Ting Shen

Antioxidant properties and neuroprotective effects of isocampneoside II on hydrogen peroxide-induced oxidative injury in PC12 cells

Oxidative stress has been considered as a major cause of cell damage in various neurodegenerative disorders. One of the reasonable strategies for delaying the diseases progression is to prevent reactive oxygen species (ROS) mediated cellular injury by dietary or pharmaceutical augmentation of free radical scavengers. Isocampneoside II (ICD) is an active phenylethanoid glycoside isolated from the medicinal hardwood genus Paulownia. This study was designed to explore free radical scavenging potential of ICD in different in vitro systems and its protective role in hydrogen peroxide (H₂O₂)-induced oxidative stress and apoptotic death in cultured rat pheochromocytoma (PC12) cells. The results showed ICD eliminated approximately 80.75% superoxide radical at the concentration of 0.1mg/ml and inhibited metal chelating by 22.07% at 8 mg/ml. Additionally, ICD showed a strong ability on reducing power and provided protection against oxidative protein damage induced by hydroxyl radicals. Pretreatment of PC12 cells with ICD prior to H₂O₂ exposure elevated cell viability, enhanced activity of superoxide dismutase and catalase, and decreased levels of malondialdehyde and intracellular ROS. Furthermore, ICD inhibited cell apoptosis and Bax/Bcl-2 ratio induced by H₂O₂. These findings suggested ICD may be considered as a potential antioxidant agent and should encourage for further research in neurodegenerative diseases.

The dichloromethane fraction from Mahonia bealei (Fort.) Carr. leaves exerts an anti-inflammatory effect both in vitro and in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE Mahonia bealei has a long history of medical use in traditional Chinese medicine for the treatment of inflammatory-associated diseases. Despite numerous phytochemical and pharmacological studies, there is a lack of systematic studies to understand the cellular and molecular mechanisms of the anti-inflammatory activity of this plant. AIM OF STUDY This study aimed to evaluate the anti-inflammatory activity of the dichloromethane fraction from M. bealei leaves (MBL-CH). MATERIALS AND METHODS RAW 264.7 cells were pretreated with different concentrations of MBL-CH for 30min prior to treatment with 1μg/ml of lipopolysaccharide (LPS). The nuclear factor κB (NF-κB) pathway and subsequent production of inflammatory mediators, such as nitric oxide (NO), prostaglandin E2 (PGE2), and tumour necrosis factor (TNF)-α were investigated. Furthermore, the in vivo mouse model of LPS-induced acute lung injury (ALI) was employed to study the anti-inflammatory effects of MBL-CH. RESULTS Pre-treatment with MBL-CH significantly inhibited the LPS-stimulated secretion of NO, PGE2, and TNF-α into the culture medium, as well as the mRNA levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and TNF-α, which were associated with a reduction in the phosphorylation of IκBα, Akt, and PI3K and inhibition of the transcriptional activity of NF-κB. Furthermore, in vivo experiments revealed that MBL-CH attenuated LPS-stimulated lung inflammation in mice. CONCLUSION Taken together, our findings indicate that MBL-CH attenuates LPS-stimulated inflammatory responses in macrophages by blocking NF-κB activation through interference with activation of the PI3K/Akt pathway, providing scientific evidence that the plant can be employed in traditional remedies.

5-Methoxyl Aesculetin Abrogates Lipopolysaccharide-Induced Inflammation by Suppressing MAPK and AP-1 Pathways in RAW 264.7 Cells.

For the first time, a pale amorphous coumarin derivative, 5-methoxyl aesculetin (MOA), was isolated from the dried bark of Fraxinus rhynchophylla Hance (Oleaceae). MOA modulates cytokine expression in lipopolysaccharide (LPS)-treated RAW 264.7 macrophages, but the precise mechanisms are still not fully understood. We determined the effects of MOA on the production of inflammatory mediators and pro-inflammatory cytokines in the LPS-induced inflammatory responses of RAW 264.7 macrophages. MOA significantly inhibited the LPS-induced production of nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor-α (TNF-α), interleukin-6, and interleukin-1β. It also effectively attenuated inducible nitric oxide (NO) synthase, cyclooxygenase-2, and TNF-α mRNA expression and significantly decreased the levels of intracellular reactive oxygen species. It inhibited phosphorylation of the extracellular signal-regulated kinase (ERK1/2), thus blocking nuclear translocation of activation protein (AP)-1. In a molecular docking study, MOA was shown to target the binding site of ERK via the formation of three hydrogen bonds with two residues of the kinase, which is sufficient for the inhibition of ERK. These results suggest that the anti-inflammatory effects of MOA in RAW 264.7 macrophages derive from its ability to block both the activation of mitogen-activated protein kinases (MAPKs) and one of their downstream transcription factors, activator protein-1 (AP-1). Our observations support the need for further research into MOA as a promising therapeutic agent in inflammatory diseases.

