Database-aided ultrahigh-performance liquid chromatography Q-Exactive-Orbitrap tandem mass spectrometry putatively identifies 16 unexpected compounds and three anticounterfeiting pharmacopoeia quality markers for Perillae Fructus.

RATIONALE: Perillae Fructus (PF) is a common traditional Chinese medicine (TCM) for the treatment of asthma. It has not been effectively characterized by rosmarinic acid (RosA), which is currently designed as the sole quality indicator in the Chinese Pharmacopoeia. METHODS: This study introduced a database-aided ultrahigh-performance liquid chromatography equipped with quadrupole-Exactive-Orbitrap mass spectrometry (UHPLC/Q-Exactive-Orbitrap MS/MS) technology to putatively identify the compounds in PF, followed by literature research, quantum chemical calculation, and molecular docking to screen potential quality markers (Q-markers) of PF. RESULTS: A total of 27 compounds were putatively identified, 16 of which had not been previously found from PF. In particular, matrine, scopolamine, and RosA showed relatively high levels of content, stability, and drug-likeness. They exhibited interactions with the asthma-related target and demonstrated the TCM properties of PF. CONCLUSIONS: The database-aided UHPLC/Q-Exactive-Orbitrap MS/MS can identify at least 27 compounds in PF. Of these, 16 compounds are unexpected, and three compounds (matrine, scopolamine, and RosA) should be considered anticounterfeiting pharmacopoeia Q-markers of PF.

Development and Evaluation of a Novel Antibacterial Wound Dressing: A Powder Preparation Based on Cross-Linked Pullulan with Polyhexamethylene Biguanide for Hydrogel-Transition in Advanced Wound Management and Infection Control.

As antibiotic resistance increasingly undermines traditional infection management strategies, there is a critical demand for innovative wound care solutions that address these emerging challenges. This study introduces a novel antibacterial wound dressing based on Cross-Linked Pullulan (Pul) and Polyhexamethylene Biguanide (PHMB) for enhanced wound management and infection control. The dressing's adsorption rate reached 200% of its original weight within 30 min, exceeded 300% after 5 h, and exhibited significant non-Newtonian fluid properties. The dressings were able to release the loaded medication completely within 20 min; additionally, the dressing demonstrated significant antibacterial activity against a broad spectrum of bacteria. Significantly, the therapeutic effects of the Pul-PHMB/GP dressing were evaluated in a mouse model. Compared to untreated wounds, wounds treated with Pul-PHMB/GP exhibited a significant gelation process within 5 min post-treatment and showed a significant increase in wound healing rate within 12 days. This powder preparation overcomes the limitations associated with liquid and gel dressings, notably in storage and precise application, preventing the premature expansion or dissolution often caused by PHMB in high-humidity environments. The powder form can transform into a gel upon contact with wound exudate, ensuring accurate coverage of irregular wounds, such as those from burns or pressure sores, and offers excellent chemical and physical stability in a dry state, which facilitates storage and transport. This makes the dressing particularly suitable for emergency medical care and precision therapy, significantly improving the efficiency and adaptability of wound treatment and providing robust support for clinical treatments and emergency responses.

Detection of Adulterated Naodesheng Tablet (Naodesheng Pian) via In-Depth Chemical Analysis and Subsequent Reconstruction of Its Pharmacopoeia Q-Markers.

