Optimization of the Water-Insoluble Procedures for <Emphasis Type="Italic">USP</Emphasis> General Chapter <Emphasis Type="Italic">Residual Solvents</Emphasis> <467>

The water-insoluble procedures in US Pharmacopeia (USP) General Chapter Residual Solvents <467>, which are based on European Pharmacopoeia procedures, were optimized and modified before their inclusion in the chapter to improve their scope, performance, and ruggedness. The optimized procedures use a static headspace introduction system with a gas chromatograph equipped with a flame ionization detector. This article describes some of the key changes made to the USP published procedures, including use of dimethyl sulfoxide (DMSO) or dimethylformamide (DMF) as the solvent, addition of 5 mL of water and 1 mL of sample (dissolved in DMSO or DMF) to the headspace vial, use of a 3:1 GC split ratio, and use of new matrix-matched system suitability solutions. These procedures were verified with two different active pharmaceutical ingredients—hydroxyzine pamoate and prednisone. In the investigation, the more polar material (hydroxyzine pamoate) showed greater recoveries for the optimized procedures when prepared in DMSO. The less polar material (prednisone) typically had greater recoveries in DMF for the optimized procedures. During experimentation, insights into sample preparation, additional types of headspace instrumentation, solvent purity, and other parameters were also gained.     

Carbohydrates,lipids and lipoproteins and islet changes in diabetes with superimposed myocardial infarction

Short-term metabolic and concomitant morphologic effects of streptozotocin diabetes on isoproterenol-induced myocardial infarction was studied in Wistar rats, Of particular significance was the observation that myocardial infarction in concert with diabetes brought about a distinctive exacerbation of the severity and complexity of the histopathological lesions. Of all the biochemical parameters, serum glucose and free fatty acids registered maximum elevation and serum lactate and cardiac glycogen levels a maximum reduction. Among the lipoproteins, an inverse relationship was found between high density lipoproteins and low density and very low density lipoproteins; while high density lipoproteins, ratio of high density lipoprotein to low density lipoprotein and the percentage of high density lipoprotein were decreased, there was a significant increase in low density lipoprotein concentration and percentage values of low density and very low density lipoproteins. In diabetes, the B cell of the endocrine pancreas depicted selective necrosis. Loss of insulin granules and wide-spread necrobiosis of cellular elements of the pancreatic islets were observed, respectively, in myocardial infarction and in diabetes plus myocardial infarction combinations. Pathological evidence of chemical-induced mild toxicity was present in the exocrine parenchyma. Mitotic features and the presence of centroacinar cells in the damaged Langerhans’ islets supposedly formed the basis of regeneration of the tissue in diabetes, with or without vascular complications     

Loss of A-type lamin expression compromises nuclear envelope integrity leading to muscular dystrophy

The nuclear lamina is a protein meshwork lining the nucleoplasmic face of the inner nuclear membrane and represents an important determinant of interphase nuclear architecture. Its major components are the A- and B-type lamins. Whereas B-type lamins are found in all mammalian cells, A-type lamin expression is developmentally regulated. In the mouse, A-type lamins do not appear until midway through embryonic development, suggesting that these proteins may be involved in the regulation of terminal differentiation. Here we show that mice lacking A-type lamins develop to term with no overt abnormalities. However, their postnatal growth is severely retarded and is characterized by the appearance of muscular dystrophy. This phenotype is associated with ultrastructural perturbations to the nuclear envelope. These include the mislocalization of emerin, an inner nuclear membrane protein, defects in which are implicated in Emery-Dreifuss muscular dystrophy (EDMD), one of the three major X-linked dystrophies. Mice lacking the A-type lamins exhibit tissue-specific alterations to their nuclear envelope integrity and emerin distribution. In skeletal and cardiac muscles, this is manifest as a dystrophic condition related to EDMD.     

