High-resolution Electrospray Ionization Research Articles

Chemical Composition, Antioxidant and Anti-Inflammatory Activity of Shiitake Mushrooms (Lentinus edodes)

Shiitake mushrooms (Lentinus edodes) are renowned as the “King of mountain treasures” in China due to their abundant nutritional and health-enhancing properties. Intensive chemical investigations of the fruiting bodies and mycelium of Shiitake mushrooms (Lentinus edodes) afforded five new compounds (1–5), named lentinmacrocycles A-C and lentincoumarins A-B, along with fifteen known compounds (6–20). Their structures and absolute configurations were elucidated by extensive spectroscopic analysis, including one-and two-dimensional (1D and 2D) NMR spectroscopy, circular dichroism (CD), and high-resolution electrospray ionization mass spectrometry (HR-ESI-MS). The anti-inflammatory activity test showed that lentincoumarins A (4), (3S)-7-hydroxymellein (9), (3R)-6-hydroxymellein (11) and succinic acid (18) exhibited strong NO inhibitory effects (IC50 < 35 μM), and that (3S)-5-hydroxymellein (10) and (3R)-6-hydroxymellein (11) exhibited potent TNF-α inhibitory effects (IC50 < 80 μM) and were more potent than the positive control, Indomethacin (IC50 = 88.5 ± 2.1 μM). The antioxidant activity test showed that (3R)-6-hydroxymellein (11) had better DPPH radical scavenging activity (IC50 = 25.2 ± 0.5 μM).

Anti-Inflammatory Effect of Xanthones from Hypericum beanii on Macrophage RAW 264.7 Cells through Reduced NO Production and TNF-α, IL-1β, IL-6, and COX-2 Expression

Hypericum beanii N. Robson, a perennial upright herb, predominantly inhabits temperate regions. This species has been utilized for the treatment of various inflammation-related diseases. One new xanthone 3,7-dihydroxy-1,6-dimethoxyxanthone (1) and twenty-three known xanthones (2–24) were isolated from the aerial parts of H. beanii. The structure of the new compound was determined based on high-resolution electrospray ionization mass spectroscopy (HR-ESIMS), nuclear magnetic resonance (NMR), Infrared Spectroscopy (IR), ultraviolet spectrophotometry (UV) spectroscopic data. The anti-inflammatory effects of all the isolates were assessed by measuring the inhibitory effect on nitric oxide (NO) production in LPS-stimulated RAW 264.7 macrophages. Compounds 3,4-dihydroxy-2-methoxyxanthone (15), 1,3,5,6-tetrahydroxyxanthone (19), and 1,3,6,7-tetrahydroxyxanthone (22) exhibited significant anti-inflammatory effects at a concentration of 10 μM with higher potency compared to the positive control quercetin. Furthermore, compounds 15, 19, and 22 reduced inducible NO synthase (iNOS), tumor necrosis factor alpha (TNF-α), interleukin-1β (IL-1β), IL-6, and cyclooxygenase 2 (COX-2) mRNA expression in the LPS-stimulated RAW 264.7 macrophages, suggesting that these compounds may mitigate the synthesis of the aforementioned molecules at the transcriptional level, provisionally confirming their anti-inflammatory efficacy.

Functional Insights into the Sphingolipids C1P, S1P, and SPC in Human Fibroblast-like Synoviocytes by Proteomic Analysis

The (patho)physiological function of the sphingolipids ceramide-1-phosphate (C1P), sphingosine-1-phosphate (S1P), and sphingosylphosphorylcholine (SPC) in articular joints during osteoarthritis (OA) is largely unknown. Therefore, we investigated the influence of these lipids on protein expression by fibroblast-like synoviocytes (FLSs) from OA knees. Cultured human FLSs (n = 7) were treated with 1 of 3 lipid species—C1P, S1P, or SPC—IL-1β, or with vehicle. The expression of individual proteins was determined by tandem mass tag peptide labeling followed by high-resolution electrospray ionization (ESI) mass spectrometry after liquid chromatographic separation (LC-MS/MS/MS). The mRNA levels of selected proteins were analyzed using RT-PCR. The 3sphingolipids were quantified in the SF of 18 OA patients using LC-MS/MS. A total of 4930 proteins were determined using multiplex MS, of which 136, 9, 1, and 0 were regulated both reproducibly and significantly by IL-1β, C1P, S1P, and SPC, respectively. In the presence of IL-1ß, all 3 sphingolipids exerted ancillary effects. Only low SF levels of C1P and SPC were found. In conclusion, the 3 lipid species regulated proteins that have not been described in OA. Our results indicate that charged multivesicular body protein 1b, metal cation symporter ZIP14, glutamine-fructose-6-P transaminase, metallothionein-1F and -2A, ferritin, and prosaposin are particularly interesting proteins due to their potential to affect inflammatory, anabolic, catabolic, and apoptotic mechanisms.