Naodesheng Tablet (Naodesheng Pian), a traditional Chinese medicine formula for stroke treatment, is made up of five herbal medicines, i.e., Sanqi, Gegen, Honghua, Shanzha, and Chuanxiong. However, the current Pharmacopoeia quality-marker (Q-marker) system cannot detect possible adulteration. Our study tried to use a new strategy, i.e., standards-library-dependent ultra-high-performance liquid chromatography-quadrupole-Orbitrap mass spectrometry (UHPLC-Q-Orbitrap MS/MS) putative identification, to reconstruct the Q-marker system. Through the strategy, 30 isomers were successfully differentiated (such as 2'-hydroxygenistein, luteolin, and kaempferol; ginsenoside Rg2 and ginsenoside Rg3; ginsenoside Rf and ginsenoside Rg1). In particular, 11 compounds were unexpectedly found in Naodesheng, including 2'-hydroxygenistein, 7,4'-dihydroxyflavone, pectolinarigenin, 7-methoxy-4'-hydroxyisoflavone, scoparone, matrine, 3,3',4',5,6,7,8-heptamethoxyflavone, 5-hydroxyflavone, diosgenin, chloesteryl acetate, and (+)-4-cholesten-3-one. In total, 68 compounds were putatively identified and fully elucidated for their MS spectra. Subsequently, relevant compounds were further investigated using UV-vis scanning experiments, semi-quantitative analysis, and quantum chemical calculation. Finally, five adulterated Naodesheng Tablets were used for validation experiments. The experiment successfully detected five adulterated ones via a lower-version LC-MS analysis. On this basis, three new candidates (hydroxy safflor yellow A (HSYA), citric acid, and levistilide A), along with puerarin and notoginsenoside R1, are re-nominated as the Q-markers for LC-MS analysis. The LC-MS analysis of puerarin, notoginsenoside R1, HSYA, citric acid, and levistilide A can clearly detect adulteration regarding all five herbal medicines mentioned above. Therefore, the reconstructed Q-markers are described as a "perfect" quality control system to detect adulteration in Naodesheng and will offer a valuable recommendation for the Pharmacopoeia Commission.

Proposing anti-counterfeiting pharmacopoeia quality markers for Shenlingbaizhu granule based on UHPLC-Q-orbitrap-MS identification.

INTRODUCTION: Shenlingbaizhu granule, a Traditional Chinese Medicine prescription comprising Renshen, Gancao, and Shanyao, is widely consumed in China nowadays. OBJECTIVE: The study tries to propose pharmacopoeia quality markers (Q-markers) to prevent counterfeiting involving Renshen, Gancao, and Shanyao. METHODOLOGY: A novel strategy, that is, library-based ultra-high-performance liquid chromatography-quadrupole-orbitrap mass spectrometry, was used to analyse the lyophilised aqueous powder of Shenlingbaizhu granule. Subsequently, quantum chemistry calculation and UV-vis spectra scanning were also performed through theoretical or experimental approaches. RESULT: Thirty-two isomers have been strictly distinguished, especially positional isomeric isochlorogenic acid B versus isochlorogenic acid C, positional isomeric schaftoside versus isoschaftoside, positional isomeric ginsenoside Rg2 versus 20S-ginsenoside Rg3, and stereoisomeric 20S-ginsenoside Rg3 versus 20R-ginsenoside Rg3. Seventeen compounds were unexpectedly observed, particularly scoparone and pectolinarigenin, while a total of 76 bioactive compounds have been putatively identified in the study. The quantum chemistry calculation and UV-vis spectra scanning results revealed that glycyrrhizic acid, ginsenoside Re, ginsenoside Rb1, and diosgenin displayed different dipole moment values and maximum absorption wavelengths from each other. CONCLUSION: The study recommends glycyrrhizic acid, ginsenoside Re, ginsenoside Rb1, and diosgenin as four anti-counterfeiting Q-markers for the pharmacopoeia. The anti-counterfeiting Q-markers can be detected using conventional HPLC. The observation of 17 unexpected compounds updates our knowledge regarding the bioactives of Shenlingbaizhu granule.

Database-aided UHPLC-Q-orbitrap MS/MS strategy putatively identifies 52 compounds from Wushicha Granule to propose anti-counterfeiting quality-markers for pharmacopoeia.

