Hong Xie

Antioxidant and Cytoprotective Effects of Tibetan Tea and Its Phenolic Components

Tibetan tea (Kangzhuan) is an essential beverage of the Tibetan people. In this study, a lyophilized aqueous extract of Tibetan tea (LATT) was prepared and analyzed by HPLC. The results suggested that there were at least five phenolic components, including gallic acid, and four catechins (i.e., (+)-catechin, (−)-catechin gallate (CG), (−)-epicatechin gallate (ECG), and (−)-epigallocatechin gallate). Gallic acid, the four catechins, and LATT were then comparatively investigated by four antioxidant assays: ferric reducing antioxidant power, 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide radical (PTIO•) scavenging, 1,1-diphenyl-2-picryl-hydrazl radical scavenging, and 2,2′-azino-bis(3-ethylbenzo-thiazoline-6-sulfonic acid) radical scavenging assays. In these assays, LATT, along with the five phenolic components, increased their antioxidant effects in a concentration-dependent manner; however, the half maximal scavenging concentrations of ECG were always lower than those of CG. Gallic acid and the four catechins were also suggested to chelate Fe2+ based on UV-visible spectral analysis. Ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UPLC−ESI−Q−TOF−MS/MS) analysis suggested that, when mixed with PTIO•, the five phenolic components could yield two types of radical adduct formation (RAF) products (i.e., tea phenolic dimers and tea phenolic-PTIO• adducts). In a flow cytometry assay, (+)-catechin and LATT was observed to have a cytoprotective effect towards oxidative-stressed bone marrow-derived mesenchymal stem cells. Based on this evidence, we concluded that LATT possesses antioxidative or cytoprotective properties. These effects may mainly be attributed to the presence of phenolic components, including gallic acid and the four catechins. These phenolic components may undergo electron transfer, H+-transfer, and Fe2+-chelating pathways to exhibit antioxidative or cytoprotective effects. In these effects, two diastereoisomeric CG and ECG showed differences to which a steric effect from the 2-carbon may contribute. Phenolic component decay may cause RAF in the antioxidant process.

π-π Conjugation Enhances Oligostilbene’s Antioxidant Capacity: Evidence from α-Viniferin and Caraphenol A

α-Viniferin and caraphenol A, the two oligostilbenes, have the sole difference of the presence or absence of an exocyclic double bond at the π-π conjugative site. In this study, the antioxidant capacity and relevant mechanisms for α-viniferin and caraphenol A were comparatively explored using spectrophotometry, UV-visible spectral analysis, and electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UPLC–ESI–Q–TOF–MS/MS) analysis. The spectrophotometric results suggested that caraphenol A always gave lower IC50 values than α-viniferin in cupric ion-reducing antioxidant capacity assay, ferric-reducing antioxidant power assay, 1,1-diphenyl-2-picryl-hydrazl radical (DPPH•)-scavenging, and 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide radical-scavenging assays. In UV-visible spectra analysis, caraphenol A was observed to show enhanced peaks at 250–350 nm when mixed with Fe2+, but α-viniferin exhibited no similar effects. UPLC–ESI–Q–TOF–MS/MS analysis revealed that α-viniferin mixed with DPPH• produced radical adduct formation (RAF) peak (m/z = 1070–1072). We conclude that the antioxidant action of α-viniferin and caraphenol A may involve both redox-mediated mechanisms (especially electron transfer and H+-transfer) and non-redox-mediated mechanisms (including Fe2+-chelating or RAF). The π-π conjugation of the exocyclic double bond in caraphenol A can greatly enhance the redox-mediated antioxidant mechanisms and partially promote the Fe2+-chelating mechanism. This makes caraphenol A far superior to α-viniferin in total antioxidant levels.

