Betulin and Its Derivatives Reduce Inflammation and COX-2 Activity in Macrophages.

Betulin is a heavily studied natural compound for its use as an anticancer or pro-regenerative agent. The structural similarity between betulin to steroids gives rise to the idea that the substance may as well act as an anti-inflammatory drug. This study is the first to describe the anti-inflammatory properties of betulinic acid, betulin, and its derivatives with amino acids 1,4-diaminebutane (Dab), 1,3-diaminepropane (Dap), Ornithine (Orn), and lysine (Lys) on murine macrophages from lymphoma site. The compounds were compared to dexamethasone. To establish the response of the macrophages to the natural compounds, we tested the viability as well as sensitivity to the inflammatory signaling (IFNγR). IL-6 secretory properties and HSP-70 content in the cells were examined. Furthermore, we characterized the effects of compounds on the inhibition of cyclooxygenase-2 (COX-2) activity both in the enzymatic assays and molecular docking studies. Then, the changes in COX-2 expression after betulin treatment were assessed. Betulin and betulinic acid are the low-cytotoxicity compounds with the highest potential to decrease inflammation via reduced IL-6 secretion. To some extent, they induce the reorganization of IFNγR with nearly no effect on COX-2 activity. Conversely, Bet-Orn and Bet-Lys are highly cytotoxic and induce the aggregation of IFNγR. Besides, Bet-Lys reduces the activity of COX-2 to a higher degree than dexamethasone. Bet-Orn is the only one to increase the HSP-70 content in the macrophages. In case of IL-6 reduction, all compounds were more potent than dexamethasone.

Non-psychotropic cannabinoids as inhibitors of TET1 protein.

Non-psychotropic cannabinoids (e.g., cannabidiol, cannabinol and cannabigerol) are contained in numerous alimentary and medicinal products. Therefore, predicting and studying their possible side effects, such as changes in DNA methylation, is an important task for assessing the safety of these products. Interference with TET enzymes by chelating ferrous ions can contribute to the altered methylation pattern. All tested cannabinoids displayed a strong affinity for Fe(II) ions. Cannabidiol and cannabinol exhibited potent inhibitory activities (IC50 = 4.8 and 6.27 µM, respectively) towards the TET1 protein, whereas cannabigerol had no effect on the enzyme activity. An in silico molecular docking study revealed marked binding potential within the catalytic cavity for CBD/CBN, but some affinity was also found for CBG, thus the total lack of activity remains unexplained. These results imply that cannabinoids could affect the activity of the TET1 protein not only due to their affinity for Fe(II) but also due to other types of interactions (e.g., hydrophobic interactions and hydrogen bonding).

A Comparative Analysis of COVID-19 Vaccines Based on over 580,000 Cases from the Vaccination Adverse Event Reporting System.

Background: The COVID-19 pandemic is being battled via the largest vaccination campaign in history, with more than eight billion doses administered thus far. Therefore, discussions about potentially adverse reactions, and broader safety concerns, are critical. The U.S. Vaccination Adverse Event Reporting System (VAERS) has recorded vaccination side effects for over 30 years. About 580,000 events have been filed for COVID-19 thus far, primarily for the Johnson & Johnson (New Jersey, USA), Pfizer/BioNTech (Mainz, Germany), and Moderna (Cambridge, USA) vaccines. Methods: Using available databases, we evaluated these three vaccines in terms of the occurrence of four generally-noticed adverse reactions­namely, cerebral venous sinus thrombosis, Guillain−Barré syndrome (a severe paralytic neuropathy), myocarditis, and pericarditis. Our statistical analysis also included a calculation of odds ratios (ORs) based on total vaccination numbers, accounting for incidence rates in the general population. Results: ORs for a number of adverse events and patient groups were (largely) increased, most notably for the occurrence of cerebral venous sinus thrombosis after vaccination with the Johnson & Johnson vaccine. The overall population OR of 10 increases to 12.5 when limited to women, and further yet (to 14.4) among women below age 50 yrs. In addition, elevated risks were found (i) for Guillain−Barré syndrome (OR of 11.6) and (ii) for myocarditis/pericarditis (ORs of 5.3/4.1, respectively) among young men (<25 yrs) vaccinated with the Pfizer/BioNTech vaccine. Conclusions: Any conclusions from such a retrospective, real-world data analysis must be drawn cautiously, and should be confirmed by prospective double-blinded clinical trials. In addition, we emphasize that the adverse events reported here are not specific side effects of COVID vaccines, and the significant, well-established benefits of COVID-19 vaccination outweigh the potential complications surveyed here.