Wushicha Granule, an over-the-counter-drug (OTC) prescription, consists of 19 traditional Chinese herbals medicines (CHMs), such as Chaihu, Hongcha, Chuanxiong, Houpo, and Gancao. The five however have not been effectively characterized by the quality-markers (Q-markers) system in current Pharmacopoeia. The study therefore established a novel database-aided ultra-high performance liquid chromatography-quadrupole-orbitrap mass spectrometry (UHPLC-Q-orbitrap MS/MS) strategy. The strategy has putatively identified 52 compounds from Wushicha Granule, mainly including flavonoids, saponins, alkaloid, lignins, and lactones. Especially, saponin "glycyrrhetinic acid" in the Granule was specifically identified as 18ß-configuration (rather than 18α-configuration). Meanwhile, two pairs of isomers were fully discriminated, including vitexin vs isovitexin and daidzein vs 7,4'-dihydroxyflavone. 8ß-Glycyrrhetinic acid, together with saponin saikosaponin A, alkaloid caffeine, lactone S-senkyunolide A, and lignin magnolol, were further studied using quantum chemical calculation, UV-vis spectra, and anti-counterfeiting validation experiment. In the validation experiment, they have successfully recognized 6 counterfeit Wushicha Granules, by means of a LC-MS equipped extraction software. Based on these results, 8ß-glycyrrhetinic acid is recommended to replace the old Q-marker "glycyrrhetinic acid"; while saikosaponin A, caffeine, S-senkyunolide A, and magnolol are recommended as new Q-markers. These recommendations can not only recognize the counterfeits regarding Chaihu, Hongcha, Chuanxiong, Houpo, and Gancao, but also prevent the possible safety-incident. All these will greatly improve the efficiency and specificity of current Pharmacopoeia.

Stimulus-responsive peptide hydrogels: a safe and least invasive administration approach for tumor treatment.

Tumours, with increasing mortality around the world, have bothered human beings for decades. Enhancing the targeting of antitumor drugs to tumour tissues is the key to enhancing their antitumor effects. The tumour microenvironment is characterised by a relatively low pH, overexpression of certain enzymes, redox imbalance, etc. Therefore, smart drug delivery systems that respond to the tumour microenvironment have been proposed to selectively release antitumor drugs. Among them, peptide hydrogels as a local drug delivery system have received much attention due to advantages such as high biocompatibility, degradability and high water-absorbing capacity. The combination of peptide segments with different physiological functions allows for tumour targeting, self-aggregation, responsiveness, etc. Morphological and microstructural changes in peptide hydrogels can occur when utilising the inherent pathological microenvironment of tumours to trigger drug release, which endows such systems with limited adverse effects and improved therapeutic efficiency. Herein, this review outlined the driving forces, impact factors, and sequence design in peptide hydrogels. We also discussed the triggers to induce the transformation of peptide-based hydrogels in the tumour microenvironment and described the advancements of peptide-based hydrogels for local drug delivery in tumour treatment. Finally, we gave a brief perspective on the prospects and challenges in this field.

Bioactive peptides derived from Radix Angelicae sinensis inhibit ferroptosis in HT22 cells through direct Keap1-Nrf2 PPI inhibition.

The development of natural peptides as direct Kelch-like ECH-associated protein 1 (Keap1)-nuclear factor erythroid2-related factor 2 (Nrf2) protein-protein interaction (PPI) inhibitors for antioxidant and anti-ferroptotic purposes has attracted increasing interest from chemists. Radix Angelicae sinensis (RAS) is a widely used traditional Chinese medicine with antioxidant capability. However, few studies have screened Keap1-Nrf2 PPI inhibitory RAS peptides (RASPs). This study optimized the extraction and hydrolysis protocols of RAS protein using response surface methodology coupled with Box-Behnken design. The molecular weight distribution of the prepared hydrolysates was analysed to obtain active fractions. Subsequently, ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry was employed to identify RASPs. Various in vitro and in silico assays were conducted to evaluate the antioxidant and anti-ferroptotic effects of RASPs. The results revealed that at least 50 RASPs could be obtained through the optimized protocols. RASPs containing active residues effectively scavenged 2,2-diphenyl-1-picrylhydrazyl radical and 2,2'-azinobis(3-ethylbenzothiazoline)-6-sulfonic acid radical cation. They also showed cytoprotective effect against erastin-induced ferroptosis in HT22 cells, which was characterized by the activation of Nrf2 and weakened under the incubation of an Nrf2 inhibitor. Moreover, RASPs could bind to Keap1 and then dissociate Nrf2 in molecular dynamics simulations. In conclusion, RASPs exhibit antioxidant activity through hydrogen atom transfer and electron transfer mechanisms. Importantly, they also inhibit ferroptosis by directly inhibiting Keap1-Nrf2 PPI.

