Stevia rebaudiana germplasm characterization using microsatellite markers and steviol glycosides quantification by HPLC

Stevia rebaudiana Bertoni, Asteraceae, is an herbaceous perennial plant native to Paraguay. This species is considered since ancient times a medicinal plant with important bioactive compounds and pharmacologic and food properties, namely diterpenes glycosides. The high natural sweetener potential stevioside and rebaudioside A produced by S. rebaudiana plants are suitable sucrose substitutes, and their obtention is influenced by environmental, phytosociological, and genetic factors. The plants’ genetic profile and sweet potential depiction are needed for suitable plant selection for improvement and deployment. Thirty-one S. rebaudiana accessions grown in the same plot where leaves samples were collected in early 2019, were genotyped using six microsatellite markers, including two steviol glycosides biosynthesis functionally involved markers. Additionally, an aqueous extract of each sample was obtained in a water bath and purified by SPE for stevioside and rebaudioside A quantification by normal phase HPLC. Stevioside and rebaudioside A contents varied between 0.53–7.36% (w w−1) and 0.37–3.60% (w w−1), respectively. Two genotypes displayed interesting ratios of rebaudioside A/stevioside (number 3 and 33). The level of genetic similarity between genotypes was tested through a pairwise similarity coefficient, and two groups of individuals had the same fingerprinting. Strong relatedness was found within genotypes, possibly due to cloning, thus, influx of new germplasm ought to be made to prevent mating between relatives, and for further selection and genetic improvement.

Normal-Phase HPLC-ELSD to Compare Lipid Profiles of Different Wheat Flours

Normal-phase high-performance liquid chromatography (HPLC) is widely used in combination with evaporative light scattering detection (ELSD) for separating and detecting lipids in various food samples. ELSD responses of different lipids were evaluated to elucidate the possibilities and challenges associated with quantification by means of HPLC-ELSD. Not only the number and type of polar functional groups but also the chain length and degree of unsaturation of (free or esterified) fatty acids (FAs) had a significant effect on ELSD responses. Tripalmitin and trilinolein yielded notably different ELSD responses, even if their constituting free FAs produced identical responses. How FA structure impacts ELSD responses of free FAs is thus not predictive for those of triacylglycerols and presumably other lipids containing esterified FAs. Because ELSD responses of lipids depend on the identity of the (esterified) FA(s) which they contain, fully accurate lipid quantification with HPLC-ELSD is challenging and time-consuming. Nonetheless, HPLC-ELSD is a good and fast technique to semi-quantitatively compare the levels of different lipid classes between samples of comparable FA composition. In this way, lipid profiles of different flours from near-isogenic wheat lines could be compared.

Determination of enantiomer impurity in Bortezomib lyo injection formulation by using normal-phase liquid chromatography

Background A highly stereo-specific liquid chromatographic technique was built up and authenticated to quantify the (1S,2R-enantiomer) impurity in Bortezomib lyo injection formulation. The separation was achieved on Chiral Pak ID-3 (3 μm, 4.6 × 250 mm) column (“amylose-based 3-chlorophenylcarbamate” chiral stationary phase) through a movable segment consisting of n-heptane, 2-propanol, ethyl alcohol, and TFA (82:15:3:0.1, v/v/v/v) at a flow rate of 0.6 mL/min. Column temperature preserved 25 °C, injection level 20 μL, sample cooler temperature ambient, and detection wavelength 270 nm. Results The retention time of (1S,2R-enantiomer) impurity and Bortezomib was determined 10.57 and 17.98 min, respectively. The resolution between (1S,2R-enantiomer) impurity and Bortezomib was found to be 4.2. The acceptance limit of the (1S,2R-enantiomer) impurity is 0.5%. The established method was authenticated as per ICH guidelines in respect of precision, accuracy, sensitivity, linearity, specificity, ruggedness, and robustness. The minimum quantity of the sample required for detection (LOD) was observed at 0.282 μg per mL and similarly the quantifying sample (LOQ) was observed to be 0.896 μg per mL. Conclusion The proposed normal phase-HPLC method that can quantify (1S,2R-enantiomer) impurity in Bortezomib lyo injection formulation at trace level concentration has been urbanized and authenticated as per ICH guidelines. The effectiveness of the technique was ensured by the specificity, exactitude, linearity, and accuracy. Hence, the method well suit for their intended purposes and can be successfully useful for regular analysis in laboratories and is suitable for the quality control.