Anti-Inflammatory Effects of Chloranthalactone B in LPS-Stimulated RAW264.7 Cells

Chloranthalactone B (CTB), a lindenane-type sesquiterpenoid, was obtained from the Chinese medicinal herb Sarcandra glabra, which is frequently used as a remedy for inflammatory diseases. However, the anti-inflammatory mechanisms of CTB have not been fully elucidated. In this study, we investigated the molecular mechanisms underlying these effects in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophages. CTB strongly inhibited the production of nitric oxide and pro-inflammatory mediators such as prostaglandin E2, tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), and IL-6 in RAW264.7 cells stimulated with LPS. A reverse-transcription polymerase chain reaction assay and Western blot further confirmed that CTB inhibited the expression of inducible nitric oxide synthase, cyclooxygenase-2, TNF-α, and IL-1β at the transcriptional level, and decreased the luciferase activities of activator protein (AP)-1 reporter promoters. These data suggest that inhibition occurred at the transcriptional level. In addition, CTB blocked the activation of p38 mitogen-activated protein kinase (MAPK) but not c-Jun N-terminal kinase or extracellular signal-regulated kinase 1/2. Furthermore, CTB suppressed the phosphorylation of MKK3/6 by targeting the binding sites via formation of hydrogen bonds. Our findings clearly show that CTB inhibits the production of inflammatory mediators by inhibiting the AP-1 and p38 MAPK pathways. Therefore, CTB could potentially be used as an anti-inflammatory agent.

Polysaccharide from wheat bran induces cytokine expression via the toll-like receptor 4-mediated p38 MAPK signaling pathway and prevents cyclophosphamide-induced immunosuppression in mice

ABSTRACT Wheat bran-derived polysaccharides have attracted particular attention due to their immunomodulatory effects. However, the molecular mechanisms underlying their functions are poorly understood. The current study was designed to examine the effect of wheat bran polysaccharide (WBP) on RAW 264.7 cells and the underlying signaling pathways, which have not been explored. In addition, we also investigated the immuno-enhancement effects of WBP on cyclophosphamide (CTX)-induced immunosuppression in mice. WBP significantly increased the concentrations of intracellular nitric oxide (NO) and cytokines such as prostaglandin E2 (PGE2) and tumor necrosis factor-α (TNF-α) in RAW 264.7 cells. The result of RT-PCR analysis indicated that WBP also enhanced inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and TNF-α expression. Further analyses demonstrated that WBP rapidly activated phosphorylated p38 mitogen-activated protein kinase (MAPK) and the transcriptional activities of activator protein-1 (AP-1) and nuclear factor (NF)-κB via toll-like receptor 4 (TLR4). Furthermore, in vivo experiments revealed that WBP increased the spleen and thymus indices significantly, and markedly promoted the production of the serum cytokines IL-2 and IFN-γ in CTX-induced immunosuppressed mice. Taken together, these results suggest that WBP can improve immunity by enhancing immune function, and could be explored as a potential immunomodulatory agent in functional food.

A cold-induced phytosulfokine peptide is related to the improvement of loquat fruit chilling tolerance

A novel peptidomics approach was used to discover cold-induced peptides in loquat fruit. Twenty unique peptides derived from 18 proproteins were identified, and they were involved in sugar signalling, protein metabolism and stress response. The quantitative analysis revealed 7 peptides with more than 2-fold upregulation, especially a 4.96-fold increase detected in the phytosulfokine (PSK) peptide. To further evaluate effects of PSK1 on fruit chilling tolerance, weight loss, firmness and internal browning were investigated in PSK1-treated loquat fruit at 0°C. By contrast, these chilling injury symptoms were effectively reduced by PSK1. PSK1 markedly delayed decreases of ATP content and energy charge. The PSK1-treated fruit exhibited significantly lower activities of cell-wall degrading enzymes and transcripts of genes related to lignin synthesis. Our results demonstrated that PSK1 improves chilling tolerance of loquat fruit by maintaining high energy status and cell integrity. Peptidomics analysis provides a promising tool to discover some key peptides.