Ultra-high-performance liquid chromatography-Quadrupole-Exactive-Orbitrap-tandem mass spectrometry-based putative identification for Eucommiae Folium (Duzhongye) and its quality-marker candidate for pharmacopeia.

Eucommiae Folium (Duzhongye) is a traditional Chinese medicine with a long history of use in China. However, its quality-marker in Chinese Pharmacopoeia is poorly defined nowadays. The study, therefore, conducted an ultra-high-performance liquid chromatography coupled with hybrid quadrupole-orbitrap tandem mass spectrometry analysis to obtain accurate data. The obtained data were then compared with the authentic standards library using Xcalibur 4.1 software package and TraceFinder General Quan. Through the comparison, the study has putatively identified 26 bioactive compounds, which include 17 flavonoid derivatives (catechin, quercetin 3-gentiobioside, quercetin 3-O-ß-D-glucose-7-O-ß-D-gentiobioside, taxifolin, myricetin 3-O-galactoside, myricitrin, hyperoside, rutin, isoquercitrin, quercetin 3-O-ß-xylopyranoside, quercitrin, isorhamnetin 3-O-ß-D-glucoside, quercetin, kaempferol, S-eriodictyol, S-naringenin, and phloridzin), four caffeoylquinic acids (neochlorogenic acid, chlorogenic acid, isochlorogenic acid A, and isochlorogenic acid C), two alkaloids (vincamine and jervine), one lignan (pinoresinol), one xanthone (cowaxanthone B), and one steroid (cholesteryl acetate). Of these, flavonoid isoquercitrin is recommended as the new and additional pharmacopeia quality-marker candidate, which can not only overcome the unreliability of old quality-marker but also recognize the possible counterfeit.

Antioxidant mechanisms and products of four 4',5,7-trihydroxyflavonoids with different structural types.

4',5,7-OHs are common substituents of natural flavonoids, a type of effective phenolic antioxidant. However, the antioxidant processes between 4',5,7-trihydroxyflavonoids with different structural types have not been compared systematically, and the antioxidant products are challenging to determine. This study compared four 4',5,7-trihydroxyflavonoids, including apigenin, genistein, kaempferol, and naringenin. In quantum chemical analyses, the four 4',5,7-trihydroxyflavonoids showed different thermodynamic properties, and the C4'-OH (or C3-OH of kaempferol) possessed the strongest activity. Moreover, the reaction rate constants were larger when a hydrogen atom was transferred from C4'-OH (or C3-OH of kaempferol) than from C5-OH. When different atoms were linked to 2,2-diphenyl-1-picrylhydrazyl radical (DPPH˙), the C3'-DPPH adducts showed the smallest energy. In experimental assays, the scavenging ability for neutral free radicals, radical cations, and radical anions was negatively correlated with the corresponding theoretical parameters. Finally, mass spectroscopy detected the apigenin-DPPH˙, genistein-DPPH˙, and naringenin-DPPH˙ adduct peaks. In conclusion, the structural type of 4',5,7-trihydroxyflavonoids can affect the antioxidant ability, site, and speed, but not the mechanism. After hydrogen abstraction at C4'-OH, 4',5,7-trihydroxyflavones, 4',5,7-trihydroxyisoflavones, and 4',5,7-trihydroxyflavanones will produce antioxidant products via C3'-radical linking.

Linkage and Stereochemistry Characters of Phenolic Antioxidant Product Formation.

