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.

Cotransplantation of NSCs and ethyl stearate promotes synaptic plasticity in PD rats by Drd1/ERK/AP-1 signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Traditional Chinese medicine views kidney shortage as a significant contributor to the aetiology of Parkinson's disease (PD), a neurodegenerative condition that is closely linked to aging. In clinical, patients with Parkinson's disease are often treated with Testudinis Carapax et Plastrum (Plastrum Testudinis, PT), a traditional Chinese medication that tonifies the kidney. Previous research has demonstrated that ethyl stearate (PubChem CID: 8122), an active component of Plastrum Testudinis Extracted with ethyl acetate (PTE), may encourage neural stem cells (NSCs) development into dopaminergic (DAergic) neurons. However, the effectiveness and mechanism of cotransplantation of ethyl stearate and NSCs in treating PD model rats still require further investigation. AIM OF THE STUDY: PD is a neurodegenerative condition marked by the loss and degradation of dopaminergic neurons in the substantia nigra of the midbrain. Synaptic damage is also a critical pathology in PD. Because of their self-renewal, minimal immunogenicity, and capacity to differentiate into dopaminergic (DAergic) neurons, NSCs are a prospective treatment option for Parkinson's disease cell transplantation therapy. However, encouraging transplanted NSCs to differentiate into dopaminergic neurons and enhancing synaptic plasticity in vivo remains a significant challenge in improving the efficacy of NSCs transplantation for PD. This investigation seeks to examine the efficacy of cotransplantation of NSCs and ethyl stearate in PD model rats and its mechanism related to synaptic plasticity. MATERIALS AND METHODS: On 6-hydroxydopamine-induced PD model rats, we performed NSCs transplantation therapy and cotransplantation therapy involving ethyl stearate and NSCs. Rotating behavior induced by apomorphine (APO) and pole climbing tests were used to evaluate behavioral changes. Using a variety of methods, including Western blotting (WB), immunofluorescence analysis, enzyme-linked immunosorbent assay, and quantitative real-time polymerase chain reaction (qRT-PCR), we examined the function and potential molecular mechanisms of ethyl stearate in combined NSCs transplantation therapy. RESULTS: In the rat PD model, cotransplantation of ethyl stearate with NSCs dramatically reduced motor dysfunction, restored TH protein levels, and boosted dopamine levels in the striatum, according to our findings. Furthermore, the expression levels of SYN1 and PSD95, markers of synaptic plasticity, and BDNF, closely related to synaptic plasticity, were significantly increased. Cotransplantation with ethyl stearate and NSCs also increased the expression levels of Dopamine Receptor D1 (Drd1), an important receptor in the dopamine neural circuit, accompanied by an increase in MMP9 levels, ERK1/2 phosphorylation levels, and c-fos protein levels. CONCLUSIONS: According to the results of our investigation, cotransplantation of ethyl stearate and NSCs significantly improves the condition of PD model rats. We found that cotransplantation of ethyl stearate and NSCs may promote the expression of MMP9 by regulating the Drd1-ERK-AP-1 pathway, thus improving synaptic plasticity after NSCs transplantation. These findings provide new experimental support for the treatment of PD with the kidney tonifying Chinese medicine Plastrum Testudinis and suggest a potential therapeutic strategy for PD based on cotransplantation therapy.

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.

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.

Atractylenolide-III alleviates osteoarthritis and chondrocyte senescence by targeting NF-κB signaling.

Atractylenolide-III (AT-III) is well known as its role in antioxidant and anti-inflammatory. Present study was aimed to figure out its effects on osteoarthritis and potential mechanisms. Rat model, human osteoarthritis cartilage explants as well as rat/human chondrocyte cultures were prepared to test AT-III's effects on osteoarthritis progression and chondrocyte senescence. Potential targeted molecules of AT-III were predicted using network pharmacology and molecular docking, assessed by Western blotting and then verified with rescue experiments. AT-III treatment alleviated osteoarthritis severity (shown by OARSI grading score and micro-CT) and chondrocyte senescence (indexed by levels of SA-ß-gal, P16, P53, MMP13, ROS and ratio of healthy/collapsed mitochondrial membrane potentials). Network pharmacology and molecular docking suggested that AT-III might play role through NF-κB pathway. Further experiments revealed that AT-III reduced phosphorylation of IKKα/ß, IκBα and P65 in NF-κB pathway. As well as nuclear translocation of p65. Both in vivo and in vitro experiments indicated that AT-III's effects on osteoarthritis and anti-senescence were reversed by an NF-κB agonist. AT-III could alleviate osteoarthritis by inhibiting chondrocyte senescence through NF-κB pathway, which indicated that AT-III is a prospective drug for osteoarthritis treatment.