Unlocking the potential of edible Ulva sp. seaweeds: Metabolomic profiling, neuroprotective mechanisms, and implications for Parkinson's disease management.

Green seaweed (Ulva sp.) is frequently used as a food component and nutraceutical agent because of its high polysaccharide and natural fiber content in Asian countries. This study investigates both metabolomic profiling of Ulva sp. and the neuroprotective efficacy of its ethanol extract and its underlying mechanisms in a rotenone-induced rat model of neurodegeneration, mimicking Parkinson's disease (PD) in humans. Metabolomic profiling of Ulva sp. extract was done using liquid chromatography high resolution electrospray ionization mass spectrometry and led to the identification of 22 compounds belonging to different chemical classes.Catenin Beta Additionally, this study demonstrated the neuroprotective properties against rotenone-induced PD, which was achieved through the suppression of elevated levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-6 together with the inhibition of reactive oxygen species (ROS) generation, apoptosis, inflammatory mediators, and the phosphoinositide 3-kinases/serine/threonine protein kinase (PI3K/AKT) pathway. Using a protein-protein interaction network, AKT1, GAPDH, TNF-α, IL-6, caspase 3, signal transducer and activator of transcription 3, Catenin Beta 1, epidermal growth factor receptor, B-cell lymphoma -2, and HSP90AA1 were identified as the top 10 most significant genes. Finally, molecular docking results showed that compounds 1, 3, and 7 might possess a promising anti-parkinsonism effect by binding to active sites of selected hub genes. Therefore, it is hypothesized that the Ulva sp. extract has the potential to be further developed as a potential therapeutic agent for the treatment of PD.

Structure, anti-inflammatory and anti-bacterial activities of novel pentacyclic triterpenoids and other constituents from the leaves of Pittosporum elevaticostatum

The investigation of the leaves of Pittosporum elevaticostatum Chang et Yan led to the isolation of fifteen pentacyclic triterpenoids (1–15), including five previously undescribed ones (1–5), and nine others (16–24). The structures of compounds 1–5 were elucidated based on comprehensive spectroscopic techniques, including one dimension (1D) and 2D nuclear magnetic resonance (NMR), high resolution electrospray ionization mass spectroscopy (HR-ESI-MS), and other methods. Compounds 2 and 13 demonstrated significant inhibitory activity against Listeria monocytogenes (L. monocytogenes) with minimum inhibitory concentration (MIC) values of 32 μM. Scanning electron microscopy (SEM) observations revealed insights into the antibacterial mechanism, indicating that compounds 2 and 13 either prevent biofilm formation of dispersed the preformed cell membranes. Additionally, compounds 1, 5, 7, and 12 exhibited anti-inflammatory activity on lipopolysaccharide (LPS)-stimulated BV-2 microglial cells with IC50 values ranging from 11.27 to 17.80 μM.

Multipocket Cage Enables the Binding of High-Order Bulky and Drug Guests Uncovered by MS Methodology.

Achieving high guest loading and multiguest-binding capacity holds crucial significance for advancement in separation, catalysis, and drug delivery with synthetic receptors; however, it remains a challenging bottleneck in characterization of high-stoichiometry guest-binding events. Herein, we describe a large-sized coordination cage (MOC-70-Zn8Pd6) possessing 12 peripheral pockets capable of accommodating multiple guests and a high-resolution electrospray ionization mass spectrometry (HR-ESI-MS)-based method to understand the solution host-guest chemistry. A diverse range of bulky guests, varying from drug molecules to rigid fullerenes as well as flexible host molecules of crown ethers and calixarenes, could be loaded into open pockets with high capacities. Notably, these hollow cage pockets provide multisites to capture different guests, showing heteroguest coloading behavior to capture binary, ternary, or even quaternary guests. Moreover, a pair of commercially applied drugs for the combination therapy of chronic lymphocytic leukemia (CLL) has been tested, highlighting its potential in multidrug delivery for combined treatment.