The present study developed a smart and novel strategy to elucidate the linkage and stereochemistry characters during phenolic antioxidant product formation. A series of phenolic isomers or analogues were treated with 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide radical, to create 16 antioxidant dimerization reactions in aqueous solution. The products were rapidly identified by ultraperformance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass-spectrometry. Through a systematic function-structure relationship analysis of these reactions and theoretical calculations, it is concluded that the phenolic antioxidant product is formed via linear linkage or furanocyclic linkage. The linear linkage is fulfilled via a radical coupling and controlled by the O-O linkage exclusion, meta-linkage exclusion, and catechol-activated principles. However, when an exocyclic π-bond conjugates with the phenolic core and is affixed at the -OH para-position, the furanocyclic linkage may occur via a subsequent intramolecular Michael addition. The intramolecular addition always lacks Re-attack to show "α,ß diastereoselectivity." The α,ß diastereoselectivity is the stereochemistry character of furanocyclic linkage during phenolic antioxidant product formation. All these novel findings can benefit not only the field food science but also other fields as well.

Phytophenol Dimerization Reaction: From Basic Rules to Diastereoselectivity and Beyond.

Phytophenol dimerization, which is a radical-mediated coupling reaction, plays a critical role in many fields, including lignin biosynthesis. To understand the reaction, 2,2-diphenyl-1-picrylhydrazyl radical was used to initiate a series of phytophenol dimerization reactions in methanol. The products were identified using ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UHPLC-ESI-Q-TOF-MS/MS) analysis in situ. The identified products mainly included biphenols, magnolol, honokiol, gingerol 6,6'-dimers, 3,6-dimethoxylcatechol ß,ß' dimer, euphorbetin, bis-eugenol, dehydrodiisoeugenol, trans-ε-viniferin, (+) pinoresinol, and (-) pinoresinol. Structure-function relationship analysis allowed four basic rules to be defined: meta-excluded, C-C bonding domination, ortho-diOH co-activation, and exocyclic C=C involvement. The exocyclic C=C involvement, however, required conjugation with the phenolic core and the para-site of the -OH group, to yield a furan-fused dimer with two chiral centers. Computational chemistry indicated that the entire process was completed via a radical coupling reaction and an intramolecular conjugate addition reaction. Similar results were also found for the horseradish peroxidase (HRP)-catalyzed coniferyl alcohol dimerization, which produced (+) and (-) pinoresinols (but no (-) epipinoresinol), suggesting that the HRP-catalyzed process was essentially an exocyclic C=C-involved phytophenol dimerization reaction. The reaction was highly diastereoselective. This was attributed to the intramolecular reaction, which prohibited Re-attack. The four basic rules and diastereoselectivity can explain and even predict the main products in various chemical and biological events, especially oxidase-catalyzed lignin cyclization.

Ferroptosis-Inhibitory Difference between Chebulagic Acid and Chebulinic Acid Indicates Beneficial Role of HHDP.

The search for a safe and effective inhibitor of ferroptosis, a recently described cell death pathway, has attracted increasing interest from scientists. Two hydrolyzable tannins, chebulagic acid and chebulinic acid, were selected for the study. Their optimized conformations were calculated using computational chemistry at the B3LYP-D3(BJ)/6-31G and B3LYP-D3(BJ)/6-311 + G(d,p) levels. The results suggested that (1) chebulagic acid presented a chair conformation, while chebulinic acid presented a skew-boat conformation; (2) the formation of chebulagic acid requires 762.1729 kcal/mol more molecular energy than chebulinic acid; and (3) the 3,6-HHDP (hexahydroxydiphenoyl) moiety was shown to be in an (R)- absolute stereoconfiguration. Subsequently, the ferroptosis inhibition of both tannins was determined using a erastin-treated bone marrow-derived mesenchymal stem cells (bmMSCs) model and compared to that of ferrostatin-1 (Fer-1). The relative inhibitory levels decreased in the following order: Fer-1 > chebulagic acid > chebulinic acid, as also revealed by the in vitro antioxidant assays. The UHPLC-ESI-Q-TOF-MS analysis suggested that, when treated with 16-(2-(14-carboxytetradecyl)-2-ethyl-4,4-dimethyl-3-oxazolidinyloxy free radicals, Fer-1 generated dimeric products, whereas the two acids did not. In conclusion, two hydrolyzable tannins, chebulagic acid and chebulinic acid, can act as natural ferroptosis inhibitors. Their ferroptosis inhibition is mediated by regular antioxidant pathways (ROS scavenging and iron chelation), rather than the redox-based catalytic recycling pathway exhibited by Fer-1. Through antioxidant pathways, the HHDP moiety in chebulagic acid enables ferroptosis-inhibitory action of hydrolyzable tannins.