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.

Protective effect of plastrum testudinis extract on dopaminergic neurons in a Parkinson's disease model through DNMT1 nuclear translocation and SNCA's methylation.

The pathological characteristics of Parkinson's disease (PD) include dopaminergic neuron damage, specifically disorders caused by dopamine synthesis, in vivo. Plastrum testudinis extract (PTE) and its bioactive ingredient ethyl stearate (PubChem CID: 8122) were reported to be correlated with tyrosine hydroxylase (TH), which is a biomarker of dopaminergic neurons. This suggests that PTE and its small-molecule active ingredient ethyl stearate have potential for development as a therapeutic drug for PD. In this study, we treated 6-hydroxydopamine (6-OHDA)-induced model rats and PC12 cells with PTE. The mechanism of action of PTE and ethyl stearate was investigated by western blotting, bisulfite sequencing PCR (BSP), real-time PCR, immunofluorescence and siRNA transfection. PTE effectively upregulated the TH expression and downregulated the alpha-synuclein expression in both the substantia nigra and the striatum of the midbrain in a PD model rat. The PC12 cell model showed that both PTE and its active monomer ethyl stearate significantly promoted TH expression and blocked alpha-synuclein, agreeing with the in vivo results. BSP showed that PTE and ethyl stearate increased the methylation level of the Snca intron 1 region. These findings suggest that some of the protective effects of PTE on dopaminergic neurons are mediated by ethyl stearate. The mechanism of ethyl stearate may involve disrupting the abnormal aggregation of DNA (cytosine-5)-methyltransferase 1 (DNMT1) with alpha-synuclein by releasing DNMT1, upregulating Snca intron 1 CpG island methylation, and ultimately, reducing the expression of alpha-synuclein.

Plastrum Testudinis Extracts Promote NSC Differentiation into Dopaminergic Neuron by Regulating the Interaction of TET1 and FoxA2.

In recent years, stem cells have gained much attention for the treatment of neurodegenerative diseases. However, inducing neural stem cell directionally differentiation is a difficult problem in the treatment of Parkinson's disease (PD) by stem cell therapy. Plastrum Testudinis (PT) can enhance the number of TH-positive neurons in the PD rat brain substantia nigra, but the underlying mechanism has not been clarified. Here, we aimed at further investigating the mechanism by which PT can promote NSC differentiation into dopaminergic neurons. A rat model of PD was used for detecting the effect of PT on the rat brain substantia nigra in vivo. The results showed the expressions of tyrosine hydroxylase (TH) and TET1 enzyme were increased after treatment with PT. Consequently, Plastrum Testudinis extracts (PTEs) were used for inducing NSC differentiation into dopaminergic neurons ex vivo. During differentiation of NSCs induced by PTE, TH expression was increased, with a concomitant increase in both TET1 and FoxA2. Next, we performed coimmunoprecipitation analysis to examine the interaction between TET1 protein and FoxA2 protein. Our results show that PTE can increase the binding rate of TET1 and FoxA2. Thus, our findings show that PTE can increase the efficiency of NSCs to directionally differentiate into dopaminergic neurons and provide experimental evidence for PT in the treatment of Parkinson's disease.

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.

Catharmus tinctorius volatile oil promote the migration of mesenchymal stem cells via ROCK2/Myosin light chain signaling.

MSC transplantation has been explored as a new clinical approach to stem cell-based therapies for bone diseases in regenerative medicine due to their osteogenic capability. However, only a small population of implanted MSC could successfully reach the injured areas. Therefore, enhancing MSC migration could be a beneficial strategy to improve the therapeutic potential of cell transplantation. Catharmus tinctorius volatile oil (CTVO) was found to facilitate MSC migration. Further exploration of the underlying molecular mechanism participating in the pro-migratory ability may provide a novel strategy to improve MSC transplantation efficacy. This study indicated that CTVO promotes MSC migration through enhancing ROCK2 mRNA and protein expressions. MSC migration induced by CTVO was blunted by ROCK2 inhibitor, which also decreased myosin light chain (MLC) phosphorylation. Meanwhile, the siRNA for ROCK2 inhibited the effect of CTVO on MSC migration ability and attenuated MLC phosphorylation, suggesting that CTVO may promote BMSC migration via the ROCK2/MLC signaling. Taken together, this study indicates that C. tinctorius volatile oil could enhance MSC migration via ROCK2/MLC signaling in vitro. C. tinctorius volatile oil-targeted therapy could be a beneficial strategy to improve the therapeutic potential of cell transplantation for bone diseases in regenerative medicine.