Identification of unusual phospholipids from bovine heart mitochondria by HPLC-MS/MS

Phospholipids, including ether phospholipids, are composed of numerous isomeric and isobaric species that have the same backbone and acyl chains. This structural resemblance results in similar fragmentation patterns by collision-induced dissociation of phospholipids regardless of class, yielding complicated MS/MS spectra when isobaric species are analyzed together. Furthermore, the presence of isobaric species can lead to misassignment of species when made solely based on their molecular weights. In this study, we used normal-phase HPLC for ESI-MS/MS analysis of phospholipids from bovine heart mitochondria. Class separation by HPLC eliminates chances for misidentification of isobaric species from different classes of phospholipids. Chromatography yields simple MS/MS spectra without interference from isobaric species, allowing clear identification of peaks corresponding to fragmented ions containing monoacylglycerol backbone derived from losing one acyl chain. Using these fragmented ions, we characterized individual and isomeric species in each class of mitochondrial phospholipids, including unusual species, such as PS, containing an ether linkage and species containing odd-numbered acyl chains in cardiolipin, PS, PI, and PG. We also characterized monolysocardiolipin and dilysocardiolipin, the least abundant but nevertheless important mitochondrial phospholipids. The results clearly show the power of HPLC-MS/MS for identification and characterization of phospholipids, including minor species.

Chiral Separation and Determination of Etoxazole Enantiomers in Vegetables by Normal-Phase and Reverse-Phase High Performance Liquid Chromatography

The chiral separation of etoxazole enantiomers on Lux Cellulose-1, Lux Cellulose-3, Chiralpak IC, and Chiralpak AD chiral columns was carefully investigated by normal-phase high performance liquid chromatography and reverse-phase high performance liquid chromatography (HPLC). Hexane/isopropanol, hexane/n-butanol, methanol/water, and acetonitrile/water were used as mobile phase at a flow rate of 0.8 mL/min. The effects of chiral stationary phase, mobile phase component, mobile phase ratio, and temperature on etoxazole separation were also studied. Etoxazole enantiomers were baseline separated on Lux Cellulose-1, Chiralpak IC, and Chiralpak AD chiral columns, and partially separated on Lux Cellulose-3 chiral column under normal-phase HPLC. However, the complete separation on Lux Cellulose-1, Chiralpak IC, and partial separation on Chiralpak AD were obtained under reverse-phase HPLC. Normal-phase HPLC presented better resolution for etoxazole enantiomers than reverse-phase HPLC. Thermodynamic parameters, including ΔH and ΔS, were also calculated based on column temperature changes from 10 °C to 40 °C, and the maximum resolutions (Rs) were not always acquired at the lowest temperature. Furthermore, the optimized method was successfully applied to determine etoxazole enantiomers in cucumber, cabbage, tomato, and soil. The results of chiral separation efficiency of etoxazole enantiomers under normal-phase and reverse-phase HPLC were compared, and contribute to the comprehensive environmental risk assessment of etoxazole at the enantiomer level.

Brain Pathology in Mucopolysaccharidoses (MPS) Patients with Neurological Forms

Mucopolysaccharidoses (MPS) are the group of lysosomal storage disorders caused by deficiencies of enzymes involved in the stepwise degradation of glycosaminoglycans. To identify brain pathology common for neurological MPS, we conducted a comprehensive analysis of brain cortex tissues from post-mortem autopsy materials of eight patients affected with MPS I, II, IIIA, IIIC, and IIID, and age-matched controls. Frozen brain tissues were analyzed for the abundance of glycosaminoglycans (heparan, dermatan, and keratan sulfates) by LC-MS/MS, glycosphingolipids by normal phase HPLC, and presence of inflammatory cytokines interleukin-6 (IL-6) and tumor necrosis factor superfamily member 10 (TNFSF10) by Western blotting. Fixed tissues were stained for the markers for microgliosis, astrogliosis, misfolded proteins, impaired autophagy, and GM2 ganglioside. Our results demonstrate that increase of heparan sulfate, decrease of keratan sulfate, and storage of simple monosialogangliosides 2 and 3 (GM2 and GM3) as well as the neutral glycosphingolipid, LacCer, together with neuroinflammation and neuronal accumulation of misfolded proteins are the hallmarks of brain pathology in MPS patients. These biomarkers are similar to those reported in the corresponding mouse models, suggesting that the pathological mechanism is common for all neurological MPS in humans and mice.

