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Miscellaneous Withanolides
Published in Amritpal Singh Saroya, Contemporary Phytomedicines, 2017
Coagulin-L (Fig. 29.23) reduces the expressions of peroxisome proliferator-activated receptor y (PPARy) and CCAAT/enhancer-binding protein a, the major transcription factors orchestrating adipocyte differentiation. Detailed analysis further proved that early exposure of coagulin-L is sufficient to cause significant inhibition during adipogenesis (Beg et al. 2014).
Increasing the Sensitivity of Adipocytes and Skeletal Muscle Cells to Insulin
Published in Christophe Wiart, Medicinal Plants in Asia for Metabolic Syndrome, 2017
Coagulin C, 17β-hydroxywithanolide K, withanolide F, (17S,20S,22R)-14α,15α,17β,20β-tetrahydroxy-1-oxowitha-2,5,24-trienolide and coagulin L isolated from the dried fruits of Withania coagulans (Stocks) Dunal given at a single oral dose of 100 mg/kg to Sprague–Dawley rats and streptozotocin-induced diabetic Sprague-Dawley rats (glycemia between 144 and 270 mg/dL) 30 minutes before sucrose loading lowered postprandial glycemia by more than 30%.274 Coagulin L given to C57BL/Ksj-db/db mice at a dose of 50 mg/kg/days for 10 days lowered postprandial blood glucose by 22.7%. This regimen lowered triglycerides by 14.7%, total cholesterol by 25.7%, increased high-density lipoprotein–cholesterol by 24.7%, lowered low-density lipoprotein–cholesterol by 21.2% and lowered very low-density lipoprotein–cholesterol by 15.6%.274 Aqueous extract of fruits of Withania coagulans (Stocks) Dunal given to albino rabbits on cholesterol-enriched diet orally at a dose of 250 mg/kg/day for 6 weeks lowered plasma cholesterol from 362.1 to 157.8 mg/dL (normal diet group: 88.2 mg/dL), low density lipoproteins and increased high-density lipoproteins.275 This regimen lowered serum lipid peroxides, increased glutathione and superoxide dismutase activity.275 In the liver of treated animals, the extract lowered lipid contents, increased 3-hydroxy-3-methylglutaryl-coenzyme A CoA reductase activity, lowered acetyl-CoA carboxylase activity, lowered lipid peroxidation and increased glutathione and increased the enzymatic activity of superoxide dismutase.275 Coagulin L at a concentration of 15 μM inhibited dexamethasone-insulin-3-isobutyl-1-methylxanthine -induced differentiation of 3T3-L1 preadipocytes into adipocytes.276 This saponin inhibited the expression of sterol regulatory element-binding protein-1c, peroxisome proliferator-activated receptor-γ, CCAAT/enhancer-binding protein-α downstream target fatty acid-binding protein, lipoprotein lipase, fatty acid synthetase, and glucose transporter-4.276
Endotoxin Detection in Body Fluids: Chemical Versus Bioassay Methodology
Published in Helmut Brade, Steven M. Opal, Stefanie N. Vogel, David C. Morrison, Endotoxin in Health and Disease, 2020
On the basis of the above-cited studies, it has been assumed that all the circulating endotoxin was detected by the LAL test. Although the methodology for removing or inactivating inhibitors from the sample varies depending upon the specifics of the assay, two major approaches have been used. These include the heat/dilution method and the chemical treatment method. The objectives of both methods are (1) to release endotoxin bound to any blood components that might interfere with its ability to activate the clotting cascade, e.g., high-density lipoprotein; (2) to inactivate any components present in the biological samples that may directly interfere positively (enhancement) or negatively (inhibition) with the LAL reagent, e.g., serine proteases color; and (3) to adjust the concentration of endotoxin and other factors, e.g., divalent cations, for optimal reaction with LAL. Dilution/heating is the easiest method, although there is currently not full agreement on the optimal dilution factor or temperature. An additional problem occurs when heat-treated plasma or other proteins, e.g., albumin, is used as a sample in the turbidimetric assay (32). This problem manifests itself as an increase in turbidity (vs. the unheated control). Thus, a heated plasma sample with a known quantity of added endotoxin will appear to have more endotoxin than was added. Although the exact reason(s) for this phenomenon is unknown, it is likely due to a nonspecific increase in turbidity caused by an interaction between denatured (plasma) protein and coagulin. Increasing dilution (greater than 10-fold) will usually result in a more accurate result, however, this is accompanied by a loss of assay sensitivity. The extent of the nonspecific increase also varies with the sample (patient), necessitating use of an internal control. These problems do not seem as pronounced with either a chemical extraction or the chromogenic assay. Dilution will also decrease the sensitivity of the chromogenic assay, although this does not appear to be a drawback since dilution is limited to 10-fold or less. Chemical treatment, while easier to control, requires rather precise attention to the preparation of the extraction solutions. These consist of either acids and/or bases coupled with detergents and buffers (26). The addition of sample to the chemical extractant also results in some dilution, however, as with the heat/dilution method, this is not considered a drawback. Problems with this approach are that not only must the reagents used be prepared strictly in an endotoxin-free environment, but their stability must be taken into consideration. Thus, extraction “kits” are, in general, not commercially available. One trial involving a kit (SepTest) was successful in demonstrating sensitive endotoxin detection and practicality for potential use in a clinical laboratory, but was not commercialized because the endotoxin detected in septic patients (enrolled in the trial to evaluate this kit) did not show a strong correlation with any of the usual symptoms associated with sepsis (27). One, therefore, should not confuse the lack of clinical utility of endotoxin in the blood with the ability of LAL to detect circulating endotoxin.