Antioxidant Mechanisms of Echinatin and Licochalcone A.

Echinatin and its 1,1-dimethyl-2-propenyl derivative licochalcone A are two chalcones found in the Chinese herbal medicine Gancao. First, their antioxidant mechanisms were investigated using four sets of colorimetric measurements in this study. Three sets were performed in aqueous solution, namely Cu2+-reduction, Fe3+-reduction, and 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide radical (PTIO•)-scavenging measurements, while 1,1-diphenyl-2-picrylhydrazyl radical (DPPH•)-scavenging colorimetric measurements were conducted in methanol solution. The four sets of measurements showed that the radical-scavenging (or metal-reduction) percentages for both echinatin and licochalcone A increased dose-dependently. However, echinatin always gave higher IC50 values than licochalcone A. Further, each product of the reactions of the chalcones with DPPH• was determined using electrospray ionization quadrupole time-of-flight tandem mass spectrometry (UPLC-ESI-Q-TOF-MS/MS). The UPLC-ESI-Q-TOF-MS/MS determination for echinatin yielded several echinatin⁻DPPH adduct peaks (m/z 662, 226, and 196) and dimeric echinatin peaks (m/z 538, 417, and 297). Similarly, that for licochalcone A yielded licochalcone A-DPPH adduct peaks (m/z 730, 226, and 196) and dimeric licochalcone A peaks (m/z 674 and 553). Finally, the above experimental data were analyzed using mass spectrometry data analysis techniques, resonance theory, and ionization constant calculations. It was concluded that, (i) in aqueous solution, both echinatin and licochalcone A may undergo an electron transfer (ET) and a proton transfer (PT) to cause the antioxidant action. In addition, (ii) in alcoholic solution, hydrogen atom transfer (HAT) antioxidant mechanisms may also occur for both. HAT may preferably occur at the 4-OH, rather than the 4'-OH. Accordingly, the oxygen at the 4-position participates in radical adduct formation (RAF). Lastly, (iii) the 1,1-dimethyl-2-propenyl substituent improves the antioxidant action in both aqueous and alcoholic solutions.

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.

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.

Two phenolic antioxidants in Suoyang enhance viability of •OH-damaged mesenchymal stem cells: comparison and mechanistic chemistry.

BACKGROUND: Suoyang originates from a psammophyte named Cynomorium songaricum Rupr and has been known as a phenolic-antioxidant-enriched traditional Chinese herbal medicine. The present study attempted to investigate the protective effect of phenolic antioxidants in Suoyang towards •OH-mediated MSCs and then further discusses the chemical mechanisms. METHODS: The lyophilized aqueous extract of Suoyang (LAS) was prepared and characterized using HPLC. Then, two phenolic antioxidant references, epicatechin and luteolin-7-O-ß-D-glucoside, along with LAS, were investigated for their effects on the viability of •OH-treated MSCs using the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl (MTT) assay. The comparison and mechanistic chemistry of epicatechin and luteolin-7-O-ß-D-glucoside were further explored using various antioxidant assays, including PTIO•-scavenging, FRAP (ferric ion reducing antioxidant power), ABTS+•-scavenging, and DPPH•-scavenging. Their Fe2+-binding capacities were also compared using ultraviolet (UV) spectra. RESULTS: The HPLC analysis indicated that there are 8 phenolic antioxidants in LAS, including epicatechin, luteolin-7-O-ß-D-glucoside, gallic acid, protocatechuic acid, catechin, isoquercitrin, phlorizin, and naringenin. The MTT assay revealed that epicatechin could more effectively increase the survival of •OH-treated MSCs than luteolin-7-O-ß-D-glucoside. Similarly, epicatechin exhibited higher antioxidant abilities than luteolin-7-O-ß-D-glucoside in the DPPH•-scavenging, ABTS+•-scavenging, FRAP, and PTIO•-scavenging assays. In the Fe2+-binding assay, luteolin-7-O-ß-D-glucoside gave a stronger UV peak at 600 nm, with ε = 2.62 × 106 M-1 cm-1, while epicatechin produced two peaks at 450 nm (ε = 8.47 × 105 M-1 cm-1) and 750 nm (ε = 9.68 × 105 M-1 cm-1). CONCLUSION: As two reference antioxidants in Suoyang, epicatechin and luteolin-7-O-ß-D-glucoside can enhance the viability of •OH-damaged MSCs. Such a beneficial effect may be from their antioxidant effects, including direct-antioxidant and indirect-antioxidant (i.e., Fe2+-binding) processes. In the direct-antioxidant process, proton (H+), one electron (e), or even hydrogen-atom (•H) transfer may occur to fulfill radical-scavenging (especially •OH-scavenging); in this aspect, epicatechin is superior to luteolin-7-O-ß-D-glucoside due to the presence of more phenolic -OHs. The additional -OHs can also be responsible for the better cytoprotective effect. In terms of indirect-antioxidant potential, however, epicatechin is inferior to luteolin-7-O-ß-D-glucoside due to the absence of a hydroxyl-keto moiety. These findings will provide new information about medicinal psammophytes for MSC transplantation.

