Boswellianols A-I, Structurally Diverse Diterpenoids from the Oleo-Gum Resin of Boswellia carterii and Their TGF-ß Inhibition Activity.

Olibanum, a golden oleo-gum resin from species in the Boswellia genus (Burseraceae family), is a famous traditional herbal medicine widely used around the world. Previous phytochemical studies mainly focused on the non-polar fractions of olibanum. In this study, nine novel diterpenoids, boswellianols A-I (1-9), and three known compounds were isolated from the polar methanolic fraction of the oleo-gum resin of Boswellia carterii. Their structures were determined through comprehensive spectroscopic analysis as well as experimental and calculated electronic circular dichroism (ECD) data comparison. Compound 1 is a novel diterpenoid possessing an undescribed prenylmaaliane-type skeleton with a 6/6/3 tricyclic system. Compounds 2-4 were unusual prenylaromadendrane-type diterpenoids, and compounds 5-9 were new highly oxidized cembrane-type diterpenoids. Compounds 1 and 5 showed significant transforming growth factor ß (TGF-ß) inhibitory activity via inhibiting the TGF-ß-induced phosphorylation of Smad3 and the expression of fibronectin and N-cadherin (the biomarker of the epithelial-mesenchymal transition) in a dose-dependent manner in LX-2 human hepatic stellate cells, indicating that compounds 1 and 5 should be potential anti-fibrosis agents. These findings give a new insight into the chemical constituents of the polar fraction of olibanum and their inhibitory activities on the TGF-ß/Smad signaling pathway.

Cevanine-type alkaloids from the bulbs of Fritillaria unibracteata var. wabuensis and their antifibrotic activities in vitro.

Steroidal alkaloids are the main bioactive components of the bulbs of Fritillaria, which have been used as traditional Chinese medicine, known as "Beimu", for the treatment of cough for thousands of years in China. Cough and dyspnea are the most common symptoms observed in patients with pulmonary fibrosis. However, the antifibrotic activity of steroidal alkaloids has not been reported yet. In this study, two previously unreported cevanine-type steroidal alkaloids (1 and 2), four previously undescribed cevanine-type alkaloid glycosides (3-6), and 19 known steroidal alkaloids (7-25) were isolated from the bulbs of Fritillaria unibracteata var. wabuensis. The structures of these compounds were elucidated by comprehensive HRESIMS and NMR spectroscopic data analysis, as well as DP4+ NMR calculations. The biological evaluation showed that compounds 2, 7-10, 14, 15, and 17 downregulated fibrotic markers induced by transforming growth factor-ß (TGF-ß) in MRC-5 cells. Moreover, compounds 14 and 17 dose dependently inhibited TGF-ß-induced epithelial-mesenchymal transition in A549 cells, alleviated TGF-ß-induced migration and proliferation of fibroblasts, and decreased the expression of fibrotic markers, fibronectin, and N-cadherin in TGF-ß-induced MRC-5 cells. The research showed the potential of cevanine-type alkaloids as a class of natural antifibrotic agents.

Atrachinenins D-S, novel meroterpenoids with geranyl hydroquinone moiety from Atractylodes chinensis by the LC/MS-based molecular decoy and targeted isolation.

To mine fascinating molecules from the rhizomes of Atractylodes chinensis, the known molecular formula of atrachinenin A was used as a bait to search LC-HRMS data in different subfractions. Sixteen new meroterpenoids, atrachinenins D-S (1-16) including three unprecedented carbon skeletons (1-5) and eleven new oxygen-bridged hybrids (6-16) were obtained by the targeted isolation. Their structures and absolute configurations were elucidated by the spectroscopic data and electronic circular dichroism (ECD) calculations. The isolated compounds were evaluated for their inhibitory activity of NO production and compounds 1, 4, 8, and 13 showed moderate anti-inflammatory activity. The proposed biosynthetic pathways of 1-5 were also discussed.

Functionalized Fe-Doped Carbon Dots Exhibiting Dual Glutathione Consumption to Amplify Ferroptosis for Enhanced Cancer Therapy.