Bioactive metabolites from the leaves of Withania adpressa
Published in Pharmaceutical Biology, 2018
Widad Ben Bakrim, Laila El Bouzidi, Jean-Marc Nuzillard, Sylvian Cretton, Noémie Saraux, Aymeric Monteillier, Philippe Christen, Muriel Cuendet, Khalid Bekkouche
Compound 5 was isolated as a white amorphous powder. HRESIMS showed a pseudo molecular ion peak at m/z 797.3853 [M + H]+ (calcd. 797.3864), indicating a molecular formula of C40H60O16. IR spectrum revealed the presence of a hydroxyl (3,420 cm−1), an α,β-unsaturated δ-lactone moiety (1715 cm−1) and a carboxyl (1690 cm−1). 1 H- and 13 C-NMR data are given in Table 1. The structure of compound 5 was partly deduced from that of coagulin L (3). The comparison of their 13 C-NMR chemical shifts (Table S1) showed that 5 contains an additional sugar unit in position 4′ compared to 3 (8 ppm deviation). The planar structure of compound 3 was determined by the analysis of 1 H, 13 C, COSY, HSQC and HMBC spectra and found to be identical to the one of coagulin L (Atta-ur-Rahman et al. 1998). The comparison of 13C-NMR chemical shifts of 2,3-dihydro-3β-hydroxywithanolide F (the aglycone of coagulin L) (Zhang and Timmermann 2016) or of the aglycone part of tetra-acetylated coagulin L (Atta-ur-Rahman et al. 1998), with those of compound 3, let us to conclude the identity of the latter was coagulin L, even though chemical shift comparison was biased by the use of different NMR solvents (CDCl3 and CD3OD) (Table 1). The absence of reported stereoisomers of coagulin L provides further support for our conclusion. The sugar unit in 5 is bound to the aglycone in position 3, as shown by the H-1′/C-3 correlation. A series of COSY correlations, starting from H-1′, revealed the chemical shifts of H-2′ to H-5′, all of these being in axial position, as deduced from the high value of their coupling constants. The HSQC spectrum indicated the chemical shift of H-6′a and H-6′b as those of a methylene group in a primary alcohol functional group whose connection with C-5′ was proven by the HMBC spectrum. The anomeric configuration of this glucose unit is β, as indicated by the high H-1′/H-2′ coupling constant (7.8 Hz).
Reactive oxygen species and immune regulation
Published in International Reviews of Immunology, 2020
Whereas in immune cells, especially in macrophage, the main origin of ROS is NADPH enzyme, whose catalytic subunit is called NADPH oxidase 2 (NOX2), which can be expressed on cytoplasmic membrane [2]. Six kinds of homo-leagues of NOX2 have been identified in different tissues: NOX1, NOX3, NOX4, NOX5, DUOX1 and DUOX2, collectively known as NOXs family. These NOXs can produce ROS through electrons transfer, and participate in the downstream signal activation of many membrane receptors [3, 4]. In respiratory epithelium, DUOX2 was demonstrated as the most abundant NOXs. Meanwhile, DUOX2-induced ROS effectively repressed influenza A virus (IAV) [5]. In diabetes model of Apoe(-/-) mice, inhibition of NOX-derived ROS prevented the diabetes-mediated increase in atherosclerotic plaque area as well as associated vascular T cell infiltration [6]. NOXs-derived ROS, which is also called reactive oxygen intermediates (ROI) further stimulates downstream molecules and pathway activation, thus promoted killing effect of lymphocytes. NF-kB is the most reported ROS-induced pathway and plays important role in immunity [7, 8]. ROS promoted the degradation of I kappa B (IkBa), the inhibitory subunit of NF-kB. Subsequently, NF-kB was rapidly transported to nuclear and activated [9]. In vitro, ROS activated NF-kB via targeting IkBa, thus promoted the expression and replication of HIV-1 in human T cell [10]. In vivo, coagulin-L repressed ROS production through inhibiting the mRNA expression of NOX2 and NOX4, followed by the activation of NF-kB as well as accumulation of NF-kB-induced inflammatory cytokines [11]. Interestingly, previous research indicated a negative feedback between ROS and NF-kB. Over-activation of NF-kB might repress the production of ROS [12]. PI3k/Akt was another important ROS related pathway. In one hand, the generation of ROS was regulated by PI3k/Akt [13]. In another hand, PI3K/Akt was also the downstream of ROS [14, 15]. H. pylori infection promoted ROS accumulation, thus activated PI3K/Akt pathway as well as DNA damage [16]. In summary, NOXs was the main origin of ROS in immune cells. NOXs-derived ROS regulated play different roles in immune response via different pathways.