Antioxidation and Cytoprotection of Acteoside and Its Derivatives: Comparison and Mechanistic Chemistry

The study tried to explore the role of sugar-residues and mechanisms of phenolic phenylpropanoid antioxidants. Acteoside, along with its apioside forsythoside B and rhamnoside poliumoside, were comparatively investigated using various antioxidant assays. In three electron-transfer (ET)-based assays (FRAP, CUPRAC, PTIO•-scavenging at pH 4.5), the relative antioxidant levels roughly ruled as: acteoside >forsythoside B > poliumoside. Such order was also observed in H+-transfer-involved PTIO•-scavenging assay at pH 7.4, and in three multiple-pathway-involved radical-scavenging assays, i.e., ABTS+•-scavenging, DPPH•-scavenging, and •O2−-scavenging. In UV-vis spectra, each of them displayed a red-shift at 335→364 nm and two weak peaks (480 and 719 nm), when mixed with Fe2+; however, acteoside gave the weakest absorption. In Ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UPLC−ESI−Q−TOF−MS/MS) analysis, no radical-adduct-formation (RAF) peak was found. MTT assay revealed that poliumoside exhibited the highest viability of oxidative-stressed bone marrow-derived mesenchymal stem cells. In conclusion, acteoside, forsythoside B, and poliumoside may be involved in multiple-pathways to exert the antioxidant action, including ET, H+-transfer, or Fe2+-chelating, but not RAF. The ET and H+-transfer may be hindered by rhamnosyl and apiosyl moieties; however, the Fe2+-chelating potential can be enhanced by two sugar-residues (especially rhamnosyl moiety). The general effect of rhamnosyl and apiosyl moieties is to improve the antioxidant or cytoprotective effects.

Antioxidant and Cytoprotective Effects of the Di-O-Caffeoylquinic Acid Family: The Mechanism, Structure–Activity Relationship, and Conformational Effect

In this study, a series of di-O-caffeoylquinic acids (di-COQs) were systematically investigated for their antioxidant and cytoprotective effects towards •OH-damaged bone marrow-derived mesenchymal stem cells (bmMSCs). Five di-COQs were measured using a set of antioxidant assays. The results show that adjacent 4,5-Di-O-caffeoylquinic acid (4,5-COQ) and 3,4-di-O-caffeoylquinic acid (3,4-COQ) always gave lower IC50 values than did non-adjacent di-COQs. In the Fe2+-chelating assay, 4,5-COQ and 3,4-COQ presented greater UV-Vis spectra and darker colors than did non-adjacent di-COQs. In the UPLC-ESI-MS/MS analysis, no corresponding radical adduct formation (RAF) peak was found in the reaction products of di-COQs with PTIO•. In the MTT assay, all di-COQs (especially 1,5-COQ, 1,3-COQ, and 4,5-COQ) dose-dependently increased the cellular viabilities of •OH-damaged bmMSCs. Based on this evidence, we conclude that the five antioxidant di-COQs can protect bmMSCs from •OH-induced damage. Their antioxidant mechanisms may include electron-transfer (ET), H+-transfer, and Fe2+-chelating, except for RAF. Two adjacent di-COQs (4,5-COQ and 3,4-COQ) always possessed a higher antioxidant ability than the non-adjacent di-COQs (1,3-COQ, 1,5-COQ, and 3,5-COQ) in chemical models. However, non-adjacent 1,3-COQ and 1,5-COQ exhibited a higher cytoprotective effect than did adjacent di-COQs. These differences can be attributed to the relative positions of two caffeoyl moieties and, ultimately, to the conformational effect from the cyclohexane skeleton.