Role of the p-Coumaroyl Moiety in the Antioxidant and Cytoprotective Effects of Flavonoid Glycosides: Comparison of Astragalin and Tiliroside.

The aim of this study was to explore the role of p-coumaroyl in the antioxidant and cytoprotective effects of flavonoid glycosides. The antioxidant effects of astragalin and tiliroside were compared using ferric ion reducing antioxidant power, DPPH• scavenging, ABTS•⁺ scavenging, •O2- scavenging, and Fe2+-chelating assays. The results of these assays revealed that astragalin and tiliroside both exhibited dose-dependent activities; however, tiliroside exhibited lower IC50 values than astragalin. In the Fe2+-chelating assay, tiliroside gave a larger shoulder-peak at 510 nm than astragalin, and was also found to be darker in color. Both of these compounds were subsequently evaluated in a Fenton-induced mesenchymal stem cell (MSC) damaged assay, where tiliroside performed more effectively as a cytoprotective agent than astragalin. Tiliroside bearing a 6''-O-p-coumaroyl moiety exhibits higher antioxidant and cytoprotective effects than astragalin. The 6''-O-p-coumaroyl moiety of tiliroside not only enhances the possibility of electron-transfer and hydrogen-atom-transfer-based multi-pathways, but also enhances the likelihood of Fe-chelating. The p-coumaroylation of the 6"-OH position could therefore be regarded as a potential approach for improving the antioxidant and cytoprotective effects of flavonoid glycosides in MSC implantation therapy.

Lyophilized aqueous extracts of Mori Fructus and Mori Ramulus protect Mesenchymal stem cells from •OH-treated damage: bioassay and antioxidant mechanism.

BACKGROUND: Mori Fructus and Mori Ramulus are two traditional Chinese herbal medicines from mulberries. The present work explores their beneficial effects on •OH-treated mesenchymal stem cells (MSCs) and discusses possible mechanisms. METHODS: Lyophilized aqueous extracts of Mori Fructus (LAMF) and Mori Ramulus (LAMR) were prepared and analyzed using HPLC. LAMF and LAMR (along with morin) were further investigated for their effects on •OH-treated MSCs using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl (MTT) assay. The direct antioxidation mechanisms were studied using 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO•)-scavenging, 2,2'-azino-bis (3-ethylbenzo-thiazoline-6-sulfonic acid (ABTS+•)-scavenging and 1,1-diphenyl-2-picryl-hydrazl (DPPH•)-scavenging, as well as Cu2+-reducing and Fe3+-reducing antioxidant power. Finally, the indirect antioxidant mechanism was investigated based on the UV-vis spectra of Fe2+-chelation. RESULTS: In each LAMF and LAMR, seven phytophenols were successfully measured by HPLC, including five flavonoids (morin, rutin, astragalin, isoquercitrin and luteolin) and two non-flavonoids (chlorogenic acid and maclurin). MTT assays revealed that LAMF, LAMR and morin could effectively increase the survival of •OH-treated MSCs at 10-100 µg/mL, and could effectively scavenge PTIO• (IC 50 6609.7 ± 756.6, 4286.9 ± 84.9 and 103.4 ± 0.9 µg/mL, respectively), DPPH• (IC 50 208.7 ± 3.0, 97.3 ± 3.1 and 8.2 ± 0.7 µg/mL, respectively) and ABTS+• (IC 50 73.5 ± 5.8, 34.4 ± 0.1 and 4.2 ± 0.2 µg/mL, respectively), and reduce Cu2+ (IC 50 212.5 ± 7.0, 123.2 ± 0.9 and 14.1 ± 0.04 µg/mL, respectively) & Fe3+ (IC 50 277.0 ± 3.1, 191.9 ± 5.2 and 5.0 ± 0.2 µg/mL, respectively). In the Fe2+-chelating assay, the five flavonoids produced much stronger shoulder-peaks than the two non-flavonoids within 420-850 nm. CONCLUSION: Mori Fructus and Mori Ramulus, can protect MSCs from •OH-induced damage. Such beneficial effects can mainly be attributed to the antioxidant action of phytophenols, which occurs via direct (ROS-scavenging) and indirect mechanism (Fe2+-chelating). The ROS-scavenging mechanism, however, include at least a H+-transfer and an electron-transfer (ET), and possibly includes a hydrogen-atom-transfer (HAT). In the Fe2+-chelating, flavonoids are more effective than non-flavonoids. This can be attributed to several adjacent planar chelating-sites between the 3-OH and 4-C = O, between the 4-C = O and 5-OH, or between the 3'-OH and 4'-OH in flavonoids. Such multiple-Fe2+-chelating reactions cause overlap in the UV-vis absorptions to deepen the complex color, enhance the peak strength, and form shoulder-peaks. By comparison, two non-flavonoids with catechol moiety produce only a weak single peak.

