Highly Oxidized Ecdysteroids from a Commercial Cyanotis arachnoidea Root Extract as Potent Blood-Brain Barrier Protective Agents.

Ecdysteroid-containing herbal extracts, commonly prepared from the roots of Cyanotis arachnoidea, are marketed worldwide as a "green" anabolic food supplement. Herein are reported the isolation and complete 1H and 13C NMR signal assignments of three new minor ecdysteroids (compounds 2-4) from this extract. Compound 4 was identified as a possible artifact that gradually forms through the autoxidation of calonysterone. The compounds tested demonstrated a significant protective effect on the blood-brain barrier endothelial cells against oxidative stress or inflammation at a concentration of 1 µM. Based on these results, minor ecdysteroids present in food supplements may offer health benefits in various neurodegenerative disease states.

Utilizing the 1H-15N NMR Methods for the Characterization of Isomeric Human Milk Oligosaccharides.

Human milk oligosaccharides (HMOs) are structurally complex unconjugated glycans that are the third largest solid fraction in human milk after lactose and lipids. HMOs are in the forefront of research since they have been proven to possess beneficial health effects, especially on breast-fed neonates. Although HMO research is a trending topic nowadays, readily available analytical methods suitable for the routine investigation of HMOs are still incomplete. NMR spectroscopy provides detailed structural information that can be used to indicate subtle structural differences, particularly for isomeric carbohydrates. Herein, we propose an NMR-based method to identify the major isomeric HMOs containing GlcNAc and/or Neu5Ac building blocks utilizing their amide functionality. Experimental conditions were optimized (H2O:D2O 9:1 v/v solvent at pH 3.0) to obtain 1H-15N HSQC and 1H-15N HSQC-TOCSY NMR spectra of the aforementioned building blocks in HMOs. Four isomeric HMO pairs, LNT/LNnT, 3'SL/6'SL, LNFP II/LNFP III, and LSTa/LSTb, were investigated, and complete NMR resonance assignments were provided. In addition, 1H and 15N NMR resonances were found to be indicative of various linkages, thereby facilitating the distinction of isomeric tri-, tetra-, and pentasaccharide HMOs. The rapid growth of HMO products (from infant formulas and dietary supplements to cosmetics) undoubtedly requires expanding the range of applicable analytical methods. Thus, our work provides a 15N NMR-based method to advance this challenging field of carbohydrate analysis.

The effects of immersion in 42℃ radon, natrium, calcium, bicarbonate content thermal-mineral water on chronic low back pain. Controlled, follow-up study.

In our minimized follow-up trial with 137 participants with chronic low back pain, one group of participants received regular outpatient care, and the other group received balneotherapy by immersion in 42℃ thermal-mineral water in addition to regular outpatient care on 15 occasions for 3 weeks. Pain on movement and at rest on the 0-100 mm visual analogue scale (VAS), Oswestry index, the number of participants evaluating the symptoms clinically acceptable (Patient Acceptable Symptom State, PASS) and the EuroQol-5D-5L (EQ-5D-5L) quality of life questionnaire were assessed at basal time (at week 0) and after balneotherapy (at weeks 3 and 12). The VAS pain scores, the Oswestry index, the EQ-5D-5L index and the EQ-VAS significantly improved in the balneotherapy group after treatment at week 3 (p < 0.001) and week 12 (p < 0.001) compared to baseline, with a significant between group difference at week 3 (p < 0.001) and week 12 (p < 0.001). The pain VAS score on movement was 66.82 ± 11.48, 26.69 ± 21.49, and 20.09 ± 23.29 in the balneotherapy group, and 63.67 ± 14.77, 67.35 ± 15.44, and 70.23 ± 18.26 in the control group at the consecutive visits. The PASS increased in both groups at week 3 and week 12 compared to the baseline, with a significant between-group difference at week 3 and week 12 for the balneotherapy group. Our results suggest the therapeutic efficacy of immersion in 42℃ thermal mineral water on chronic low back pain.ClinicalTrials.gov Identifier: NCT05342051.

Oxidized Resveratrol Metabolites as Potent Antioxidants and Xanthine Oxidase Inhibitors.

