Anti-Apoptotic and Anti-Inflammatory Effects of an Ethanolic Extract of Lycium chinense Root against Particulate Matter 10-Induced Cell Death and Inflammation in RBL-2H3 Basophil Cells and BALB/c Mice.

Particulate matters (PMs) from polluted air cause diverse pulmonary and cardiovascular diseases, including lung inflammation. While the fruits (Goji) of Lycium trees are commonly consumed as traditional medicine and functional food ingredients, the majority of their roots are discarded as by-products. To enhance the industrial applicability of Lycium roots, we prepared an ethanol extract (named GR30) of L. chinense Miller roots and evaluated its potential protective effects against particulate matter 10 (PM10)-induced inflammation and immune cell death. The GR30 treatment (0-500 µg/mL) significantly attenuated the PM10-induced cell cycle arrest, DNA fragmentation and mitochondria-dependent apoptosis in RBL-2H3 basophil cells. GR30 also significantly antagonized the PM10-induced expression of proinflammatory cytokines (IL-4, IL-13, and TNF-α) and COX2 expression through downregulation of MAPKs (ERK and JNK) signalling pathway. Oral administration of GR30 (200-400 mg/kg) to PM10 (20 mg/mL)-challenged mice significantly reduced the serum levels of IgE and the expression of TNF-α and Bax in lung tissues, which were elevated by PM10 exposure. These results revealed that the ethanolic extract (GR30) of L. chinense Miller roots exhibited anti-inflammatory and cyto-protective activity against PM10-induced inflammation and basophil cell death, and thus, it would be useful in functional food industries to ameliorate PM-mediated damage to respiratory and immune systems.

Protective Effect of a Mixture of Astragalus membranaceus and Lithospermum erythrorhizon Extract against Hepatic Steatosis in High Fat Diet-Induced Nonalcoholic Fatty Liver Disease Mice.

The present study aimed to evaluate the potential synergistic and protective effects of ALM16, a mixture of Astragalus membranaceus (AM) and Lithospermum erythrorhizon (LE) extract in a ratio of 7 : 3, against hepatic steatosis in high fat diet (HFD)-induced nonalcoholic fatty liver disease (NAFLD) mice. Forty-eight mice were randomly divided into eight groups and orally administered daily for 6 weeks with a normal diet (ND) or high fat diet alone (HFD), HFD with AM (HFD + 100 mg/kg AM extract), HFD with LE (HFD + 100 mg/kg LE extract), HFD with ALM16 (HFD + 50, 100, and 200 mg/kg ALM16), or HFD with MT (HFD + 100 mg/kg Milk thistle extract) as a positive control. ALM16 significantly decreased the body and liver weight, serum and hepatic lipid profiles, including triglyceride (TG), total cholesterol (TC), high-density lipoprotein-cholesterol (HDL), and low-density lipoprotein-cholesterol (LDL), and serum glucose levels, compared to the HFD group. Moreover, ALM16 significantly ameliorated the HFD-induced increased hepatic injury markers, including aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), and gamma-glutamyltransferase (GGT)-1. Furthermore, as compared to the mice fed HFD alone, ALM16 increased the levels of phosphorylated AMP-activated protein kinase (p-AMPK) and acetyl-CoA carboxylase (p-ACC), thereby upregulating the expression of carnitine palmitoyltransferase (CPT)-1 and downregulating the expression of sterol regulatory element-binding protein (SREBP)-1c and fatty acid synthase (FAS). These results demonstrated that ALM16 markedly inhibited HFD-induced hepatic steatosis in NAFLD mice by modulating AMPK and ACC signaling pathways, and may be more effective than the single extracts of AM or LE.

Platelet-Like Gold Nanostars for Cancer Therapy: The Ability to Treat Cancer and Evade Immune Reactions.