Reaction Discovery Using Spectroscopic Insights from an Enzymatic C-H Amination Intermediate.

Engineered hemoproteins can selectively incorporate nitrogen from nitrene precursors like hydroxylamine, O-substituted hydroxylamines, and organic azides into organic molecules. Although iron-nitrenoids are often invoked as the reactive intermediates in these reactions, their innate reactivity and transient nature have made their characterization challenging. Here we characterize an iron-nitrosyl intermediate generated from NH2OH within a protoglobin active site that can undergo nitrogen-group transfer catalysis, using UV-vis, electron paramagnetic resonance (EPR) spectroscopy, and high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) techniques. The mechanistic insights gained led to the discovery of aminating reagents─nitrite (NO2-), nitric oxide (NO), and nitroxyl (HNO)─that are new to both nature and synthetic chemistry. Based on the findings, we propose a catalytic cycle for C-H amination inspired by the nitrite reductase pathway. This study highlights the potential of engineered hemoproteins to access natural nitrogen sources for sustainable chemical synthesis and offers a new perspective on the use of biological nitrogen cycle intermediates in biocatalysis.

Isolation and biological activity of six new polyketide and terpenoid derivatives from Neopestalotiopsis Clavispora AL01

A fungus strain, Neopestalotiopsis clavispora AL01, was isolated from the leaf spot of the plant Phoenix dactylifera. Further chemical investigation of the fermentation extract of this strain afforded six new secondary metabolites (1–6), along with 11 known compounds (7–17) which included a new natural compound (7). Their structures were determined by extensive spectroscopic analysis including one-and two-dimensional (1D and 2D) NMR spectroscopy, high-resolution electrospray ionization mass spectrometry (HRESIMS), and ECD and NMR calculations. All compounds were evaluated for their phytotoxic activities. Among them, compounds 10, 12 and 13 exhibited phytotoxic activities against Nicotiana tabacum. Compound 3 exhibited weak antibacterial activity against methicillin-resistant Staphylococcus aureus, Micrococcus luteus and Vibrio harveyi. Taken collectively, these findings establish a solid research foundation for future investigations on bioactive natural products derived from phytopathogenic fungi.

H3trica: Versatile Macrocyclic Chelator for [225Ac]Ac3+ and [155/161Tb]Tb3+ Theranostics.

H3trica is a nonadentate chelating ligand intended for coordinating large radiometal ions, such as those used in nuclear medicine. This chelator, featuring a triaza-18-crown-6 macrocycle with three pendant carboxylic acid functional groups, was synthesized and characterized. Complementary nuclear magnetic resonance (NMR) spectroscopy and high-resolution electrospray-ionization mass spectroscopy (HR-ESI-MS) studies were used to explore the coordination of H3trica with metal ions such as La3+, Y3+ (as a model for Tb3+), and Lu3+ at the bulk scale. Thermodynamic solution studies provided valuable insights, highlighting robust metal complexation of H3trica with La3+, Tb3+, and Lu3+, with the most noteworthy log KML value observed for Tb3+ (log KTbL = 17.08), followed by La3+ (log KLaL = 16.64) and Lu3+ (log KLuL = 16.25). Concentration-dependent radiolabeling studies with [225Ac]Ac3+, [155Tb]Tb3+, and [161Tb]Tb3+ demonstrated rapid complexation (5-30 min) under mild conditions (pH 6-7, 25 °C). Importantly, the radiolabeled complexes exhibited stability during incubation in human serum for one-half-life of the corresponding radiometal. Thus, H3trica emerges as a valuable chelator, demonstrating its potential to coordinate the theranostic couple [225Ac]Ac3+/[155Tb]Tb3+ as well as the powerful terbium quartet ([149/152/155/161Tb]Tb3+) with efficiency and stability.

Zwitterionic Thiolate-Protected Ag22(0/I) and Ag20(I) Clusters: Assembly, Structural Characterization, and Antibacterial Activity.