Resveratrol is a well-known natural polyphenol with a plethora of pharmacological activities. As a potent antioxidant, resveratrol is highly oxidizable and readily reacts with reactive oxygen species (ROS). Such a reaction not only leads to a decrease in ROS levels in a biological environment but may also generate a wide range of metabolites with altered bioactivities. Inspired by this notion, in the current study, our aim was to take a diversity-oriented chemical approach to study the chemical space of oxidized resveratrol metabolites. Chemical oxidation of resveratrol and a bioactivity-guided isolation strategy using xanthine oxidase (XO) and radical scavenging activities led to the isolation of a diverse group of compounds, including a chlorine-substituted compound (2), two iodine-substituted compounds (3 and 4), two viniferins (5 and 6), an ethoxy-substituted compound (7), and two ethoxy-substitute,0d dimers (8 and 9). Compounds 4, 7, 8, and 9 are reported here for the first time. All compounds without ethoxy substitution exerted stronger XO inhibition than their parent compound, resveratrol. By enzyme kinetic and in silico docking studies, compounds 2 and 4 were identified as potent competitive inhibitors of the enzyme, while compound 3 and the viniferins acted as mixed-type inhibitors. Further, compounds 2 and 9 had better DPPH scavenging activity and oxygen radical absorbing capacity than resveratrol. Our results suggest that the antioxidant activity of resveratrol is modulated by the effect of a cascade of chemically stable oxidized metabolites, several of which have significantly altered target specificity as compared to their parent compound.

Diversity-Oriented Synthesis Catalyzed by Diethylaminosulfur-Trifluoride-Preparation of New Antitumor Ecdysteroid Derivatives.

Fluorine represents a privileged building block in pharmaceutical chemistry. Diethylaminosulfur-trifluoride (DAST) is a reagent commonly used for replacement of alcoholic hydroxyl groups with fluorine and is also known to catalyze water elimination and cyclic Beckmann-rearrangement type reactions. In this work we aimed to use DAST for diversity-oriented semisynthetic transformation of natural products bearing multiple hydroxyl groups to prepare new bioactive compounds. Four ecdysteroids, including a new constituent of Cyanotis arachnoidea, were selected as starting materials for DAST-catalyzed transformations. The newly prepared compounds represented combinations of various structural changes DAST was known to catalyze, and a unique cyclopropane ring closure that was found for the first time. Several compounds demonstrated in vitro antitumor properties. A new 17-N-acetylecdysteroid (13) exerted potent antiproliferative activity and no cytotoxicity on drug susceptible and multi-drug resistant mouse T-cell lymphoma cells. Further, compound 13 acted in significant synergism with doxorubicin without detectable direct ABCB1 inhibition. Our results demonstrate that DAST is a versatile tool for diversity-oriented synthesis to expand chemical space towards new bioactive compounds.

A Commercial Extract of Cyanotis arachnoidea Roots as a Source of Unusual Ecdysteroid Derivatives with Insect Hormone Receptor Binding Activity.

Ecdysteroids act as molting hormones in insects and as nonhormonal anabolic agents and adaptogens in mammals. A wide range of ecdysteroid-containing herbal extracts are available worldwide as food supplements. The aim of this work was to study such an extract as a possible industrial source of new bioactive ecdysteroids. A large-scale chromatographic isolation was performed from an extract of Cyanotis arachnoidea roots. Ten ecdysteroids (1-10) including eight new compounds were isolated and characterized by extensive nuclear magnetic resonance studies. Highly unusual structures were identified, including a H-14ß (1, 2, 4, and 10) moiety, among which a 14ß(H)17ß(H) phytosteroid (1) is reported for the first time. Compounds with an intact side chain (4-10) and 11 other natural or semisynthetic ecdysteroids (11-21) were tested for insect ecdysteroid receptor (EcR) binding activity. Two new compounds, i.e., 14-deoxydacryhainansterone (5) and 22-oxodacryhainansterone (6), showed strong EcR binding activity (IC50 = 41.7 and 380 nM, respectively). Six compounds were identified as EcR agonists and another two as antagonists using a transgenic ecdysteroid reporter gene assay. The present results demonstrate that commercial C. arachnoidea extracts are rich in new, unusual bioactive ecdysteroids. Because of the lack of an authentic plant material, the truly biosynthetic or artifactual nature of these compounds cannot be confirmed.