Effect of Double Bond Position on 2-Phenyl-benzofuran Antioxidants: A Comparative Study of Moracin C and Iso-Moracin C

Two 2-phenyl-benzofurans, moracin C {2-[3′,5′-dihydroxy-4′-(3-methlbut-2-enyl)phenyl]-6-hydroxybenzofuran} and its isomer iso-moracin C{2-[3′,5′-dihydroxy-4′-(3-methlbut-1-enyl)phenyl]-6-hydroxybenzofuran}, were comparatively studied using redox-related antioxidant assays and non-redox antioxidant assays. Moracin C always resulted in higher IC50 values than iso-moracin C in the redox-related antioxidant assays, including •O2−-inhibition, Cu2+-reducing power, DPPH•-inhibition, and ABTS+•-inhibition assays. In the non-redox antioxidant assay, moracin C and iso-moracin C underwent similar radical-adduct-formation (RAF), evidenced by the peaks at m/z 704 and m/z 618 in HPLC-MS spectra. In conclusion, both moracin C and iso-moracin C can act as 2-phenyl-benzofuran antioxidants; their antioxidant mechanisms may include redox-related ET and H+-transfer, and non-redox RAF. A double bond at the conjugation position can enhance the redox-related antioxidant potential, but hardly affects the RAF potential.

Protective Mechanism of the Antioxidant Baicalein toward Hydroxyl Radical-Treated Bone Marrow-Derived Mesenchymal Stem Cells

Our study explores the antioxidant and cytoprotective effects of baicalein and further discusses the possible mechanisms. A methyl thiazolyl tetrazolium (MTT) assay revealed that baicalein could considerably enhance the viability of hydroxyl radical-treated bone marrow-mesenchymal stem cells (bmMSCs) at 37–370 µM. The highest viability rate was 120.4%. In subsequent studies, baicalein was observed to effectively scavenge hydroxyl radical and PTIO• radicals, reducing Fe3+ and Cu2+ ions. In the Fe2+-chelating UV-vis spectra, mixing of baicalein with Fe2+ yielded two evident redshifts (275 → 279 nm and 324 → 352 nm) and a broad absorption peak (λmax ≈ 650 nm, ε = 1.6 × 103 L mol−1·cm−1). Finally, we compared the Fe2+-chelating UV-vis spectra of baicalein and its analogues, including 5-hydroxyflavone, 6-hydroxyflavone, 7-hydroxyflavone, catechol, pyrogallol, and chrysin. This analysis revealed that the 4-keto group of the C-ring played a role. The 5,6,7-trihydroxy-group (pyrogallol group) in the A-ring served as an auxochrome, enhancing the absorbance of the UV-vis spectra and deepening the color of the Fe2+-complex. We concluded that baicalein, as an effective hydroxyl radical-scavenger, can protect bmMSCs from hydroxyl radical-mediated oxidative stress. Its hydroxyl radical-scavenging effects are likely exerted via two pathways: direct scavenging of hydroxyl radicals, possibly through electron transfer, and indirect inhibition of hydroxyl radical generation via Fe2+ chelation through the 4-keto-5,6,7-trihydroxy groups.

Antioxidant Structure–Activity Relationship Analysis of Five Dihydrochalcones

The study determined the comparative antioxidant capacities of five similar dihydrochalcones: phloretin, phloridzin, trilobatin, neohesperidin dihydrochalcone, and naringin dihydrochalcone. In the ferric-reducing antioxidant power (FRAP) assay, the antioxidant activities of pairs of dihydrochalcones had the following relationship: phloretin > phloridzin, phloretin > trilobatin, trilobatin > phloridzin, trilobatin > naringin dihydrochalcone, and neohesperidin dihydrochalcone > naringin dihydrochalcone. Similar relative antioxidant levels were also obtained from 1,1-diphenyl-2-picryl-hydrazl radical (DPPH•)-scavenging, 2,2′-azino-bis(3-ethylbenzo-thiazoline-6-sulfonic acid) (ABTS•+)-scavenging, and superoxide radical (•O2−)-scavenging assays. Using ultra-performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UPLC−ESI−Q−TOF−MS/MS) analysis for the reaction products with DPPH•, phloretin, phloridzin, and trilobatin were found to yield both dihydrochalcone-DPPH adduct and dihydrochalcone-dihydrochalcone dimer, whereas naringin dihydrochalcone gave a naringin dihydrochalcone-DPPH adduct, and neohesperidin dihydrochalcone gave a dimer. In conclusion, the five dihydrochalcones may undergo redox-based reactions (especially electron transfer (ET) and hydrogen atom transfer (HAT)), as well as radical adduct formation, to exert their antioxidant action. Methoxylation at the ortho-OH enhances the ET and HAT potential possibly via p-π conjugation, whereas the glycosylation of the –OH group not only reduces the ET and HAT potential but also hinders the ability of radical adduct formation. The 2′,6′-di-OH moiety in dihydrochalcone possesses higher ET and HAT activities than the 2′,4′-di-OH moiety because of its resonance with the adjacent keto group.

