containing the elements carbon, hydrogen, nitrogen and oxygen. A Micromass Waters Q-TOF Ultima Global mass spectrometer was used to measure the molecular masses of precursor and fragment ions. Our software assigned correct molecular formulas for 91 compounds, incorrect molecular formulas for 3 compounds, and no molecular formula for 2 compounds. The obtained 95% success rate indicates high reliability of the software. The mass accuracy of the precursor ion and the fragment ions, which is critical for the success of the analysis, was high, i.e. the accuracy and the precision of 850 data were 0.0012 Da and 0.0016 Da, respectively. For the precursor and fragment ions below 500 Da, 60% and 90% of the data showed accuracy within < or = 0.001 Da and < or = 0.002 Da, respectively. The precursor and fragment ions above 500 Da showed slightly lower accuracy, i.e. 40% and 70% of them showed accuracy within < or = 0.001 Da and < or = 0.002 Da, respectively. The molecular formulas of the precursor and the fragments were further used to analyze possible mass spectrometric fragmentation pathways, which would be a powerful tool in structural analysis and identification of small molecules. The method is valuable in the rapid screening and identification of small molecules such as the dereplication of natural products, characterization of drug metabolites, and identification of small peptide fragments in proteomics. The analysis was also extended to compounds that contain a chlorine or bromine atom.

One-step purification of palmatine and its derivative dl-tetrahydropalmatine from Enantia chlorantha using high-performance displacement chromatography.

Palmatine and its reduced form, dl-tetrahydropalmatine are a group of isoquinoline alkaloids that have been reported to display a variety of biological and pharmacological activities. Both drugs are hydrophilic and are difficult to be purified by conventional purification methods of natural products. A high-performance displacement chromatography (HPDC) method successfully purified palmatine and its semi-synthetic derivative dl-tetrahydropalmatine from crude extract of the African medicinal plant Enantia chlorantha. The crude extract from the root bark of E. chlorantha was fractionated on an analytical reversed-phase C(18) column by using 0.1% trifluoroacetic acid (TFA) or acetic acid/H2O as a carrier and cetylpyridinium trifluoroacetate (or acetate) (1.9mg/mL) in 0.1% TFA (or acetic acid)/H2O as a displacer. Palmatine was quantitatively purified at >98% purity in the fully developed displacement mode. dl-Tetrahydropalmatine was semi-synthesized by NaBH4 reduction from crude palmatine and directly purified by HPDC. Both palmatine and dl-tetrahydropalmatine were identified by high-resolution electrospray tandem mass spectrometry, (1)H NMR and (13)C NMR. This is the first report of one-step HPDC purification of natural and semi-synthetic products from a complex crude extract.

Molecular formula analysis by an MS/MS/MS technique to expedite dereplication of natural products.

A facile and sensitive mass spectrometric method has been developed for the dereplication of natural products. The method provides information about the molecular formula and substructure of a precursor molecule and its fragments, which are invaluable aids in dereplication of natural products at their early stages of purification and characterization. Collision-induced MS/MS technique is used to fragment a precursor ion into several product ions, and individual product ions are selected and subjected to collision-induced MS/MS/MS analysis. This method enables the identification of the fragmentation pathway of a precursor molecule from its first-generation fragments (MS/MS), through to the nth generation product ions (MSn). It also allows for the identification of the corresponding neutral products released (neutral losses). Elements used in the molecular formula analysis include C, H, N, O, and S, as most natural products are constituted by these five elements. High-resolution mass separation and accurate mass measurements afforded the unique identification of molecular formula of small neutral products. Through sequential add-up of the molecular formulas of the small neutral products, the molecular formula of the precursor ion and its productions were uniquely determined. The molecular formula of the precursor molecule was then reversely used to identify or confirm the molecular formula of the neutral products and that of the productions. The molecular formula of the neutral fragments allowed for the identification of substructures, leading to a rapid and efficient characterization of precursor natural product. The method was applied to paclitaxel (C47H51NO14; 853 amu) to identify its molecular formula and its substructures, and to characterize its potential fragmentation pathways. The method was further validated by correctly identifying the molecular formula of minocycline (C23H27N3O7; 457 amu) and piperacillin (C23H27N5O7S; 517 amu).