Squalenoylated Nanoparticle Pro-Drugs of Adjuvant Antitumor 11α-Hydroxyecdysteroid 2,3-Acetonides Act as Cytoprotective Agents Against Doxorubicin and Paclitaxel.

Several ecdysteroid acetonides act as adjuvant chemo-sensitizing agents against various cancer cell lines, and they can be formulated to self-assembling nanoparticle (NP) pro-drugs through a hydrolysable conjugation with squalene. In the bloodstream such squalenoylated nanoparticles dissolve into low-density lipoprotein (LDL) that allows targeting tissues containing high levels of LDL-receptors. In this work, ajugasterone C 2,3;20,22-diacetonide (3) and 11α-hydroxypoststerone 2,3-acetonide (4) were squalenoylated to obtain two new ecdysteroid pro-drugs (6 and 7) and their nano-assemblies (6NP and 7NP ). A complete NMR signal assignment of 6 and 7 was achieved. Interaction of compounds 3 and 4 with chemotherapeutics was studied by the Chou-Talalay method. Compound 3 showed strong synergism with doxorubicin on a multi-drug resistant lymphoma cell line. In contrast, its nanoassembly 6NP significantly decreased the cytotoxicity of doxorubicin on these MDR cells, strongly suggesting that at least the 2,3-acetonide group was cleaved by the acidic pH of lysosomes after endocytosis of the prodrug. Further, compound 4 acted in strong antagonism with paclitaxel on MCF-7 cells and its nanoassemby 7NP also protected MCF-7 cells from the effect of paclitaxel. Our results suggest that acid-resistant A-ring substitution would be crucial to design adjuvant antitumor squalenoylated ecdysteroid prodrugs. Additionally, our results may be considered as a serendipitous discovery of a novel way to deliver cytoprotective, adaptogen ecdysteroids to healthy tissues with upregulated LDL-R.

A multicentre randomized controlled follow-up study of the effects of the underwater traction therapy in chronic low back pain.

Low back pain (LBP) is one of the most costly diseases in the developed world. This study aimed to investigate the effects of underwater traction therapy on chronic low back pain. The primary objective was to prove that underwater traction therapy has favorable effects on LBP. Our secondary objective was to evaluate whether it also leads to improvement in the quality of life. This is a prospective, multicenter, follow-up study. A total of 176 patients with more than 3 months of low back pain enrolled from outpatient clinics were randomized into three groups: underwater weight bath traction therapy and non-steroidal anti-inflammatory drugs (NSAIDs); weight bath; and only NSAIDs. The following parameters were measured before, right after, and 9 weeks after the 3-week therapy: levels of low back pain in rest and during activity were tested using the visual analogue scale (VAS), the Oswestry Low Back Disability Questionnaire, and the EuroQol-5D-5L Questionnaire.The VAS levels improved significantly (p < 0.05) in both underwater weight bath traction therapy groups by the end of the treatment, whereas the improvement in the third group was not statistically significant. Furthermore, the improvements measured in the groups receiving traction therapy were persistent during the follow-up period. There were no significant changes in the Oswestry Index or the EuroQol-5D-5L without VAS parameters in any of the groups.Based on our results, for patients suffering from LBP pain who underwent underwater weight bath traction therapy, there were favorable impacts on the pain levels at rest or during activity. Clinical trial registration ID: NCT03488498, April 5, 2018.

Chemoselective Strategy for the Direct Formation of Tetrahydro-2,5-methanobenzo[ c]azepines or Azetotetrahydroisoquinolines via Regio- and Stereoselective Reactions.

The present study reports regio- and highly diastereoselective preparative methods for the synthesis of versatile alkaloid-type compounds from oxiranylmethyl tetrahydroisoquinolines. 2,5-Methanobenzo[ c]azepines or azetidine-fused heterocycles were synthesized in tandem reactions depending on the absence or presence of a BF3 co-reagent. A high functional group tolerance has also been demonstrated. DFT calculations with an explicit solvent model confirmed the proposed reaction mechanisms and the role of kinetic controls on the stereochemical outcome of the reported new methods.