Comparative Transcriptional Analysis of Loquat Fruit Identifies Major Signal Networks Involved in Fruit Development and Ripening Process

Loquat (Eriobotrya japonica Lindl.) is an important non-climacteric fruit and rich in essential nutrients such as minerals and carotenoids. During fruit development and ripening, thousands of the differentially expressed genes (DEGs) from various metabolic pathways cause a series of physiological and biochemical changes. To better understand the underlying mechanism of fruit development, the Solexa/Illumina RNA-seq high-throughput sequencing was used to evaluate the global changes of gene transcription levels. More than 51,610,234 high quality reads from ten runs of fruit development were sequenced and assembled into 48,838 unigenes. Among 3256 DEGs, 2304 unigenes could be annotated to the Gene Ontology database. These DEGs were distributed into 119 pathways described in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. A large number of DEGs were involved in carbohydrate metabolism, hormone signaling, and cell-wall degradation. The real-time reverse transcription (qRT)-PCR analyses revealed that several genes related to cell expansion, auxin signaling and ethylene response were differentially expressed during fruit development. Other members of transcription factor families were also identified. There were 952 DEGs considered as novel genes with no annotation in any databases. These unigenes will serve as an invaluable genetic resource for loquat molecular breeding and postharvest storage.

Chemical Constituents of Leaves of Mahonia bealei

Mahonia bealei (Fortune) Pynaert, an evergreen shrub in the family Berberidaceae, is widely distributed in Asia, Europe, and America [1]. M. bealei has long been used in folk medicines and was officially included in the Chinese Pharmacopoeia (from Version 2010). Its leaves, stems, and roots can be used for treating dysentery and diarrhea, clearing heat, diminishing inflammation, and expelling toxin [2–6]. Moreover, dried leaves of M. bealei are extensively used to produce bitter teas in the Qinling Mountain areas of China [7]. Previous phytochemical investigations revealed that M. bealei stems and roots were rich in alkaloids and cerebrosides [8]. In an earlier work, we studied the antioxidant and antiproliferative properties of M. bealei leaves, as well as the isolation of a known isoquinoline alkaloid named berberine. However, to date no publications have concentrated on the secondary metabolites of M. bealei leaves. In the current work, investigations on the chemical constituents of M. bealei leaves led to the isolation and purification of six known compounds, including two triterpene, ursolic acid (1) and oleanolic acid (2), two alkaloids, berberine (3) and jatrorrhizine (4), and two flavonoids, quercetin (5) and quercetin-3-O-D-xylopyranoside (6). Their structures were elucidated by MS and 1D and 2D NMR spectroscopy, as well as comparison with the literature data. Compounds 1, 2, and 4–6 were isolated from M. bealei leaves for the first time, and compounds 1, 2, 5, and 6 have never been reported from the genus Mahonia previously. Mahonia bealei leaves were obtained from Guizhou Province, China in May of 2013 and authenticated by Haifeng Wu at Peking Union Medical College and Chinese Academy of Medical Sciences. A voucher specimen (MBL-2013) has been deposited at the School of Life Sciences Laboratory, Huaiyin Normal University. The air-dried M. bealei leaves (3.5 kg) were finely powdered and extracted three times (each for 0.5 h) with EtOH by an ultrasonic-assisted method. The extract was suspended in water and partitioned with n-hexane, CH2Cl2, EtOAc, and n-BuOH successively. A portion of the EtOAc soluble extract (30.41 g) was subjected to vacuum liquid chromatography (VLC) on silica gel (mesh 100–200) and eluted with petroleum ether–EtOAc and CH2Cl2–MeOH to give eight fractions (Fr. A1–A8) on the basis of TLC analysis. Fraction A2 was first subjected to repeated silica gel (mesh 200–300) chromatography and eluted with CH2Cl2–MeOH (50:1 and 10:1) to give five fractions (Fr. B1–B5). Fractions B2–B5 was then purified by Sephadex LH-20 and eluted with MeOH to afford compounds 1 (61.0 mg), 2 (72.0 mg), 3 (21 mg), and 4 (14 mg). Fraction A5 was also purified by repeated Sephadex LH-20 column chromatography using MeOH as mobile solvents to give three fractions (Fr. B7–B9). Fraction B8 was purified by preparative HPLC to obtain compounds 5 (33 mg) and 6 (41.0 mg). All the isolated compounds were identified by spectral analysis (ESI-MS, 1H and 13C NMR) and by comparison with the literature data [9–14]. Ursolic Acid (1). C30H48O30, white amorphous powder. ESI-MS m/z 455 [M – H] –. 1H NMR (400 MHz, DMSO-d6, , ppm, J/Hz): 11.92 (1H, s, COOH-28), 5.13 (1H, s, H-12), 4.27 (1H, d, J = 4.9, 3-OH), 2.67 (1H, s, H-3), 2.15 (1H, d, H-18), 1.04 (3H, s, CH3-24), 0.92 (3H, s, CH3-29), 0.90 (3H, s, CH3-27), 0.87 (3H, s, CH3-23), 0.81 (3H, d, J = 6.3, CH3-30), 0.75