Comparison of Ferroptosis-Inhibitory Mechanisms between Ferrostatin-1 and Dietary Stilbenes (Piceatannol and Astringin).

Synthetic arylamines and dietary phytophenolics could inhibit ferroptosis, a recently discovered regulated cell death process. However, no study indicates whether their inhibitory mechanisms are inherently different. Herein, the ferroptosis-inhibitory mechanisms of selected ferrostatin-1 (Fer-1) and two dietary stilbenes (piceatannol and astringin) were compared. Cellular assays suggested that the ferroptosis-inhibitory and electron-transfer potential levels decreased as follows: Fer-1 >> piceatannol > astringin; however, the hydrogen-donating potential had an order different from that observed by the antioxidant experiments and quantum chemistry calculations. Quantum calculations suggested that Fer-1 has a much lower ionization potential than the two stilbenes, and the aromatic N-atoms were surrounded by the largest electron clouds. By comparison, the C4'O-H groups in the two stilbenes exhibited the lowest bond disassociation enthalpies. Finally, the three were found to produce corresponding dimer peaks through ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry analysis. In conclusion, Fer-1 mainly depends on the electron transfer of aromatic N-atoms to construct a redox recycle. However, piceatannol and astringin preferentially donate hydrogen atoms at the 4'-OH position to mediate the conventional antioxidant mechanism that inhibits ferroptosis, and to ultimately form dimers. These results suggest that dietary phytophenols may be safer ferroptosis inhibitors for balancing normal and ferroptotic cells than arylamines with high electron-transfer potential.

Ferroptosis-Inhibitory Effect and Possible Mechanisms of Ellagitannin Geraniin.

The search for safe and effective ferroptosis-inhibitors has become an important topic. Geraniin, an ellagitannin bearing hexahydroxydiphenoyl (HHDP) and dehydrohexahydroxydiphenoyl (DHHDP) groups, was observed to inhibit erastin-induced ferroptosis in bone marrow-derived mesenchymal stem cells (bmMSCs). To determine the mechanism, geraniin was further analyzed using UV-vis spectra and several colorimetric assays, where its IC50 values were always much lower than that of the Trolox positive control. When interacted with several free radicals, geraniin gave no radical adduct formation (RAF) peak in the ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry. In conclusion, geraniin exhibits ferroptosis-inhibitory potential towards erastin-treated bmMSCs; such potential may mainly stem from its strong lipid peroxidation (LPO)-inhibition, Fe2+ -chelating, and antioxidant actions. Geraniin gives neither dimer nor radical adduct, owing to the bulky HHDP (or DHHDP) group; thus, it is considered as a safe and effective ferroptosis-inhibitor.

Covalent Bridging of Corilagin Improves Antiferroptosis Activity: Comparison with 1,3,6-Tri-O-galloyl-ß-d-glucopyranose.

The ellagitannin corilagin and its analogue 1,3,6-tri-O-galloyl-ß-d-glucopyranose (TGG) were found to protect bone marrow-derived mesenchymal stem cells (bmMSCs) against erastin-induced ferroptosis by cellular assays. However, the antiferroptosis bioactivity of corilagin was higher than that of TGG. Corilagin also exhibited higher antioxidant and Fe2+-chelation levels than TGG. Treated with 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals, corilagin and TGG yielded a corilagin- and a TGG-DPPH adduct, respectively. The corilagin-DPPH adduct retained the covalent bridge throughout the ultrahigh-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UHPLC-ESI-Q-TOF-MS) analysis. The strength of the covalent bridge is attributable to enhancement of its partial π-π conjugation. Thus, the bridge has sufficient strength to twist the chair conformation of the glucopyranosyl ring and to assemble two large aromatic rings, thereby improving the antioxidant (including Fe2+-chelation) reactivities. The bridge can also stabilize the product intermediate via partial π-π conjugation. Hence, corilagin is a superior ferroptosis inhibitor and antioxidant compared to TGG.