Nonapoptotic ferroptosis is a promising cancer treatment which offers a solution to the multidrug resistance of conventional apoptosis-induced programmed cancer cell death therapies. Reducing intracellular glutathione (GSH) is essential for inducing excess ROS and has been considered a crucial process to trigger ferroptosis. However, treatments reducing GSH alone have not produced satisfactory effects due to their restricted target. In this regard, FeCDs (Fe3+-modified l-histidine -sourced carbon dots) with dual GSH-consumption capabilities were constructed to engineer ferroptosis by self-amplifying intratumoral oxidative stress. Carbon dots have the ability to consume GSH, and the introduction of Fe3+ can amplify the GSH-consuming ability of CDs, reacting with excess H2O2 in the tumor microenvironment to generate highly oxidized •OH. This is a novel strategy through synergistic self-amplification therapy combining Fe3+ and CDs with GSH-consuming activity. The acid-triggered degradation material (FeCDs@PAE-PEG) was prepared by encapsulating FeCDs in an oil-in-water manner. Compared with other ferroptosis-triggering nanoparticles, the established FeCDs@PAE-PEG is targeted and significantly enhances the consumption efficiency of GSH and accumulation of excess iron without the involvement of infrared light and ultrasound. This synergistic strategy exhibits excellent ferroptosis-inducing ability and antitumor efficacy both in vitro and in vivo and offers great potential for clinical translation of ferroptosis.

Serum sphingolipids aid in diagnosing adult HIV-negative patients with pulmonary cryptococcosis: a clinical cohort study.

Background: Pulmonary cryptococcosis (PC) contributes to the ongoing global disease burden in human immunodeficiency virus (HIV)-negative populations. Since some PC patients are misdiagnosed under existing diagnostic guidelines, new diagnostic markers are needed to improve diagnostic accuracy and therapeutic efficacy and reduce disease risk. Methods: Our previously established sphingolipidomic approach was employed to explore the use of serum sphingolipids (SPLs) in diagnosing HIV-negative patients with PC. A clinical cohort of PC, pulmonary aspergillosis (PA), and tuberculosis (TB) patients and healthy controls was assessed to identify SPL biomarkers. Results: A total of 47 PC, 27 PA, and 18 TB patients and 40 controls were enrolled. PC and TB patients had similar clinical features, laboratory test results and radiological features, excluding plural effusion. The serum ceramide [Cer (d18:1/18:0)] level showed a significant increase in PC patients compared to controls and PA and TB patients (P<0.05). Cer (d18:1/18:0) was identified as a specific diagnostic biomarker for PC. The optimal cut-off value of greater than 18.00 nM showed a diagnostic sensitivity of 76.60% and a specificity of 95.00% and better distinguished PC patients from PA and TB patients. Furthermore, the serum Cer (d18:1/18:0) level gradually decreased after 3 and 6 months of treatment, suggesting the prediction potential for therapeutic efficacy of this biomarker. In addition, Cer (d18:1/18:0) analysis presented a higher sensitivity than the cryptococcal antigen (CrAg) assay. Conclusions: This is the first study to report the use of the SPL Cer (d18:1/18:0) as a serum biomarker for diagnosing Cryptococcus spp. infection in HIV-negative patients.

Hydroxyapatite-based nano-drug delivery system for nicotinamide mononucleotide (NMN): significantly enhancing NMN bioavailability and replenishing in vivo nicotinamide adenine dinucleotide (NAD+) levels.

OBJECTIVES: This study addresses the bioavailability challenges associated with oral nicotinamide mononucleotide (NMN) administration by introducing an innovative NMN formulation incorporated with hydroxyapatite (NMN-HAP). METHODS: The NMN-HAP was developed using a wet chemical precipitation and physical adsorption method. To assess its superiority over conventional free NMN, we examined NMN, nicotinamide adenine dinucleotide (NAD+), and nicotinamide riboside (NR) levels in mouse plasma and tissues following oral administration of NMN-HAP. KEY FINDINGS: NMN-HAP nanoparticles demonstrated a rod-shaped morphology, with an average size of ~50 nm, along with encapsulation efficiency and drug loading capacity exceeding 40%. In vitro, drug release results indicated that NMN-HAP exhibited significantly lower release compared with free NMN. In vivo studies showed that NMN-HAP extended circulation time, improved bioavailability compared with free NMN, and elevated plasma levels of NMN, NAD+, and NR. Moreover, NMN-HAP administration displayed tissue-specific distribution with a substantial accumulation of NMN, NAD+, and NR in the brain and liver. CONCLUSION: NMN-HAP represents an ideal formulation for enhancing NMN bioavailability, enabling tissue-specific delivery, and ultimately elevating in vivo NAD+ levels. Considering HAP's biocompatible nature and versatile characteristics, we anticipate that this system has significant potential for various future applications.