SuperTCM: A biocultural database combining biological pathways and historical linguistic data of Chinese Materia Medica for drug development.

AIM OF THE STUDY: Botanicals used in Traditional Chinese Medicine (TCM) are a rich source for drug discovery and provide models for multi-component drug development. To facilitate the studies of the actions of TCM drugs and expand their applications, a comprehensive database is urgently required. METHODS: One online resource connects all the relevant data from multiple scientific sources and languages. Drug information from published TCM databases and the official Chinese Pharmacopoeia as well as specialized meta-websites such as Kew's Medicinal Plant Names Service was integrated on a higher level. RESULTS: Our database, SuperTCM, covers the aspects of TCM derived from medicinal plants, encompassing pharmacological recipes up to chemical compounds. It provides the information for 6516 TCM drugs (or "herbs") with 5372 botanical species, 55,772 active ingredients against 543 targets in 254 KEGG pathways associated with 8634 diseases. SuperTCM is freely available at http://tcm.charite.de/supertcm.

Reynoutria Rhizomes as a Natural Source of SARS-CoV-2 Mpro Inhibitors-Molecular Docking and In Vitro Study.

More than a year has passed since the world began to fight the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) responsible for the Coronavirus disease 2019 (COVID-19) pandemic, and still it spreads around the world, mutating at the same time. One of the sources of compounds with potential antiviral activity is Traditional Chinese Medicinal (TCM) plants used in China in the supportive treatment of COVID-19. Reynoutria japonica is important part of the Shu Feng Jie Du Granule/Capsule-TCM herbal formula, recommended by China Food and Drug Administration (CFDA) for treatment of patients with H1N1- and H5N9-induced acute lung injury and is also used in China to treat COVID-19, mainly combined with other remedies. In our study, 25 compounds from rhizomes of R. japonica and Reynoutria sachalinensis (related species), were docked into the binding site of SARS-CoV-2 main protease. Next, 11 of them (vanicoside A, vanicoside B, resveratrol, piceid, emodin, epicatechin, epicatechin gallate, epigallocatechin gallate, procyanidin B2, procyanidin C1, procyanidin B2 3,3'-di-O-gallate) as well as extracts and fractions from rhizomes of R. japonica and R. sachalinensis were tested in vitro using a fluorescent peptide substrate. Among the tested phytochemicals the best results were achieved for vanicoside A and vanicoside B with moderate inhibition of SARS-CoV-2 Mpro, IC50 = 23.10 µM and 43.59 µM, respectively. The butanol fractions of plants showed the strongest inhibition of SARS-CoV-2 Mpro (IC50 = 4.031 µg/mL for R. sachalinensis and IC50 = 7.877 µg/mL for R. japonica). As the main constituents of butanol fractions, besides the phenylpropanoid disaccharide esters (e.g., vanicosides), are highly polymerized procyanidins, we suppose that they could be responsible for their strong inhibitory properties. As inhibition of SARS-CoV-2 main protease could prevent the replication of the virus our research provides data that may explain the beneficial effects of R. japonica on COVID-19 and identify the most active compounds worthy of more extensive research.

Allosteric Modulation of the CB1 Cannabinoid Receptor by Cannabidiol-A Molecular Modeling Study of the N-Terminal Domain and the Allosteric-Orthosteric Coupling.

The CB1 cannabinoid receptor (CB1R) contains one of the longest N termini among class A G protein-coupled receptors. Mutagenesis studies suggest that the allosteric binding site of cannabidiol (CBD) involves residues from the N terminal domain. In order to study the allosteric binding of CBD to CB1R we modeled the whole N-terminus of this receptor using the replica exchange molecular dynamics with solute tempering (REST2) approach. Then, the obtained structures of CB1R with the N terminus were used for ligand docking. A natural cannabinoid receptor agonist, Δ9-THC, was docked to the orthosteric site and a negative allosteric modulator, CBD, to the allosteric site positioned between extracellular ends of helices TM1 and TM2. The molecular dynamics simulations were then performed for CB1R with ligands: (i) CBD together with THC, and (ii) THC-only. Analyses of the differences in the residue-residue interaction patterns between those two cases allowed us to elucidate the allosteric network responsible for the modulation of the CB1R by CBD. In addition, we identified the changes in the orthosteric binding mode of Δ9-THC, as well as the changes in its binding energy, caused by the CBD allosteric binding. We have also found that the presence of a complete N-terminal domain is essential for a stable binding of CBD in the allosteric site of CB1R as well as for the allosteric-orthosteric coupling mechanism.