Structure-activity relationship and mechanism of four monostilbenes with respect to ferroptosis inhibition.

Erastin-treated bone marrow-derived mesenchymal stem cells (bmMSCs) were prepared and used to compare the ferroptosis inhibitory bioactivities of four monostilbenes, including rhapontigenin (1a), isorhapontigenin (1b), piceatannol-3'-O-glucoside (1c), and rhapontin (1d). Their relative levels were 1c ≈ 1b > 1a ≈ 1d in 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid (C11-BODIPY), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and flow cytometric assays. The comparison highlighted two 4'-OH-containing monostilbenes (1c and 1b) in ferroptosis inhibitory bioactivity. Similar structure-activity relationships were also observed in antioxidant assays, including 1,1-diphenyl-2-picryl-hydrazl radical (DPPH˙)-trapping, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide radical (PTIO˙)-trapping, and Fe3+-reducing assays. UPLC-ESI-Q-TOF-MS analysis of the DPPH˙-trapping reaction of the monostilbenes revealed that they can inhibit ferroptosis in erastin-treated bmMSCs through a hydrogen donation-based antioxidant pathway. After hydrogen donation, these monostilbenes usually produce the corresponding stable dimers; additionally, the hydrogen donation potential was enhanced by the 4'-OH. The enhancement by 4'-OH can be attributed to the transannular resonance effect. This effect can be used to predict the inhibition potential of other π-π conjugative phenolics.

Antioxidant and Cytoprotective effects of Pyrola decorata H. Andres and its five phenolic components.

BACKGROUND: Pyrola decorata H. Andres, is exclusively distributed in China and a source of traditional Chinese herbal medicine Luxiancao for more than 2000 years. Here, we evaluated the antioxidant and cytoprotective effects of P. decorata and its five phenolic components (protocatechuic acid, gallic acid, hyperoside, 2''-O-galloylhyperin, and quercetin), and discussed their antioxidant chemistry. METHODS: A lyophilized aqueous extract of P. decorata (LAEP) was prepared and analyzed with high-performance liquid chromatography (HPLC). LAEP and its five phenolic components were comparatively investigated using five antioxidant assays, including ferric-reducing antioxidant power, cupric ion-reducing antioxidant capacity, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide radical (PTIO•)-scavenging, 1,1-diphenyl-2-picryl-hydrazl radical (DPPH•)-scavenging, and 2,2'-azino-bis(3-ethylbenzo-thiazoline-6-sulfonic acid) radical (ABTS+•)-scavenging activities. The reaction products of the five phenolic components with 4-methoxy-2,2,6,6-tetramethylpiperidine-1-oxyl radical (4-methoxy-TEMPO•) were determined with ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UPLC-ESI-Q-TOF-MS/MS) analysis. LAEP and its five phenolic components were incubated with bone marrow-derived mesenchymal stem cells (bmMSCs) subjected to oxidative stress to demonstrate their cytoprotective effects with a flow cytometry assay. RESULTS: In the five antioxidant assays, LAEP and its five phenolic components dose-dependently increased the radical-scavenging (or reducing power) activities. However, the IC50 values of hyperoside were consistently higher than those of 2''-O-galloylhyperin. UPLC-ESI-Q-TOF-MS/MS analysis results indicated that the five phenolics could yield dimer products in the presence of 4-methoxy-TEMPO• via the radical adduct formation (RAF) pathway. Flow cytometry assay results confirmed the cytoprotective activity of LAEP and its five phenolic components toward stressed bmMSCs. In particular, 2''-O-galloylhyperin could more effectively reduce the percentage of damaged bmMSCs than hyperoside. CONCLUSION: LAEP and its five phenolic components may undergo redox-based pathways (such as electron transfer and H+ transfer) and covalent-based pathway (i.e., RAF) to exhibit antioxidant activity. One consequence of RAF is the generation of phenolic-phenolic dimer. In both organic and aqueous media, 2''-O-galloylhyperin exhibited higher redox-based antioxidant levels (or cytoprotective levels) than those with hyperoside. The differences could be attributed to 2''-O-galloylation reaction.