Antioxidant and Cytoprotective Effects of Kukoamines A and B: Comparison and Positional Isomeric Effect

In this study, two natural phenolic polyamines, kukoamine A and B, were comparatively investigated for their antioxidant and cytoprotective effects in Fenton-damaged bone marrow-derived mesenchymal stem cells (bmMSCs). When compared with kukoamine B, kukoamine A consistently demonstrated higher IC50 values in PTIO•-scavenging (pH 7.4), Cu2+-reducing, DPPH•-scavenging, •O2−-scavenging, and •OH-scavenging assays. However, in the PTIO•-scavenging assay, the IC50 values of each kukoamine varied with pH value. In the Fe2+-chelating assay, kukoamine B presented greater UV-Vis absorption and darker color than kukoamine A. In the HPLC–ESI–MS/MS analysis, kukoamine A with DPPH• produced radical-adduct-formation (RAF) peaks (m/z 922 and 713). The 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl (MTT) assay suggested that both kukoamines concentration-dependently increased the viabilities of Fenton-damaged bmMSCs at 56.5–188.4 μM. However, kukoamine A showed lower viability percentages than kukoamine B. In conclusion, the two isomers kukoamine A and B can protect bmMSCs from Fenton-induced damage, possibly through direct or indirect antioxidant pathways, including electron-transfer, proton-transfer, hydrogen atom transfer, RAF, and Fe2+-chelating. Since kukoamine B possesses higher potentials than kukoamine A in these pathways, kukoamine B is thus superior to kukoamine A in terms of cytoprotection. These differences can ultimately be attributed to positional isomeric effects.

Steric Effect of Antioxidant Diels-Alder-Type Adducts: A Comparison of Sanggenon C with Sanggenon D

Sanggenons C and D are two Diels-Alder-type adducts from Chinese crude drug Sang-bai-pi. Structurally, both sanggenons construct stereoisomers. In the study, they were comparatively determined using four antioxidant assays, including ferric ion reducing antioxidant power (FRAP) assay, Cu2+-reducing assay, 1,1-diphenyl-2-picryl-hydrazl (DPPH•)-scavenging assay, and 2,2′-azino-bis (3-ethylbenzo-thiazoline-6-sulfonic acid radical (ABTS•+)-scavenging assay. Their Fe2+-binding reactions were explored using UV-Vis spectra. Finally, their cytoprotective effects were evaluated using flow cytometry. In electron transfer (ET)-based FRAP and Cu2+-reducing assays, sanggenon D was found to have lower IC50 values than sanggenon C; however, in multi-pathway-based DPPH•-scavenging and ABTS•+-scavenging assays, sanggenon C possessed lower IC50 values than sanggenon D. UV-Vis spectra suggested that sanggenon C generated a bathochromic-shift (286 nm → 302 nm) and displayed stronger UV absorption than sanggenon D. In flow cytometry, sanggenon C and sanggenon D, respectively, exhibited 31.1% and 42.0% early apoptosis-percentages towards oxidative-stressed mesenchymal stem cells (MSCs). In conclusion, both sanggenons may undergo multiple pathways (e.g., ET and Fe2+-binding) to protect MSCs against oxidative stress. In the mere ET aspect, sanggenon D possesses a higher level than sanggenon C, while in multi-pathway-based radical-scavenging, Fe2+-binding, and cytoprotection aspects, sanggenon C is more active than sanggenon D. These discrepancies can conclusively be attributed to the steric effect.