Sarcandra glabra (Caoshanhu) protects mesenchymal stem cells from oxidative stress: a bioevaluation and mechanistic chemistry.

BACKGROUND: Sarcandra glabra (Caoshanhu) is a traditional Chinese herbal medicine used for treating various oxidative-stressed diseases. The present work evaluated its protective effect on mesenchymal stem cells (MSCs) from oxidative stress and then discussed possible mechanisms underlying this observation. METHODS: Ethanolic extract of S. glabra (ESG) was investigated by chemical methods for its content of total phenolics, rosmarinic acid, and astilbin. ESG, along with rosmarinic acid and astilbin, was investigated for the effect on the viability of Fenton-treated MSCs using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl (MTT) assay. The observed cell protective effect was further explored by mechanistic chemistry using various antioxidant assays, including DNA protection, •OH-scavenging, •O2--scavenging, FRAP (ferric ion reducing antioxidant power), ABTS+•-scavenging, DPPH•-scavenging, and Fe2+-chelating assays. RESULTS: Analysis of ESG revealed a content of 46.31 ± 0.56 mg quercetin/g total phenolics, 0.78 ± 0.01 % rosmarinic acid, and 3.37 ± 0.01 % astilbin. Results from the MTT assay revealed that three compounds (rosmarinic acid>astilbin>ESG) could effectively increase the survival of Fenton-treated MSCs. Similarly, in •O2--scavenging, DPPH•-scavenging, and Fe2+-chelating assays, rosmarinic acid exhibited more activity than astilbin; while in FRAP, ABTS+•-scavenging assays, astilbin was stronger than rosmarinic acid. CONCLUSION: S. glabra can prevent MSCs from •OH-induced oxidative stress. Such protective effect can be attributed to its antioxidant ability and the presence of two kinds of phytophenols, i.e. caffeoyl derivatives and flavonoids. As the respective representatives of caffeoyl derivatives and flavonoids, rosmarinic acid and astilbin may exert the antioxidant action via direct ROS-scavenging and indirect ROS-scavenging (i.e. Fe2+-chelating). The direct ROS-scavenging ability involves hydrogen atom transfer (HAT) and/or electron transfer (ET) pathway. Astilbin engages the latter pathway more, which can be attributed to the larger planar conjugation in A/C fused rings. Rosmarinic acid, on the other hand, shows more HAT and Fe2+-chelating potential, which may be due to rosmarinic acid bearing one more catechol moiety whereas astilbin has steric-hindrance from 3-α-L-rhamnose and an H-bonding between 4,5 sites. The antioxidant features of rosmarinic acid can be generalized to other caffeoyl derivatives, while that of astilbin cannot be generalized to other flavonoids because of the difference in chemical structures.

Protective Effect against Hydroxyl-induced DNA Damage and Antioxidant Activity of Citri reticulatae Pericarpium.