Oxidative testicular injury: effect of L-leucine on redox, cholinergic and purinergic dysfunctions, and dysregulated metabolic pathways.

The antioxidant and anti-proinflammatory activities of L-leucine were investigated on oxidative testicular injury, ex vivo. In vitro analysis revealed L-leucine to be a potent scavenger of free radicals, while inhibiting acetylcholinesterase activity. Oxidative injury was induced in testicular tissues using FeSO4. Treatment with L-leucine led to depletion of oxidative-induced elevated levels of NO, MDA, and myeloperoxidase activity, with concomitant elevation of reduced glutathione and non-protein thiol levels, SOD and catalase activities. L-leucine caused a significant (p < 0.05) alteration of oxidative-elevated acetylcholinesterase and chymotrypsin activities, while concomitantly elevating the activities of ATPase, ENTPDase and 5'-nucleotidase. L-leucine conferred a protective effect against oxidative induced DNA damage. Molecular docking revealed molecular interactions with COX-2, IL-1 beta and iNOS. Treatment with L-leucine led to restoration of oxidative depleted ascorbic acid-2-sulfate, with concomitant depletion of the oxidative induced metabolites: D-4-Hydroxy-2-oxoglutarate, L-cystine, adenosine triphosphate, maleylacetoacetic acid, cholesteryl ester, and 6-Hydroxy flavin adenine dinucleotide. Treatment with L-leucine reactivated glycolysis while concomitantly deactivating oxidative-induced citrate cycle and increasing the impact-fold of purine metabolism pathway. L-leucine was predicted not to be an inhibitor of CYP1A2, CYP2C19, CYP2C9, CYP2D6, and CYP3A4, with a predicted LD50 value of 5000 mg/Kg and toxicity class of 5. Additionally, L-leucine showed little or no in vitro cytotoxicity in mammalian cells. These results suggest the therapeutic potentials of L-leucine on oxidative testicular injury, as evident by its ability to attenuate oxidative stress and proinflammation, while stalling cholinergic dysfunction and modulating nucleotide hyrolysis; as well as modulate oxidative dysregulated metabolites and their pathways.

Cytotoxic Effect of Vanicosides A and B from Reynoutria sachalinensis Against Melanotic and Amelanotic Melanoma Cell Lines and in silico Evaluation for Inhibition of BRAFV600E and MEK1.

Vanicosides A and B are the esters of hydroxycinnamic acids with sucrose, occurring in a few plant species from the Polygonaceae family. So far, vanicosides A and B have not been evaluated for anticancer activity against human malignant melanoma. In this study, we tested these two natural products, isolated from Reynoutria sachalinensis rhizomes, against two human melanoma cell lines (amelanotic C32 cell line and melanotic A375 cell line, both bearing endogenous BRAFV600E mutation) and two normal human cell lines-keratinocytes (HaCaT) and the primary fibroblast line. Additionally, a molecular docking of vanicoside A and vanicoside B with selected targets involved in melanoma progression was performed. Cell viability was studied using an MTT assay. A RealTime-Glo™ Annexin V Apoptosis and Necrosis assay was used for monitoring programmed cell death (PCD). Vanicoside A demonstrated strong cytotoxicity against the amelanotic C32 cell line (viability of the C32 cell line was decreased to 55% after 72 h incubation with 5.0 µM of vanicoside A), significantly stronger than vanicoside B. This stronger cytotoxic activity can be attributed to an additional acetyl group in vanicoside A. No significant differences in the cytotoxicity of vanicosides were observed against the less sensitive A375 cell line. Moreover, vanicosides caused the death of melanoma cells at concentrations from 2.5 to 50 µM, without harming the primary fibroblast line. The keratinocyte cell line (HaCaT) was more sensitive to vanicosides than fibroblasts, showing a clear decrease in viability after incubation with 25 µM of vanicoside A as well as a significant phosphatidylserine (PS) exposure, but without a measurable cell death-associated fluorescence. Vanicosides induced an apoptotic death pathway in melanoma cell lines, but because of the initial loss of cell membrane integrity, an additional cell death mechanism might be involved like permeability transition pore (PTP)-mediated necrosis that needs to be explored in the future. Molecular docking indicated that both compounds bind to the active site of the BRAFV600E kinase and MEK-1 kinase; further experiments on their specific inhibitory activity of these targets should be considered.