Quantitative 1H NMR with global spectral deconvolution approach for quality assessment of natural and cultured Cordyceps sinensis.

Cordyceps sinensis is a precious medicinal food which has been successfully cultivated indoors. It remains to be investigated for a simultaneous comparison on aqueous components of natural and cultivated samples. Herein, an approach of quantitative nuclear magnetic resonance (qNMR) analysis combined with global spectral deconvolution (GSD) was established for simultaneous quantification of 26 aqueous components in C. sinensis. Processed by GSD, the distorted baselines of 1H NMR spectra were greatly improved, and overlapped signals were also well separated so as to achieve accurate identification and quantitation of components in C. sinensis. Method validation by UHPLC-QTOF-MS and TOF-SIMS analysis revealed that qNMR combined with GSD is a reliable approach for simultaneous quantification of multiple components including characteristic markers of glutamine, GABA and trehalose in authentic and fake C. sinensis. The well-established qNMR approach can be used for quality assessment of natural and cultivated C. sinensis as well as differentiation from fake ones.

Sesquiterpenoids from the roots of Aucklandia costus and their anti-inflammatory activities.

Five undescribed sesquiterpenoid dimers, aucklandiolides A-E (1-5), one new sesquiterpenoid glycoside, ß-cyclocostunolide-15-ß-D-glucopyranoside (6), and seventeen known analogues (7-23) were isolated from the roots of Aucklandia costus. Their structures were elucidated by comprehensive HRESIMS and NMR spectroscopic data analysis, and their configurations were confirmed by the computational calculations of ECD and NMR chemical shifts. Aucklandiolides A and B are the first examples of dimeric sesquiterpenoids with a unique 6/6/6/5/6/6 ring system originated from a proposed Diels-Alder cycloaddition between two eudesmane sesquiterpenoids. Besides, compounds 9-11, 20, and 22 showed significant inhibition of nitric oxide production in LPS-stimulated RAW 264.7 cells at a concentration of 20 µM.

Discrimination of Baphicacanthis Cusiae Rhizoma et radix and its adulterant species and establishment of an assay method for quality control.

BACKGROUND: Baphicacanthis Cusiae Rhizoma et Radix, commonly known as Nan-Ban-Lan-Gen (NBLG), is an essential traditional Chinese medicine that possesses diverse bioactivities, particularly noteworthy for its antiviral properties. Although NBLG has been listed in the Chinese Pharmacopoeia as an independent Chinese medicine, the establishment of a comprehensive quality standard for NBLG remains elusive. The absence of assay for marker compound in its quality standards has led to the lack of corresponding quality control measures for NBLG-containing preparations, and its discrimination from adulterant species in the market which thereby can significantly impact the efficacy and safety of NBLG-containing products. METHODS: Ultra-high performance liquid chromatography (UHPLC) coupled with quadrupole-time-of-flight mass spectrometry (Q-TOF-MS) was employed for comprehensive profiling of the chemical constituents of NBLG, the stem of Baphicacanthus cusia (Nees) Bremek (NBLJ), and the roots of Isatis indigotica Fort. (Bei-Ban-Lan-Gen, BBLG). Additionally, multivariate analysis was conducted to compare the chemical components of NBLG with those of NBLJ and BBLG. Furthermore, we established an optimized and validated HPLC method to obtain the fingerprint of NBLG and quantify the content of 2-benzoxazolinone and acteoside in the samples. RESULTS: A total of 73 compounds belonging to six classes were assigned in NBLG, with alkaloids being the most abundant and diverse species. High compositional similarities with significant differences in content were observed between NBLG and NBLJ. Moreover, the chemical profile of BBLG markedly differed from that of NBLG. An informative high performance liquid chromatography (HPLC) fingerprint of NBLG comprising seven characteristic peaks that can be used for quality assessment was established. Notably, we propose a quality control standard for NBLG, stipulating that the limit of content in dry weight for both 2-benzoxazolinone and acteoside should not be less than 0.010%. CONCLUSION: This study provides the most comprehensive chemical information to date on NBLG, offering valuable insights into its authentication and quality control. Our findings highlight the importance of comprehensive chemical profiling to differentiate potential substitutions and adulterations of herbal medicines, particularly when the original source is scarce or unavailable. These results can aid in the development of quality control measures for NBLG-containing preparations, ensuring their safety and efficacy.