Flow-chemistry enabled efficient synthesis of ß-peptides: backbone topology vs. helix formation.

Enantiodiscriminative helix formation was observed for ß-peptide H14 helices. This observation is caused by the synperiplanar orientation of H-O atoms which is more unfavorable than those for H-H interaction. The 1,2 H-O interaction leads to the destruction of the helical structure. The introduction of a double C-C bond in the backbone rules out helix formation.

Side-chain cleaved phytoecdysteroid metabolites as activators of protein kinase B.

Phytoecdysteroids exert their non-hormonal anabolic and adaptogenic effects in mammals, including humans, through a partially revealed mechanism of action involving the activation of protein kinase B (Akt). We have recently found that poststerone, a side-chain cleaved in vivo metabolite of 20-hydroxyecdysone, exerts potent anabolic activity in rats. Here we report the semi-synthetic preparation of a series of side-chain cleaved ecdysteroids and their activity on the Akt phosphorylation in murine skeletal muscle cells. Twelve C-21 ecdysteroids including 8 new compounds were obtained through the oxidative side-chain cleavage of various phytoecdysteroids, or through the base-catalyzed autoxidation of poststerone. The complete 1H and 13C NMR spectroscopic assignments of the new compounds are presented. Among the tested compounds, 9 could activate Akt stronger than poststerone revealing that side-chain cleaved derivatives of phytoecdysteroids other than 20-hydroxyecdysone are valuable bioactive metabolites. Thus, our results suggest that the expectable in vivo formation of such compounds should contribute to the bioactivity of herbal preparations containing ecdysteroid mixtures.

Correction to: The effects of the calcium-magnesium-bicarbonate content in thermal mineral water on chronic low back pain: a randomized, controlled follow-up study.

The original article mistakenly displays each set of author names in the wrong order, i.e., first names as last names and vice versa. The author correct names are: Tamás Gáti, Ildikó Katalin Tefner, Lajos Kovács, Katalin Hodosi, Tamás Bender. The original article has been corrected.

The effects of the calcium-magnesium-bicarbonate content in thermal mineral water on chronic low back pain: a randomized, controlled follow-up study.

The aim of this study was to investigate the effects of balneotherapy on chronic low back pain. This is a minimized, follow-up study evaluated according to the analysis of intention to treat. The subjects included in the study were 105 patients suffering from chronic low back pain. The control group (n = 53) received the traditional musculoskeletal pain killer treatment, while the target group (n = 52) attended thermal mineral water treatment for 3 weeks for 15 occasions on top of the usual musculoskeletal pain killer treatment. The following parameters were measured before, right after, and 9 weeks after the 3-week therapy: the level of low back pain in rest and the level during activity are tested using the Visual Analog Scale (VAS); specific questionnaire on the back pain (Oswestry); and a questionnaire on quality of life (EuroQual-5D). All of the investigated parameters improved significantly (p < 0.001) in the target group by the end of the treatment compared to the base period, and this improvement was persistent during the follow-up period. There were no significant changes in the measured parameters in the control group. Based on our results, balneotherapy might have favorable impact on the clinical parameters and quality of life of patients suffering from chronic low back pain.

Nitrogen-containing ecdysteroid derivatives vs. multi-drug resistance in cancer: Preparation and antitumor activity of oximes, oxime ethers and a lactam.

Multidrug resistance is a widespread problem among various diseases and cancer is no exception. We had previously described the chemo-sensitizing activity of ecdysteroid derivatives with low polarity on drug susceptible and multi-drug resistant (MDR) cancer cells. We have also shown that these molecules have a marked selectivity towards the MDR cells. Recent studies on the oximation of various steroid derivatives indicated remarkable increase in their antitumor activity, but there is no related bioactivity data on ecdysteroid oximes. In our present study, 13 novel ecdysteroid derivatives (oximes, oxime ethers and a lactam) and one known compound were synthesized from 20-hydroxyecdysone 2,3;20,22-diacetonide and fully characterized by comprehensive NMR techniques revealing their complete 1H and 13C signal assignments. The compounds exerted moderate to strong in vitro antiproliferative activity on HeLa, SiHa, MCF-7 and MDA-MB-231 cell lines. Oxime and particularly oxime ether formation strongly increased their inhibitory activity on the efflux of rhodamine 123 by P-glycoprotein (P-gp), while the new ecdysteroid lactam did not interfere with the efflux function. All compounds exerted potent chemo-sensitizing activity towards doxorubicin on a mouse lymphoma cell line and on its MDR counterpart, and, on the latter, the lactam was found the most active. Because of its MDR-selective chemo-sensitizing activity with no functional effect on P-gp, this lactam is of high potential interest as a new lead for further antitumor studies.