Substitution rates of organelle and nuclear genes in sharks: implicating metabolic rate (again)

Rates of nucleotide substitution for nuclear genes are thought to be governed primarily by the number of germ line replication events (the so-called "generation time" hypothesis). In contrast, rates of mitochondrial DNA evolution appear to be set primarily by DNA damage pathways of mutation mediated by mutagenic by-products of oxidative phosphorylation (the so-called "metabolic-rate" hypothesis). Comparison of synonymous substitution rates estimated for the mitochondrial cytochrome b gene and nuclear-encoded dlx, hsp70, and RAG-1 genes in mammals and sharks shows that rates of molecular evolution for sharks are approximately an order of magnitude slower than those for mammals for both nuclear and mitochondrial genes. In addition, there is significant positive covariation of substitution rate for mitochondrial and nuclear genes within sharks. These results, interpreted in light of the pervasiveness of DNA damage by mutagenic by-products of oxygen metabolism to both nuclear and mitochondrial genes and coupled with increasing evidence for cross-genome activity of DNA repair enzymes, suggest that molecular clocks for mitochondrial and nuclear genes may be set primarily by common mutational mechanisms.      

Influence of high-pressure homogenization,ultrasonication, and supercritical fluid on free astaxanthin extraction from β-glucanase-treated Phaffia rhodozyma cells

In this study astaxanthin production by Phaffia rhodozyma was enhanced by chemical mutation using ethyl methane sulfonate. The mutant produces a higher amount of astaxanthin than the wild yeast strain. In comparison to supercritical fluid technique, high-pressure homogenization is better for extracting astaxanthin from yeast cells. Ultrasonication of dimethyl sulfoxide, hexane, and acetone-treated cells yielded less astaxanthin than β-glucanase enzyme-treated cells. The combination of ultrasonication with β-glucanase enzyme is found to be the most efficient method of extraction among all the tested physical and chemical extraction methods. It gives a maximum yield of 435.71 ± 6.55 µg free astaxanthin per gram of yeast cell mass.     

Acetate supplementation increases brain phosphocreatine and reduces AMP levels with no effect on mitochondrial biogenesis

Acetate supplementation in rats increases plasma acetate and brain acetyl-CoA levels. Although acetate is used as a marker to study glial energy metabolism, the effect that acetate supplementation has on normal brain energy stores has not been quantified. To determine the effect(s) that an increase in acetyl-CoA levels has on brain energy metabolism, we measured brain nucleotide, phosphagen and glycogen levels, and quantified cardiolipin content and mitochondrial number in rats subjected to acetate supplementation. Acetate supplementation was induced with glyceryl triacetate (GTA) by oral gavage (6 g/kg body weight). Rats used for biochemical analysis were euthanized using head-focused microwave irradiation at 2, and 4 h following treatment to immediately stop metabolism. We found that acetate did not alter brain ATP, ADP, NAD, GTP levels, or the energy charge ratio [ECR, (ATP + ½ ADP)/(ATP + ADP + AMP)] when compared to controls. However, after 4 h of treatment brain phosphocreatine levels were significantly elevated with a concomitant reduction in AMP levels with no change in glycogen levels. In parallel studies where rats were treated with GTA for 28 days, we found that acetate did not alter brain glycogen and mitochondrial biogenesis as determined by measuring brain cardiolipin content, the fatty acid composition of cardiolipin and using quantitative ultra-structural analysis to determine mitochondrial density/unit area of cytoplasm in hippocampal CA3 neurons. Collectively, these data suggest that an increase in brain acetyl-CoA levels by acetate supplementation does increase brain energy stores however it has no effect on brain glycogen and neuronal mitochondrial biogenesis.     

The Escherichia coli CydX Protein Is a Member of the CydAB Cytochrome bd Oxidase Complex and Is Required for Cytochrome bd Oxidase Activity

Cytochrome bd oxidase operons from more than 50 species of bacteria contain a short gene encoding a small protein that ranges from ∼30 to 50 amino acids and is predicted to localize to the cell membrane. Although cytochrome bd oxidases have been studied for more than 70 years, little is known about the role of this small protein, denoted CydX, in oxidase activity. Here we report that Escherichia coli mutants lacking CydX exhibit phenotypes associated with reduced oxidase activity. In addition, cell membrane extracts from ΔcydX mutant strains have reduced oxidase activity in vitro. Consistent with data showing that CydX is required for cytochrome bd oxidase activity, copurification experiments indicate that CydX interacts with the CydAB cytochrome bd oxidase complex. Together, these data support the hypothesis that CydX is a subunit of the CydAB cytochrome bd oxidase complex that is required for complex activity. The results of mutation analysis of CydX suggest that few individual amino acids in the small protein are essential for function, at least in the context of protein overexpression. In addition, the results of analysis of the paralogous small transmembrane protein AppX show that the two proteins could have some overlapping functionality in the cell and that both have the potential to interact with the CydAB complex.     