Norditerpenoids with Selective Anti-Cholinesterase Activity from the Roots of Perovskia atriplicifolia Benth.

Inhibition of cholinesterases remains one of a few available treatment strategies for neurodegenerative dementias such as Alzheimer's disease and related conditions. The current study was inspired by previous data on anticholinesterase properties of diterpenoids from Perovskia atriplicifolia and other Lamiaceae species. The acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibition by the three new natural compounds-(1R,15R)-1-acetoxycryptotanshinone (1), (1R)-1-acetoxytanshinone IIA (2), and (15R)-1-oxoaegyptinone A (3)-as well as, new for this genus, isograndifoliol (4) were assessed. Three of these compounds exhibited profound inhibition of butyrylcholinesterase (BChE) and much weaker inhibition of acetylcholinesterase (AChE). All compounds (1-4) selectively inhibited BChE (IC50 = 2.4, 7.9, 50.8, and 0.9 µM, respectively), whereas only compounds 3 and 4 moderately inhibited AChE (IC50 329.8 µM and 342.9 µM). Molecular docking and in silico toxicology prediction studies were also performed on the active compounds. Natural oxygenated norditerpenoids from the traditional Central Asian medicinal plant P. atriplicifolia are selective BChE inhibitors. Their high potential makes them useful candidate molecules for further investigation as lead compounds in the development of a natural drug against dementia caused by neurodegenerative diseases.

Computational Prediction of Potential Inhibitors of the Main Protease of SARS-CoV-2.

The rapidly developing pandemic, known as coronavirus disease 2019 (COVID-19) and caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has recently spread across 213 countries and territories. This pandemic is a dire public health threat-particularly for those suffering from hypertension, cardiovascular diseases, pulmonary diseases, or diabetes; without approved treatments, it is likely to persist or recur. To facilitate the rapid discovery of inhibitors with clinical potential, we have applied ligand- and structure-based computational approaches to develop a virtual screening methodology that allows us to predict potential inhibitors. In this work, virtual screening was performed against two natural products databases, Super Natural II and Traditional Chinese Medicine. Additionally, we have used an integrated drug repurposing approach to computationally identify potential inhibitors of the main protease of SARS-CoV-2 in databases of drugs (both approved and withdrawn). Roughly 360,000 compounds were screened using various molecular fingerprints and molecular docking methods; of these, 80 docked compounds were evaluated in detail, and the 12 best hits from four datasets were further inspected via molecular dynamics simulations. Finally, toxicity and cytochrome inhibition profiles were computationally analyzed for the selected candidate compounds.

Analysis of Antioxidant Polyphenols in Loquat Leaves using HPLC-based Activity Profiling.

Leaves of Eriobotryajaponica (loquat) have been used in Traditional Chinese Medicine with beneficial effects in numerous diseases. Extracts from loquat leaves are rich in antioxidants, containing among others: triterpenes, sesquiterpenes, flavonoids, tannins, and megastigmane glycosides. However, there is no conclusive study revealing which of these compounds are the main bioactive principles. The goal of this study was to pinpoint compounds responsible for strong antioxidant activity. Eriobotryaefolium was extracted and fractionated between solvents of increasing polarity. All extracts and fractions were screened for total polyphenols and tannins, and antioxidant activity was checked by DPPH, phosphomolybdenum and linoleic acid tests. The ethyl acetate fraction demonstrated the highest antioxidant activity and contained the largest amount of polyphenols. Applying HPLC-based activity profiling to localize antioxidants revealed that cinchonain IIb, as well as flavonoid glycosides such as hyperoside, isoquercitrin, kaempferol glycosides, quercetin-rhamnoside, as well as two tentatively identified protocatechuic acid derivatives are the main substances responsible for the strong antioxidant activity of the ethyl acetate fraction.