Reprogramming of the tumor microenvironment using a PCN-224@IrNCs/D-Arg nanoplatform for the synergistic PDT, NO, and radiosensitization therapy of breast cancer and improving anti-tumor immunity.

The low X-ray attenuation coefficient of tumor soft tissue and the hypoxic tumor microenvironment (TME) during radiation therapy (RT) of breast cancer result in RT resistance and thus reduced therapeutic efficacy. In addition, immunosuppression induced by the TME severely limits the antitumor immunity of radiation therapy. In this paper, we propose a PCN-224@IrNCs/D-Arg nanoplatform for the synergistic radiosensitization, photodynamic, and NO therapy of breast cancer that also boosts antitumor immunity (PCN = porous coordination network, IrNCs = iridium nanocrystals, D-Arg = D-arginine). The local tumors can be selectively ablated via reprogramming the tumor microenvironment (TME), photodynamic therapy (PDT) and NO therapy, and the presence of the high-Z element Ir that sensitizes radiotherapy. The synergistic execution of these treatment modalities also resulted in adapted antitumor immune response. The intrinsic immunomodulatory effects of the nanoplatform also repolarize macrophages toward the M1 phenotype and induce dendritic cell maturation, activating antitumor T cells to induce immunogenic cell death as demonstrated in vitro and in vivo. The nanocomposite design reported herein represents a new regimen for the treatment of breast cancer through TME reprogramming to exert a synergistic effect for effective cancer therapy and antitumor immunity.

An integrated approach to evaluate acetamiprid-induced oxidative damage to tRNA in human cells based on oxidized nucleotide and tRNA profiling.

Acetamiprid is poisonous to mammals due to severe acetamiprid-induced oxidative stress that could cause mitochondrial dysfunctions, lipid and protein oxidation, inflammation, apoptosis, and DNA damage. Evidence has accumulated for the role of oxidative stress in changing structures and functions of transfer RNAs (tRNAs) by inducing tRNA cleavage, reprogramming tRNA modifications and impairing aminoacyl-tRNA synthetase editing sites. However, the impact of acetamiprid-induced oxidative stress on tRNA is still unknown. Here, we investigated the effects of acetamiprid on cell viability, reactive oxygen species (ROS) levels, DNA damage, cellular oxidized nucleotide concentrations, and oxidative damage to tRNA in HepG2 cells and LO2 cells. Acetamiprid can cause the significant increment of ROS and DNA oxidative damage. In this study, an integrated approach was established to simultaneously study the network of oxidized nucleotides and explore the tRNA oxidative damage after acetamiprid exposure. A simple and high-throughput liquid chromatography with tandem mass spectrometry (LC-MS/MS) method coupled with (trimethylsilyl)diazomethane (TMSD) derivatization was successfully developed to quantify 12 cellular oxidized nucleotides that cannot be detected using traditional detection methods because of the huge interferences from naturally abundant nucleotides. Meanwhile, the accumulation rate and the locating sites of 8-oxo-2, 7-dihydro-guanine (8-oxo-G) in tRNA were inspected using the established N-(tert-Butyldimethylsilyl)-N-methyl-trifluoroacetamide (MTBSTFA) labeling-based tRNA profiling method. After acetamiprid treatment, the increment of oxidized nucleoside triphosphates is smaller than that of their corresponding mono- and diphosphates, as well as the dephosphorylated nucleosides, on account of the existence of sanitization enzymes. Several tRNA fragments, CUC[m1A]Gp, CACGp, [Cm]C[m2G]p, and DDGp, are significantly downregulated in acetamiprid-treated HepG2 cells, while only [Cm]C[m2G]p in acetamiprid-treated LO2 cells. According to the profiling results, the significantly changed fragment CUC[m1A]Gp might be caused by the oxidation of guanine (G) to form 8-oxo-G at position 15 in human tRNAphe([Gm]AA), providing more information about the effect of oxidized nucleobases on tRNA's functions.