Zwitterionic thiolate ligands have the potential to introduce novel assembly modes and functions for noble metal clusters. However, their utilization in the synthesis of silver clusters remains understudied, particularly for the clusters containing reductive Ag(0) species. In this article, we report the first synthesis of a mixed-valence silver(0/I) cluster protected by zwitterionic Tab as thiolate ligands (Tab = 4-(trimethylammonio)benzenethiolate), denoted as [Ag22(Tab)24](PF6)20·16CH3OH·6Et2O (Ag22·16CH3OH·6Et2O), alongside an Ag(I) cluster [Ag20(Tab)12(PhCOO)10(MeCN)2(H2O)](PF6)10·11MeCN (Ag20·11MeCN). Ag22 has a distinct hierarchical supratetrahedral structure with a central {Ag6} kernel surrounded by four [Ag4(Tab)6]4+ units. High-resolution electrospray ionization mass spectra demonstrate that Ag22 has two free electrons, indicating a superatomic core. Ag20 has a drum-like [Ag12(Tab)6(PhCOO)6(H2O)]6+ inner core capped by two tetrahedral-like [Ag4(Tab)3(PhCOO)2(MeCN)]2+ units. Ag20 can be transformed into Ag22 after its reaction with NaBH4 in solution. Antibacterial measurements reveal that Ag22 has a significantly lower minimum inhibitory concentration than that of the Ag20 cluster. This work not only extends the stabilization of silver(0/I) clusters to neutral thiol ligands but also offers new materials for the development of novel antibacterial materials.

Three new chemical constituents of Senecio raphanifolius

Three new constituents, 4a-hydroxy-1-methyl-4,4a,8,8a-tetrahydro-1 H-isochromene-3,7-dione (1), 1-(2-(prop-1-en-2-yl)-3-((3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2 H-pyran-2-yl)oxy)-2,3-dihydrobenzofuran-5-yl)ethan-1-one (2), and 5-(hydroxymethyl)-2,3-dihydro-1 H-pyrrolizin-1-one (3), together with 3 known flavonoids (4-6), 6 organic acids (7-12), 1 glycoside (13), and 2 terpenoids (14-15) were isolated from the 50 % ethanol extract of the whole plant of Senecio raphanifolius Wall. ex DC. The structures of these novel compounds were determined using high-resolution electrospray ionization–mass spectrometry and NMR, mainly using 2D NMR. The anti-inflammatory activities of compounds 1-3 were evaluated based on their ability to inhibit nitric oxide (NO) production in lipopolysaccharide-stimulated RAW264.7 cells. Compound 1 could reduce NO levels and had an inhibition rate of 42.86 % at 100 µM.

Oleanane-type triterpenoids from Sabia limoniacea and their anti-inflammatory activities

Eighteen new oleanane-type triterpenoids were isolated from the stems of Sabia limoniacea, including sabialimon A (1), a triterpenoid with an unprecedented 6/6/6/7/7 pentacyclic skeleton and seventeen undescribed triterpenoids, sabialimons B−R (2 − 18), along with six previously described analogs (19 − 24). Their structures were fully elucidated via extensive spectroscopic analysis including 1D and 2D NMR, high-resolution electrospray ionization mass spectrometry (HRESIMS), experimental electronic circular dichroism measurements and X-ray crystallographic studies. Compound 1 is the first triterpenoid that possesses a rare ring system (6/6/6/7/7) with an oxygen-bearing bridge between C-17 and C-18 and a hemiketal form at C-17, which is generated a larger ring by the degradation of C-28 and D/E-ring expansion. Biological evaluation revealed that sabialimon I (9), sabialimon K (11), sabialimon P (16) and 11,13(18)-oleanadien-28-hydroxymethyl 3-one (20) exhibited significantly inhibitory activities against nitric oxide (NO) release with IC50 values of 29.65, 23.41, 18.12 and 26.64 μM, respectively, as compared with the positive control (dexamethasone, IC50 value: 40.35 μM). Furthermore, sabialimon P markedly decreased the secretion of TNF-α, iNOS, IL-6 and NF-κB and inhibited the expression of COX–2 and NF-κB/p65 in LPS-induced RAW264.7 cells in a dose-dependent manner.

Ten ring-B aromatized ergosterols from Aspergillus spectabilis

Spectasterols F−O (1−10), ten interesting ergosterols with an aromatized B ring, were obtained from Aspergillus spectabilis. Their structures and absolute configurations were determined using a combination of high-resolution electrospray ionization mass spectrometry (HR-ESI-MS), nuclear magnetic resonance (NMR) spectroscopy, single-crystal X-ray diffraction analyses, and electronic circular dichroism (ECD) calculations. Structurally, these aromatic ergosterols feature versatile side chains. Notably, compound aromatic ergosterols featured versatile side chains, and compound 4 is an unusual C23 ergosterol characterized by a shorter side chain due to oxidative cleavage between C-23 and C-24. All compounds were evaluated for their neuroprotective activities, with compound 8 showing a dose-dependent ability to reduce apoptosis and protect mitochondrial function in glutamate-induced SH-SY5Y cells.