Regulatory B cells in rheumatoid arthritis: Alterations in patients receiving anti-TNF therapy.

Cytokines, including tumor necrosis factor alpha (TNFα) are involved in Rheumatoid arthritis (RA) pathogenesis by augmenting autoimmunity, sustaining long term inflammation in the synovium, and promoting joint damage. Anti-TNF therapy is one of the most efficient and widely used therapies for RA, although its mechanism is not clarified yet. Earlier we demonstrated that RA patients have a reduced number of IL-10 producing regulatory B cells (B10 cells) as compared to healthy individuals and they are functionally impaired. Our aim was to study the influence of anti-TNF therapy on B10 cells in RA, to follow the alteration of B cell activation markers (CD25, CD69) and to monitor the level of citrullinated peptid-specific antibodies and the secreted IL-10 in patients' sera during the therapy. We have observed that at six month after starting the therapy the frequency of B10 cells remarkably increased, while the expression of the activation marker, CD69 decreased on B cells. In contrast, serum levels of IL-10 and anti-citrullinated peptide antibodies did not change post-treatment. CONCLUSION: The reduced activation state of B cells and the increasing number of regulatory B10. cells might contribute to the therapeutic efficacy of anti-TNF agents in RA.

Induction and Differentiation of IL-10-Producing Regulatory B Cells from Healthy Blood Donors and Rheumatoid Arthritis Patients.

The most important feature of B cells is the production of Abs upon activation; additionally, B cells produce pro- and anti-inflammatory cytokines in response to certain stimuli. IL-10-producing B cells represent a major subset of regulatory B cells (Bregs) that suppress autoimmune and inflammatory responses. B cells play a crucial role in the development and maintenance of the chronic inflammatory autoimmune disease rheumatoid arthritis (RA); however, controversial data are available on IL-10- producing Bregs in RA. Our aim was to identify the optimal conditions that induce IL-10+ Bregs and, furthermore, to shed light on the signaling pathways that are responsible for their expansion. The results show that dual stimulation by CpG and CD40L for 48 h is optimal for IL-10 induction, and this can be synergistically boosted by IL-21. We identified the CD19+CD27+ memory B cell population as the major source of IL-10+ Bregs. We detected significantly fewer CD19+CD27+IL-10+ cells in RA patients compared with healthy controls, and these were functionally defective in suppressing IFN-γ production by CD4+ T cells in coculture. IL-21 drastically increased the number of IL-10+ Bregs within the CD19+CD27+ and CD19+CD27- populations; furthermore, it induced the appearance of IL-10+Blimp-1+ plasmablasts. Monitoring the phosphorylation of key signaling molecules revealed that activation of ERK, p38, and CREB is indispensable for the induction of IL-10 production, whereas phosphorylation of STAT3 further enhances IL-10 expression in human Bregs. We conclude that CREB and STAT3 are the key transcription factors responsible for the expansion and differentiation of human IL-10-producing Bregs.

Expedient and Diastereodivergent Assembly of Terpenoid Decalin Subunits having Quaternary Stereocenters through Organocatalytic Robinson Annulation of Nazarov Reagent.

We report an expedient approach to highly functionalized cis- and trans-decalines that could function as key structural subunits toward the synthesis of various classes of terpenoids. Key to the strategy is an organocatalyzed Robinson annulation reaction of the Nazarov reagent that affords chiral enone building blocks with high enantioselectivities. The quaternary carbon stereogenic center can direct the subsequent reactions and allow the rapid and diastereoconvergent assembly of complex decalines with contiguous stereocenters.