Chemical Constituents from the Fruits of Amomum kravanh and Their Role in Activating Alcohol Dehydrogenase.

Alcoholism is a worldwide health problem, and diseases caused by alcoholism are killing people every year. Amomum kravanh is a traditional Chinese medicine used to relieve hangovers. However, whether its bioactive components improve alcohol metabolism is not clear. In this study, ten new (amomumols A-J, 1-10) and thirty-five known (11-45) compounds were isolated from the fruits of Amomum kravanh by an activity-guided separation. Ten novel compounds were identified as four sesquiterpenoids (1-4), three monoterpene derivatives (5-7), two neolignans (8, 9), and a novel norsesquiterpenoid (10) with a new C14 nor-bisabolane skeleton. Their structures were determined by the comprehensive analysis of high-resolution electrospray ionization mass spectrometry (HRESIMS), nuclear magnetic resonance (NMR), and electronic circular dichroism (ECD) calculation. The effects of all isolated compounds on the activity of alcohol dehydrogenase were evaluated in vitro, and it was found that eight compounds (11, 12, 15, 18, 26, and 36-38) exhibited significant activation effects on the alcohol dehydrogenase at 50 µM.

Di- and Triterpenoids from the Rhizomes of Isodon amethystoides and Their Anti-inflammatory Activities.

Amethystoidesic acid (1), a triterpenoid with an unprecedented 5/6/6/6 tetracyclic skeleton, and six undescribed diterpenoids, amethystoidins A-F (2-7), were isolated from the rhizomes of Isodon amethystoides along with 31 known di- and triterpenoids (8-38). Their structures were fully elucidated via extensive spectroscopic analysis including 1D and 2D NMR, high-resolution electrospray ionization mass spectrometry (HRESIMS), and electronic circular dichroism (ECD) calculations. Compound 1 is the first example of a triterpenoid possessing a rare ring system (5/6/6/6) derived from a contracted A-ring and the 18,19-seco-E-ring of ursolic acid. Compounds 6, 16, 21, 22, 24, and 27 significantly inhibited nitric oxide (NO) production in lipopolysaccharide (LPS)-stimulated RAW264.7 cells, which could be partly mediated by the downregulation of LPS-induced inducible nitric oxide synthase (iNOS) protein expression.

Therapeutic potency of compound RMY-205 for pulmonary fibrosis induced by SARS-CoV-2 nucleocapsid protein.

Pulmonary fibrosis is a typical sequela of coronavirus disease 2019 (COVID-19), which is linked with a poor prognosis for COVID-19 patients. However, the underlying mechanism of pulmonary fibrosis induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is unclear. Here, we demonstrated that the nucleocapsid (N) protein of SARS-CoV-2 induced pulmonary fibrosis by activating pulmonary fibroblasts. N protein interacted with the transforming growth factor ß receptor I (TßRI), to disrupt the interaction of TßRI-FK506 Binding Protein12 (FKBP12), which led to activation of TßRI to phosphorylate Smad3 and boost expression of pro-fibrotic genes and secretion of cytokines to promote pulmonary fibrosis. Furthermore, we identified a compound, RMY-205, that bound to Smad3 to disrupt TßRI-induced Smad3 activation. The therapeutic potential of RMY-205 was strengthened in mouse models of N protein-induced pulmonary fibrosis. This study highlights a signaling pathway of pulmonary fibrosis induced by N protein and demonstrates a novel therapeutic strategy for treating pulmonary fibrosis by a compound targeting Smad3.

Robust quantitation of gangliosides and sulfatides in human brain using UHPLC-MRM-MS: Method development and application in Alzheimer's disease.