The cell membrane-coating strategy has opened new opportunities for the development of biomimetic and multifunctional drug delivery platforms. Recently, a variety of gold nanoparticles, which can combine with blood cell membranes, have been shown to provide an effective approach for cancer therapy. Meanwhile, this class of hybrid nanostructures can deceive the immunological system to exhibit synergistic therapeutic effects. Here, we synthesized red blood cell (RBC) and platelet membrane-coated gold nanostars containing curcumin (R/P-cGNS) and evaluated whether R/P-cGNS had improved anticancer efficacy. We also validated a controlled release profile under near-infrared irradiation for the ability to target melanoma cells and to have an immunomodulatory effect on macrophages. RBC membrane coating provided self-antigens; therefore, it could evade clearance by macrophages, while platelet membrane coating provided targetability to cancer cells. Additionally, the nutraceutical curcumin provided anticancer and anti-inflammatory effects. In conclusion, the results presented in this study demonstrated that R/P-cGNS can deliver drugs to the target region and enhance anticancer effects while avoiding macrophage phagocytosis. We believe that R/P-cGNS can be a new design of the cell-based hybrid system for effective cancer therapy.

Immunostimulating activity of Lycium chinense Miller root extract through enhancing cytokine and chemokine production and phagocytic capacity of macrophages.

Whereas the fruits and a small portion of root bark of Lycium trees are commonly marketed in Korea as traditional medicine or functional foods, majority of their whole roots have been largely discarded. To develop the whole root of these plants as more value-added materials, this study aimed to evaluate the potential immunostimulating activity of a water extract (GTR-101) from L. chinense Miller roots using macrophages. The GTR-101 (0-500 µg/ml) significantly, dose-dependently increased the secretion of pro-inflammatory cytokines (TNF-α and IL-6), chemokines (RANTES and MIP-1α), nitric oxide, and the expression of inducible nitric oxide synthase, and activated the Akt, NF-κB, and MAPKs (ERK and p38) signaling proteins. GTR-101 also significantly enhanced the phagocytic activity of RAW 264.7 cells and bone marrow-derived macrophages. These results suggest that GTR-101 stimulates the early innate immunity via inducing the pro-inflammatory cytokine and chemokine secretion and enhancing the phagocytic activity of macrophages. PRACTICAL APPLICATIONS: The GTR-101 prepared from L. chinense Miller roots may be useful for enhancing body's defense systems especially in the elderly and cancer patients with an impaired or reduced immune response and may thus be effectively used as a natural immunostimulating ingredient in health foods or complementary medicine.

Quercetin 3-O-xyloside ameliorates acute pancreatitis in vitro via the reduction of ER stress and enhancement of apoptosis.

BACKGROUND AND PURPOSE: Glycosylation of phenolic compounds has been reported to increase water-solubility, reduce toxicity, and sometimes give improved or novel pharmacological activities. Present study was aimed to evaluate and compare the beneficial effects of quercetin aglycone (Quer) and its glycosylated derivative, quercetin 3-O-xyloside (Quer-Xyl), against acute pancreatitis (AP). METHODS: The cellular acute pancreatitis model was established by treating the rat pancreatic acinar cells (AR42J) with lipopolysaccharide (10 µg/ml) and cerulein (10-7 M). The cytotoxicity of Quer or Quer-Xyl on AR42J cells was assessed by MTT assay. Calcium and ROS levels were fluorometrically determined. The ER stress levels (PERK, GRP78), expression levels of amylase and lipase, and apoptotic markers (caspase-3 and -9) were measured by RT-PCR, western blotting, or fluorometric assay. RESULTS: While Quer increased the mRNA expressions of AP marker enzymes, amylase and lipase, Quer-Xyl dose-dependently reversed their expressions. Quer-Xyl suppressed intracellular ROS production and both mRNA and protein levels of GRP78 and PERK, which were significantly elevated in cerulein and LPS-treated AR42J cells. Further, RT-PCR and fluorescence assay revealed that Quer-Xyl dose-dependently augmented the mRNA expressions and activities of caspase-3 and -9. CONCLUSION: These results showed that Quer-Xyl, but not Quer, has a significant anti-pancreatitis activity through attenuating intracellular ROS production and ER stress response and enhancing apoptotic cell death, suggesting that it might be useful as a potent functional ingredient in health-beneficial foods or as a therapeutic agent to prevent or treat AP.

Shape-Controlled Synthesis of Au Nanostructures Using EDTA Tetrasodium Salt and Their Photothermal Therapy Applications.