Anthraquinones Derivatives and Its Antibacterial Properties from Paederia foetida Stems

Background: Paederia foetida (Rubiaceae) locally known as Chinese fever vine is a prominent plant species in the east and south Asia. The extract of paederia foetida Linn. has been used for the treatment of gastric infections or other digestive disorders in Chinese Traditional Medicine. Objective: The main aim of the study was to isolate bioactive constituents of P. foetida stem through a bio-guided assay, then evaluate their antibacterial activity and compare them with standard agents. Materials and Methods: The stems of P. foetida were extracted by methanol and successively partitioned with ethyl acetate and n-butanol. The ethyl acetate layer further fractionated using column chromatography and normal phase HPLC. The chemical structures of the isolated compounds were elucidated through comparison of the 1H and 13C NMR and MS spectral data with the literature. The antibacterial activity of P. foetida stem was evaluated using agar well diffusion assay and resazurin based micro-dilution technique. Results: Ten compounds were isolated from the Chinese fever vine stem including four anthraquinones, morindaparvin A (1), 1,3-dihydroxy-2-methoxyanthraquinone (2), digiferrol (3), and alizarin (4); two steroids, β-sitosterol (5), and stigmastan-3-one (6); two coumarins, scopoletin (7) and fraxidin (8); two aromatics, ferulic acid (9) and vanillic acid (10). The four anthraquinones 1-4 were isolated for the first time from Chinese fever vine stem. Compound 2 and 3 significantly inhibited staphylococcus aureus with MIC values 18.75 and 9.37 μg/mL respectively, and streptomycin (1.8 μg/mL) was used as a positive control. Conclusion: Compound 2 and 3 can be considered as a prospective candidate for the treatment of staphylococcal bacterial infections in both human and animals.

Reduced sphingolipid hydrolase activities, substrate accumulation and ganglioside decline in Parkinson’s disease

Background Haploinsufficiency in the Gaucher disease GBA gene, which encodes the lysosomal glucocerebrosidase GBA, and ageing represent major risk factors for developing Parkinson’s disease (PD). Recently, more than fifty other lysosomal storage disorder gene variants have been identified in PD, implicating lysosomal dysfunction more broadly as a key risk factor for PD. Despite the evidence of multiple lysosomal genetic risks, it remains unclear how sphingolipid hydrolase activities, other than GBA, are altered with ageing or in PD. Moreover, it is not fully known if levels of glycosphingolipid substrates for these enzymes change in vulnerable brain regions of PD. Finally, little is known about the levels of complex gangliosides in substantia nigra which may play a significant role in ageing and PD. Methods To study sphingolipid hydrolase activities and glycosphingolipid expression in ageing and in PD, two independent cohorts of human substantia nigra tissues were obtained. Fluorescent 4-methylumbelliferone assays were used to determine multiple enzyme activities. The lysosomal GBA and non-lysosomal GBA2 activities were distinguished using the inhibitor NB-DGJ. Sensitive and quantitative normal-phase HPLC was performed to study glycosphingolipid levels. In addition, glycosphingolipid levels in cerebrospinal fluid and serum were analysed as possible biomarkers for PD. Results The present study demonstrates, in two independent cohorts of human post-mortem substantia nigra, that sporadic PD is associated with deficiencies in multiple lysosomal hydrolases (e.g. α-galactosidase and β-hexosaminidase), in addition to reduced GBA and GBA2 activities and concomitant glycosphingolipid substrate accumulation. Furthermore, the data show significant reductions in levels of complex gangliosides (e.g. GM1a) in substantia nigra, CSF and serum in ageing, PD, and REM sleep behaviour disorder, which is a strong predictor of PD. Conclusions These findings conclusively demonstrate reductions in GBA activity in the parkinsonian midbrain, and for the first time, reductions in the activity of several other sphingolipid hydrolases. Furthermore, significant reductions were seen in complex gangliosides in PD and ageing. The diminished activities of these lysosomal hydrolases, the glycosphingolipid substrate accumulation, and the reduced levels of complex gangliosides are likely major contributors to the primary development of the pathology seen in PD and related disorders with age.