Gangliosides (GAs) and sulfatides (STs) are acidic glycosphingolipids that are particularly abundant in the nervous system and are closely related to aging and neurodegenerative disorders. To explore their roles in brain diseases, in-depth molecular profiling, including structural variations of sphingoid backbone, fatty acyl group, and sugar chain of GAs and STs was performed. A total of 210 GAs and 38 STs were characterized in the inferior frontal gyrus (IFG) of human brain, with 90 GAs discovered in brain tissues for the first time. Influential MS parameters for detecting GAs and STs in multiple reaction monitoring (MRM) mode were systematically examined and optimized to minimize in-source fragmentation, resulting in remarkable signal intensity enhancement for GAs and STs, especially for polysialylated species. To eliminate analytical variations, isotopic interference-free internal standards were prepared by simple and fast reduction reaction. The final established method facilitated the simultaneous quantitation of 184 GAs and 30 STs from 25 subtypes, which represents the highest number of GAs quantitated among all quantitation methods recorded in literature so far. The method was further validated and applied to reveal the aberrant change of GAs and STs in the IFG of 12 Alzheimer's disease (AD) patients. Four GAs exhibited high classification capacity for AD (AUC ≥0.80) and were thereby considered the most promising signatures for AD. These findings suggested the close correlation between GAs and the pathogenesis of AD, highlighting the achievements of our robust method for investigating the roles of GAs and STs in various physiological states and diseases.

Selective Chemical Labeling Strategy for Oligonucleotides Determination: A First Application to Full-Range Profiling of Transfer RNA Modifications.

To date, the extremely high polarity and poor signal intensity of macromolecular nucleic acids are greatly impeding the progress of mass spectrometry technology in the quality control of nucleic acid drugs and the characterization of DNA oxidation and RNA modifications. We recently described a general N-(tert-butyldimethylsilyl)-N-methyl-trifluoroacetamide (MTBSTFA) labeling method for oligonucleotide determination and applied it to the full-range profiling of tRNA in vitro and in vivo studies for the first time. The primary advantages of this method include strong retention, no observable byproducts, predictable and easily interpreted MS2 data, and the circumvention of instrument harmful reagents that were necessary in previous methods. Selective labeling of N-(tert-butyldimethylsilyl)-N-methyl-trifluoroacetamide to the terminal phosphate groups of oligonucleotides endows it broadly applicable for DNA/RNA profiling. Moreover, the improvement of sequence coverage was achieved in yeast tRNAphe(GAA) analysis owing to this method's good detection capability of 1-12 nucleotides in length. We also extended this strategy to determine the abundance of modified bases and discover new modifications via digesting RNA into single-nucleotide products, promoting the comprehensive mapping of RNA. The easy availability of derivatization reagent and the simple, rapid one-step reaction render it easy to operate for researchers. When applied in characterizing tRNAs in HepG2 cells and rats with nonalcoholic fatty liver disease, a fragment of U[m1G][m2G], specific for tRNAAsn(QUU) in cells, was significantly upregulated, indicating a possible clue to nonalcoholic fatty liver disease pathogenesis.

Fractional extraction and structural characterization of glycogen particles from the whole cultivated caterpillar fungus Ophiocordyceps sinensis.

Ophiocordyceps sinensis (syn. Cordyceps sinensis) is a valuable medicinal fungus in traditional Chinese medicine, and one or more polysaccharides are the key constituents with important medical effects. Glycogen as a functional polysaccharide is widely identified in eukaryotes including fungi. However, there is no definitive report of glycogen presence in O. sinensis. In this study, we carefully fractionated polysaccharides from cultivated caterpillar fungus O. sinensis, which were then characterized via methods for glycogen analysis. According to the results, 1.03 ± 0.43 % of polysaccharides were quantified via amyloglucosidase digestion in the whole cultivated caterpillar fungus, which had a typical spherical shape under transmission electron microscope with an average peak radius of 37.63 ± 0.57 nm via size exclusion chromatography and an average chain length of 12.47 ± 0.94 degree of polymerization via fluorophore-assisted capillary electrophoresis. Taken together, this study confirmed that the polysaccharides extracted form O. sinensis were mostly glycogen.

The role of post-transcriptional modification on a new tRNAIle(GAU) identified from Ganoderma lucidum in its fragments' cytotoxicity on cancer cells.