CRM197-Coupled Der p 2 Peptides Suppress Allergic Airway Inflammation in a Der p 2-Induced Asthma Mouse Model.

Allergic diseases affect more than 25% of the global population. Der p 2 is the major allergen of the house dust mite (HDM) Dermatophagoides pteronyssinus. Allergen-specific immunotherapy is the only treatment to change the course of allergic diseases. In this study, two synthesized Der p 2 peptides coupled to cross-reacting material 197 (CRM197) showed reduced IgE reactivity and allergenic activity. CRM197-coupled Der p 2 peptides induced rDer p 2-specific IgG1 antibodies in mice, which could inhibit HDM-allergic patients' IgE binding to rDer p 2. The immunity effects of CRM197-coupled Der p 2 peptides were studied in an rDer p 2-induced asthma mouse model. CRM197-coupled Der p 2 peptides can suppress asthmatic airway inflammation in this model. Analysis of IL-4, IL-5, and IFN-γ levels in bronchoalveolar lavage fluid revealed that the suppression was associated with a shift from a Th2 to a Th1 response. Thus, CRM197-bound Der p 2 peptides exhibited less allergenic activity than the rDer p 2 allergen, which preserved immunogenicity and may be candidates for mite allergy vaccines.

Structure-Activity Relationship and Prediction of the Electron-Transfer Potential of the Xanthones Series.

The structure-activity relationships of 31 xanthones were analyzed by using the ferric reducing antioxidant power (FRAP) assay to determine their electron-transfer (ET) potential. It was proven that the ET potential of xanthones was dominated by four moieties (i.e. hydroquinone moiety, 5,6-catechol moiety, 6,7-catechol moiety, and 7,8-catechol moiety) and was only slightly affected by other structural features, including a single phenolic OH group, the resorcinol moiety, the transannular dihydroxy moiety, a methoxy group, a sugar residue, an isoprenyl group, a cyclized isoprenyl group, and an isopentanol group. The results could be used to predict the ET potentials of other antioxidant xanthones.

2-Phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide Radical (PTIO•) Trapping Activity and Mechanisms of 16 Phenolic Xanthones.

This study used the 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide radical (PTIO•) trapping model to study the antioxidant activities of 16 natural xanthones in aqueous solution, including garcinone C, γ-mangostin, subelliptenone G, mangiferin, 1,6,7-trihydroxy-xanthone, 1,2,5-trihydroxyxanthone, 1,5,6-trihydroxyxanthone, norathyriol, 1,3,5,6-tetrahydroxy-xanthone, isojacareubin, 1,3,5,8-tetrahydroxyxanthone, isomangiferin, 2-hydroxyxanthone, 7-O-methylmangiferin, neomangiferin, and lancerin. It was observed that most of the 16 xanthones could scavenge the PTIO• radical in a dose-dependent manner at pH 4.5 and 7.4. Among them, 12 xanthones of the para-di-OHs (or ortho-di-OHs) type always exhibited lower half maximal inhibitory concentration (IC50) values than those not of the para-di-OHs (or ortho-di-OHs) type. Ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UPLC-ESI-Q-TOF-MS/MS) analysis revealed that most of these xanthones gave xanthone-xanthone dimers after incubation with PTIO•, except for neomangiferin. Based on these data, we concluded that the antioxidant activity of phenolic xanthone may be mediated by electron-transfer (ET) plus H⁺-transfer mechanisms. Through these mechanisms, some xanthones can further dimerize unless they bear huge substituents with steric hindrance. Four substituent types (i.e., para-di-OHs, 5,6-di-OHs, 6,7-di-OHs, and 7,8-di-OHs) dominate the antioxidant activity of phenolic xanthones, while other substituents (including isoprenyl and 3-hydroxy-3-methylbutyl substituents) play a minor role as long as they do not break the above four types.