A new phenylethanoid glycoside from Incarvillea compacta

Abstract A new phenylethanoid glycoside, 3′′′-O-methylcampneoside I (1), was isolated from the 90% ethanolic extract of the roots of Incarvillea compacta, together with three known compounds, campneoside I (2), ilicifolioside A (3), and campneoside II (4). Their structures were determined spectroscopically and compared with previously reported spectral data. Compound 1 existed as epimers and displayed better 1,1-diphenyl-2-picrylhydrazyl (DPPH)-free radical scavenging activity using di-tert-butyl-4-methylphenol (BHT) as the positive control. In addition, pretreatment of human HepG2 cells with compound 1 significantly increased the viability on CCl4-induced cell death.

Secondary Metabolites of Mahonia bealei Branches

Mahonia bealei (Berberidaceae), an evergreen shrub, is widely grown in China as well as in many countries in Asia, Europe, and America [1–3]. The leaves, roots, and stems of M. bealei have long been used for the treatment of dysentery and diarrhea, clearing heat, expelling of toxin, and reducing inflammation [4–6]. Earlier literature reported that the leaves, stems, and roots of M. bealei were rich in cerebrosides and alkaloids [7, 8]. In our previous works, we studied the antiproliferative and antioxidant activities of M. bealei leaves, as well as the isolation and structural elucidation of two triterpenes (ursolic acid and oleanolic acid), two alkaloids (berberine and jatrorrhizine), and two flavonoids (quercetin and quercetin 3-O-D-xylopyranoside) from the leaves of M. bealei [1, 9]. However, to the best of our knowledge, no investigation was carried out to study the secondary metabolites of M. bealei branches. In the current work, we describe the isolation and purification of 10 secondary metabolites (1–10) from M. bealei branches. The chemical structures of the compounds, elucidated by interpretation of their spectroscopic data, including NMR, MS, and by comparison of their spectroscopic data with literature values, were characterized as oleanolic acid 3-O-D-glucopyranoside (1), tamarixetin 3-O-D-glucopyranoside (2), quercetin 3-O-D-xylopyranoside (3), isorhamnetin 3-O-L-rhamnopyranoside (4), gallic acid (5), trans-cinnamic acid (6), luteolin (7), isorhamnetin (8), tricin (9), and chryseriol (10). It is worth noting that all the 10 compounds were isolated here for the first time from M. bealei branches, and 1–4 and 7–10 have never been found in Mahonia genus previously. M. bealei branches were collected in August of 2013 from Guizhou Province, China and identified by Dr. Haifeng Wu at Peking Union Medical College and Chinese Academy of Medical Sciences. A voucher specimen (MBB-201310) has been deposited at the School of Life Sciences Laboratory, Huaiyin Normal University. The branches M. bealei (5.26 kg) were air-dried, finely powdered, then extracted four times (each for 0.5 h) with EtOH by ultrasonic-assisted method. After concentration and removal of the solvents, the crude extract was suspended in water and partitioned with n-hexane, CHCl3, EtOAc, and n-BuOH successively. A portion of the n-BuOH fraction (100.40 g) was separated by vacuum liquid chromatography (VLC) on macroporous resin D 101 and eluted with water and 30%, 60%, and 90% EtOH. The 30% EtOH part (35.6 g) was subjected to column chromatography (CC) over Sephadex LH-20 eluted with MeOH–H2O (4:1) to afford five fractions (Frs. A1–A5). Fraction A2 was further separated on CC by Sephadex LH-20 eluted with gradient MeOH–H2O (from 3:2 to 2:3) to afford six subfractions (Frs. B1–B6). Subfractions B2 and B3 were combined and further purified by repeated ODS CC with MeOH–H2O (from 1:2 to 3:1) to give compound 1 (32.6 mg). Fraction A3 was further separated on CC by Sephadex LH-20 eluted with gradient MeOH–H2O (from 1:3 to 3:1) to afford compounds 2 (22.0 mg), 3 (25.5 mg), and 4 (31.2 mg). Fraction A4 was subjected to Sephadex LH-20 CC eluting with MeOH–H2O (from 1:2 to 5:1) and ODS CC with MeOH–H2O (from 1:3 to 1:0) successively to yield compounds 5 (28.2 mg) and 6 (12.8 mg).