PURPOSE: As a typical Chinese herbal medicine, Citri reticulatae pericarpium (CRP) possesses various pharmacological effects involved in antioxidant ability. However, its antioxidant effects have not been reported yet. The objective of this work was to investigate its antioxidant ability, then further discuss the antioxidant mechanism. METHODS: CRP was extracted by ethanol to obtain ethanol extract of Citri reticulatae pericarpium (ECRP). ECRP was then measured by various antioxidant methods, including DNA damage assay, DPPH assay, ABTS assay, Fe(3+)-reducing assay and Cu(2+)-reducing assay. Finally, the content of total flavonoids was analyzed by spectrophotometric method. RESULTS: Our results revealed that ECRP could effectively protect against hydroxyl-induced DNA damage (IC50 944.47±147.74 µg/mL). In addition, it could also scavenge DPPH· radical (IC50349.67±1.91 µg/mL) and ABTS(+)• radical (IC5011.33±0.10 µg/mL), reduce Fe(3+) (IC50 140.95±2.15 µg/mL) and Cu(2+) (IC50 70.46±1.77 µg/mL). Chemical analysis demonstrated that the content of total flavonoids in ECRP was 198.29±12.24 mg quercetin/g. CONCLUSION: Citri reticulatae pericarpium can effectively protect against hydroxyl-induced DNA damage. One mechanism of protective effect may be radical-scavenging which is via donating hydrogen atom (H·), donating electron (e). Its antioxidant ability can be mainly attributed to the flavonoids, especially hesperidin and narirutin.

Protective Effect against Hydroxyl-induced DNA Damage and Antioxidant Activity of Radix Glycyrrhizae (Liquorice Root).

PURPOSE: As a typical Chinese herbal medicine, Radix Glycyrrhizae (RG) possesses various pharmacological effects involved in antioxidant ability. However, its antioxidant has not been explored so far. The aim of the study was to investigate its antioxidant ability, then further discuss the antioxidant mechanism. METHODS: RG was extracted by ethanol to obtain ethanolic extract of Radix Glycyrrhizae (ERG). ERG was then determined by various antioxidant methods, including DNA damage assay, DPPH assay, ABTS assay, Fe(3+)-reducing assay and Cu(2+)-reducing assay. Finally, the contents of total phenolics and total flavonoids were analyzed by spectrophotometric methods. RESULTS: Our results revealed that ERG could effectively protect against hydroxyl-induced DNA damage (IC50 517.28±26.61µg/mL). In addition, ERG could scavenge DPPH· radical (IC50165.18±6.48µg/mL) and ABTS(+)• radical (IC507.46±0.07µg/mL), reduce Fe(3+) (IC50 97.23±2.88 µg/mL) and Cu(2+) (IC50 59.21±0.18 µg/mL). Chemical analysis demonstrated that the contents of total phenolics and flavonoids in ERG were 111.48±0.88 and 218.26±8.57 mg quercetin/g, respectively. CONCLUSION: Radix Glycyrrhizae can effectively protect against hydroxyl-induced DNA damage. One mechanism of protective effect may be radical-scavenging which is via donating hydrogen atom (H·), donating electron (e). Its antioxidant ability can be mainly attributed to the flavonoids or total phenolics.

Antioxidant ability and mechanism of rhizoma Atractylodes macrocephala.

Rhizoma Atractylodes macrocephala (AM) has been used in Traditional Chinese Medicine (TCM) for about 2,000 years. In the study, we firstly determined the antioxidant levels of five AM extracts by •OH-scavenging, •O2−-scavenging, Fe2+-chelating, Cu2+-chelating, DPPH·-scavenging, and ABTS+·-scavenging assays. After measurement of the chemical contents in five AM extracts, we quantitatively analyzed the correlations between antioxidant levels and chemical contents. It was observed that total phenolics and total flavonoids had significant positive correlations with antioxidant levels (R = 0.685 and 0.479, respectively). In contrast, total sugars and total saponins presented lower correlations with antioxidant levels (R=−0.272 and 0.244, respectively). It means that antioxidant activity of AM should be attributed to total phenolics (including phenolic acids and flavonoids), and not total sugars and total saponins. Further analysis indicated that phenolic acids exhibited higher R values with radical-scavenging assays (R=0.32­1.00), while flavonoids showed higher R values with metal-chelating assays (R=0.86 and 0.90). In conclusion, AM exerts its antioxidant effect through metal-chelating, and radical-scavenging which is via donating hydrogen atom and donating electron. Its metal-chelating may result from flavonoids, while its radical-scavenging can be attributed to phenolic acids, especially caffeic acid, ferulic acid, and protocatechuic acid.