Determination and quantification of asiaticoside in endophytic fungus from <Emphasis Type="Italic">Centella asiatica</Emphasis> (L.) Urban

Centella asiatica (L.) Urban is a highly considered medicinal plant owing to its secondary metabolites asiaticoside, madecassoside, asiatic acid, and madecassic acid. The asiaticoside, one of the most important constituents of the plant, is a triterpenoid saponin having memory enhancement property. Given its medicinal properties, we isolated and characterized endophytic fungi from this plant with the aim to screen these microorganisms for asiaticoside production. In total, we isolated 13 endophytic fungi from the leaves of the plant, out of which one of the isolates produced asiaticoside. This asiaticoside producing isolate was identified as Colletotrichum gloeosporioides by internal transcribed spacer-based rDNA sequencing. The presence of asiaticoside in ethyl acetate extract of C. gloeosporioides was confirmed by LC–MS. The production of asiaticoside measured in relation to incubation time and subculture generation revealed presence of 62.29?±?3.36 µg/100 mL of asiaticoside by C. gloeosporioides on the 15th day in first subculture generation followed by a decrease in subsequent generations. A similar trend was also shown by yield and growth curve of C. gloeosporioides. The asiaticoside production and yield were found to be positively correlated. This paper reported the production of asiaticoside by an endophytic fungus C. gloeosporioides for the first time. The present findings definitely provide an impetus to the production of asiaticoside by utilizing the endophytic source.

Graphical Abstract

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Chemical compound studied in this article: Asiaticoside (PubChemCID: 108062)

     

Morphological,phytochemical and genetic diversity of threatened Polygonatum verticillatum (L.) All. populations of different altitudes and habitat types in Himalayan region

Polygonatum verticillatum (L.) All. is an important medicinal herb that belongs to the family Asparagaceae. The rhizome of the species is used in Chyavanprash preparation and several other ayurvedic formulations. Numerous active constituents like saponins, alkaloids, phytohormones, flavonoids, antioxidants, lysine, serine, aspartic acid, diosgenin, β-sitosterol, etc. have been reported from this species. In this study, morphological, phytochemical, antioxidant and genetic variations of 11 distant populations of P. verticillatum were measured. Considerably (P < 0.05) higher variations were recorded among different populations of P. verticillatum using morphological, phytochemical and genetic diversity parameters. AGFW (above ground fresh weights); flavonols, FRAP (Ferric ion reducing antioxidant power) and NO (Nitric Oxide scavenging activity) were recorded maximum in Kafni population. Similarly, a significantly higher above and below ground dry weight was recorded in Mayawati and Surmoli populations respectively. Maximum phenolic content, tannins, and DPPH (2,2-diphenyl-1-picrylhydrazyl) activity were recorded in Milam population. A total of 165 individuals from 11 populations were assessed for genetic diversity using inter-simple sequence repeats (ISSR) marker. High genetic diversity (He = 0.35) was recorded in Himkhola and Surmoli populations while it was observed minimum (0.28) in the Mayawati population. Altitude showed a significant positive correlation with tannins (r = 0.674; P < 005) and DPPH (r = 0.820; P < 0.01). Phenol content exhibited a considerably positive relationship with He (r = 0.606; P < 0.05) and BGFW (r = 0.620; P < 0.05), flavonol displayed a positive correlation with Pp% (r = 0.606; P < 0.05). The population structure of P. verticillatum, exhibited that the optimal value of the K was 3 for its populations as determined by the ΔK statistic structure. Among populations, the amount of gene flow is higher (Nm = 1.717) among all sites. Hence, it can be concluded that P. verticillatum populations possess considerable variability in the collected populations. Likewise, the populations from Kafni, Satbunga and Himkhola with higher morphological, phytochemicals and genetic variability were prioritized and therefore recommended for cultivation and mass multiplication to meet the industrial demand for target species.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12298-021-01044-9.