Three neo-Clerodane Diterpenoids from Tinospora cordifolia Stems and Their Arginase Inhibitory Activities.

Three neo-clerodane diterpenoids, including two new tinocordifoliols A (1) and B (2) and one known tinopanoid R (3), were isolated from the ethyl acetate-soluble fraction of the 70% ethanol extract of Tinospora cordifolia stems. The structures were elucidated by various spectroscopic methods, including one dimensional (1D) and 2D-NMR, high resolution-electrospray ionization (HR-ESI)-MS, and electronic circular dichroism (ECD) data. The T. cordifolia extract and all isolated compounds 1-3 possessed arginase I inhibitory activities. Among them, 3 exhibited moderate competitive inhibition of human arginase I (IC50 = 61.9 µM). Furthermore, docking studies revealed that the presence of a β-substituted furan in 3 may play a key role in the arginase I inhibitory activities.

Magterpenes A-C: Three Meroterpenoids with an Unprecedented 6/6/6/6/6 Polycyclic Skeleton Extracted from Magnolia officinalis Rehd. et Wils.

Magterpenes A-C (1-3), three unprecedented meroterpenoids featuring a unique 6/6/6/6/6 polycyclic skeleton, were isolated from the ethanol extract of Magnolia officinalis Rehd. et Wils. The compounds were obtained as racemic mixtures that were completely resolved through chiral columns. Their structures were elucidated by extensive analyses of one-dimensional (1D) and 2D nuclear magnetic resonance, high-resolution electrospray ionization mass spectrometry, chemical calculations of 1H/13C NMR, and electronic circular dichroism calculations. The compounds were constructed via two Diels-Alder reactions in the proposed biosynthetic pathway. All isolates were evaluated for their nephroprotective and hepatoprotective activities. The results demonstrated that (+)-1 and (-)-1 possessed promising nephroprotective activities in a dose-dependent manner, while (-)-2 and (+)-3 exhibited moderate hepatoprotective activities.

Metal and solvent dependent formation of imidazolidine or hemiaminal ether complexes

The coordination chemistry of the imidazolidinylpyridine ligand with Ni(II), Cu(II), Cd(II), and Zn(II) ions was investigated in both protic and aprotic solvents. Interestingly, the coordination environment around the metal center varied significantly depending upon the nature of metal ion and solvent used for synthesis. In acetonitrile the imidazolidinylpyridine bound to Cu(II) and Ni(II) remains intact, while in methanol it formed ring-opened hemiaminal ether. The Cd(II) and Zn(II) ion stabilized the imidazolidinylpyridine as a face-capping tridentate ligand regardless of the nature of the solvent. The imidazolidinylpyridine and the complexes formed were characterized by NMR spectroscopy, high resolution electrospray ionization mass spectrometry and single-crystal X-ray crystallography, appropriately. The structural features in the solid-state structures were particularly intriguing, shedding light on the complex interplay between the ligand, metal ions, and solvent environments in coordination chemistry.

Lipidomics reveals the reshaping of the mitochondrial phospholipid profile in cells lacking OPA1 and mitofusins

Depletion or mutations of key proteins for mitochondrial fusion, like optic atrophy 1 (OPA1) and mitofusins 1 and 2 (Mfn 1 and 2), are known to significantly impact the mitochondrial ultrastructure, suggesting alterations of their membranes’ lipid profiles. In order to make an insight into this issue, we used hydrophilic interaction liquid chromatography coupled with electrospray ionization–high resolution MS to investigate the mitochondrial phospholipid (PL) profile of mouse embryonic fibroblasts knocked out for OPA1 and Mfn1/2 genes. One hundred sixty-seven different sum compositions were recognized for the four major PL classes of mitochondria, namely phosphatidylcholines (PCs, 63), phosphatidylethanolamines (55), phosphatidylinositols (21), and cardiolipins (28). A slight decrease in the cardiolipin/PC ratio was found for Mfn1/2-knockout mitochondria. Principal component analysis and hierarchical cluster analysis were subsequently used to further process hydrophilic interaction liquid chromatography–ESI-MS data. A progressive decrease in the incidence of alk(en)yl/acyl species in PC and phosphatidylethanolamine classes and a general increase in the incidence of unsaturated acyl chains across all the investigated PL classes was inferred in OPA1 and Mfn1/2 knockouts compared to WT mouse embryonic fibroblasts. These findings suggest a reshaping of the PL profile consistent with the changes observed in the mitochondrial ultrastructure when fusion proteins are absent. Based on the existing knowledge on the metabolism of mitochondrial phospholipids, we propose that fusion proteins, especially Mfns, might influence the PL transfer between the mitochondria and the endoplasmic reticulum, likely in the context of mitochondria-associated membranes.