Ganoderma lucidum (Ganoderma) is a famous Chinese herbal medicine which has been used clinically for thousands of years in China. Despite numerous studies on triterpenes and polysaccharides, the bioactivity of RNAs abundant in Ganoderma remains unknown. Here, based on LC-MS techniques, dihydrouracil, 5-methyluridine (m5U) and pseudouridine were identified at position 19, 52 and 53 of a new tRNAIle(GAU) which was isolated as the most abundant tRNA species in Ganoderma, and is the first purified tRNA from fungus. Cytotoxic screening of tRNA-half (t-half) and tRNA fragment (tRF) derived from this tRNA, as well as their mimics (t-half or tRF as antisense strand), demonstrated that the double-stranded form, i.e., tRF and t-halve mimics, exhibited stronger cytotoxicity than their single-stranded form, and the cytotoxicity of t-half mimic is significantly stronger than that of tRF mimic. Notably, the cytotoxicity of 3'-t-half mimic is not only much more potent than that of taxol, but also is much more potent than that of ganoderic acids, the major bioactive components in Ganoderma. Furthermore, 3'-t-half mimic_M2 (m5U modified) exhibited significantly stronger cytotoxicity than unmodified 3'-t-half mimic, which is consistent with the computational simulation showing that m5U modification enhances the stability of the tertiary structure of 3'-t-half mimic. Overall, the present study not only indicates t-halves are bioactive components in Ganoderma which should not be neglected, but also reveals an important role of post-transcriptional modification on tRNA in its fragments' cytotoxicity against cancer cells, which benefits the design and development of RNAi drugs from natural resource.

Anti-Entry Activity of Natural Flavonoids against SARS-CoV-2 by Targeting Spike RBD.

COVID-19 is still a global public health concern, and the SARS-CoV-2 mutations require more effective antiviral agents. In this study, the antiviral entry activity of thirty-one flavonoids was systematically evaluated by a SARS-CoV-2 pseudovirus model. Twenty-four flavonoids exhibited antiviral entry activity with IC50 values ranging from 10.27 to 172.63 µM and SI values ranging from 2.33 to 48.69. The structure-activity relationship of these flavonoids as SARS-CoV-2 entry inhibitors was comprehensively summarized. A subsequent biolayer interferometry assay indicated that flavonoids bind to viral spike RBD to block viral interaction with ACE2 receptor, and a molecular docking study also revealed that flavonols could bind to Pocket 3, the non-mutant regions of SARS-CoV-2 variants, suggesting that flavonols might be also active against virus variants. These natural flavonoids showed very low cytotoxic effects on human normal cell lines. Our findings suggested that natural flavonoids might be potential antiviral entry agents against SARS-CoV-2 via inactivating the viral spike. It is hoped that our study will provide some encouraging evidence for the use of natural flavonoids as disinfectants to prevent viral infections.

Bioassay-Guided Isolation of Anthelmintic Components from Semen pharbitidis, and the Mechanism of Action of Pharbitin.

Parasitic helminths continue to pose problems in human and veterinary medicine, as well as in agriculture. Semen pharbitidis, the seeds of Pharbitis nil (Linn.) Choisy (Convolvulaceae), is a well-known traditional Chinese medicinal botanical preparation widely used for treating intestinal parasites in China owing to its desirable efficacy. However, the anthelmintic compounds in Semen pharbitidis and their mechanism of action have not been investigated yet. This study aimed to identify the compounds active against helminths from Semen pharbitidis, and to establish the mechanism of action of these active compounds. Bioassay-guided fractionation was used to identify the anthelmintic compounds from Semen pharbitidis. The anthelmintic assay was performed by monitoring Caenorhabditis elegans (C. elegans) motility with a WMicrotracker instrument. Active compounds were identified by high-resolution mass spectrometry. Several (analogues of) fragments of the anthelmintic compounds were purchased and tested to explore the structure-activity relationship, and to find more potent compounds. A panel of C. elegans mutant strains resistant to major currently used anthelmintic drugs was used to explore the mechanism of action of the active compounds. The bioassay-guided isolation from an ethanol extract of Semen pharbitidis led to a group of glycosides, namely pharbitin (IC50: 41.0 ± 9.4 µg/mL). Hit expansion for pharbitin fragments yielded two potent analogues: 2-bromohexadecanoic acid (IC50: 1.6 ± 0.7 µM) and myristoleic acid (IC50: 35.2 ± 7.6 µM). One drug-resistant mutant ZZ37 unc-63 (x37) demonstrated a ~17-fold increased resistance to pharbitin compared with wild-type worms. Collectively, we provide further experimental scientific evidence to support the traditional use of Semen pharbitidis for the treatment of intestinal parasites. The anthelmintic activity of Semen pharbitidis is due to pharbitin, whose target could be UNC-63 in C. elegans.