Structurally Diverse Phenylpropanamides from Cannabis Fructus and Their Potential Neuroprotective Effects.

This study aimed to investigate the chemical components and potential health benefits of the fruits of Cannabis sativa L. Fourteen new phenylpropanamides designated as cannabisin I-XIV (1-14) and 40 known analogs were isolated and characterized via nuclear magnetic resonance spectroscopy, high-resolution electrospray ionization mass spectrometry, and electronic circular dichroism. In vitro bioassay using H2O2-induced PC12 cell damage models demonstrated that hempseeds extract and compounds 1, 3, 15, 26, 30, 36, 41, and 48 exhibited neuroprotective properties. 3,3'-Demethylgrossamide (30) displayed encouraging protection activity, which was further investigated to relieve the oxidative stress and apoptosis of PC12 cells treated with H2O2. The isolation and characterization of these neuroprotective phenylpropanamides from the fruits of C. sativa provide insights into its health-promoting properties as a healthy food and herbal medicine for preventing and treating neurodegenerative diseases, especially Alzheimer's disease.

Comparison of the Secondary Metabolism of the Basidiomycetes Armillaria mellea and Desarmillaria ectypa.

During the course of our ongoing studies on the secondary metabolism of cultures of Basidiomycota, a new meroterpenoid named 10, 15-dihydroxydihydromelleolide (1) was isolated along with the known armillaridin (2) and arnamiol (3) from cultures of the rare saprotrophic species, Desarmillaria ectypa. These are the first secondary metabolites that were ever isolated from the latter species. A concurrently studied strain of the common pathogenic A. mellea yielded other melleolides, with 5'-O-methylmelledonal (4), melledonal C (5), 10 α-hydroxydihydromelleolide (6) and melledonal (7). The chemical structures were elucidated using 1D and 2D NMR spectroscopy and high-resolution electrospray ionization mass spectrometry (HR-ESI-MS). All compounds were studied for their antimicrobial and cytotoxic effects against a panel of microbes and mammalian cell lines, and the results are also reported.

Undescribed secondary metabolites derived from Cinnamomum migao H. W. Li, showcasing anti-inflammatory, antioxidant, and in silico properties

Inflammatory processes are renowned as the fundamental basis for numerous pathological conditions. The objective of this study was to assess the antioxidant, anti-inflammatory, molecular docking, and molecular dynamics simulation properties of 3 previously undescribed and 1 known guaiane-type sesquiterpenoids isolated from Cinnamomum migao. The structures of these compounds were confirmed using numerous chromatographic and spectroscopic methods, such as high-resolution electrospray ionization mass spectrometry (HRESIMS), one-dimensional nuclear magnetic resonance (1D NMR), two-dimensional nuclear magnetic resonance (2D NMR), and experimental circular dichroism (ECD). Additionally, in oxidatively stressed mouse hepatocytes, all tested compounds at a concentration of 50 µM increased the levels of natural antioxidant enzymes such as CAT, SOD, POD, and Nrf-2 compared to those in untreated cells. Compounds 1 and 2 strongly suppressed NO generation and proinflammatory cytokine production, with IC50 values of 8.76 and 5.45 µM, respectively, against TNF-α mRNA. Furthermore, the molecular docking and simulations explore the high affinity of the selected compounds and best fit in the TNF-α active site. Compounds 1 and 2 show prominent binding energies and bring significant confirmational changes in the TNF-α protein. The results of this study provide further validation for the traditional use of C. migao in the treatment of inflammation and pain, providing novel evidence regarding the therapeutic potential of the identified guaiane-type sesquiterpenoids.