Tuning the optical properties of Au nanostructures is of paramount importance for scientific interest and has a wide variety of applications. Since the surface plasmon resonance properties of Au nanostructures can be readily adjusted by changing their shape, many approaches for preparing Au nanostructures with various shapes have been reported to date. However, complicated steps or the addition of several reagents would be required to achieve shape control of Au nanostructures. The present work describes a facile and effective shape-controlled synthesis of Au nanostructures and their photothermal therapy applications. The preparation procedure involved the reaction of HAuCl4 and ethylenediaminetetraacetic acid (EDTA) tetrasodium salt, which acted as a reducing agent and ligand, at room temperature without the need for any toxic reagent or additives. The morphology control from spheres to branched forms and nanowire networks was easily achieved by varying the EDTA concentration. Detailed investigations revealed that the four carboxylic groups of the EDTA tetrasodium salt are essential for effective growth and stabilization. The produced Au nanowire networks exhibited a broad absorption band in the near-infrared (NIR) region, thereby showing efficient cancer therapeutic performance by inducing the selective photothermal destruction of cancerous glioblastoma cells (U87MG) under NIR irradiation.

Enzyme Hydrolysates of Ginseng Marc Polysaccharides Promote the Phagocytic Activity of Macrophages Via Activation of TLR2 and Mer Tyrosine Kinase.

Although ginseng marc is a by-product obtained during manufacturing of various commercial ginseng products and has been routinely discarded as a waste, it still contains considerable amounts of potential bioactive compounds, including saponins and polysaccharides. Previously, we reported that ginseng oligosaccharides derived from ginseng marc polysaccharides by enzymatic hydrolysis exert immunostimulatory activities in macrophages and these activated macrophages are in turn able to inhibit the growth of skin melanoma cells by inducing apoptosis. In the present study, a more detailed investigation of the immunostimulatory activity and underlying action mechanisms of an enzymatic hydrolysate (GEH) containing these oligosaccharides derived from ginseng marc polysaccharides was performed. The levels of proinflammatory cytokines and anti-inflammatory cytokines were measured in GEH-stimulated RAW264.7 macrophages using RT-PCR analysis and ELISA. The expression levels of Toll-like receptor 2 (TLR2) and TLR4, Dectin-1, and MerTK were measured by RT-PCR analysis or western blot analysis, and the phagocytic activities of GEH-challenged bone marrow-derived macrophages toward apoptotic Jurkat cells were assayed using fluorescence microscopy. GEH induced the production of both proinflammatory cytokines TNF-α and IL-6, and anti-inflammatory cytokine IL-10 in RAW 264.7 cells. The expression of the TLR2 and MerTK mRNAs was increased upon GEH treatment. Phagocytosis of apoptotic Jurkat cells was enhanced in GEH-treated macrophages. Based on the results, this enzymatic hydrolysate (GEH) containing oligosaccharides exerts immunostimulatory effects by maintaining the balance between M1 and M2 cytokines, facilitating macrophage activation and contributing to the efficient phagocytosis of apoptotic cells. Therefore, the GEH could be developed as value-added, health-beneficial food materials with immunostimulatory effects.

Structural analysis and anti-obesity effect of a pectic polysaccharide isolated from Korean mulberry fruit Oddi (Morus alba L.).

A water-soluble polysaccharide JS-MP-1 was isolated from Korean mulberry fruits Oddi (Morus alba L.). Sugar linkage analysis and NMR data confirmed that it is a rhamnogalacturonan type I (RG I) polymer carrying arabinan and arabinogalactan (AG II) side chains. JS-MP-1 reduced dose-dependently the viability of 3T3-L1 pre-adipocyte cells, significantly stimulated the cleavage of caspases 9 and 3 and poly (ADP-ribose) polymerase (PARP) and decreased the ratio of Bcl-2 to Bax expression level that led to mitochondrial dysfunction and apoptosis in pre-adipocyte cells. The apoptotic death was mediated by stimulation of MAPKs (ERK and p38) signalling pathway. These results suggest that JS-MP-1 is able to reduce the number of fat cells and the mass of adipose tissue via inhibition of pre-adipocyte proliferation and thus JS-MP-1 itself or a crude aqueous Oddi extract containing this polysaccharide can be used as functional ingredient of health-beneficial food supplements for the treatment or prevention of obesity disorders.

Neuroprotective effects of phytosterols and flavonoids from Cirsium setidens and Aster scaber in human brain neuroblastoma SK-N-SH cells.

AIMS: We investigated the neuroprotective effects and action mechanism of three major compounds [daucosterol (Dau), pectolinarin (Pec), and astragalin (Ast)] isolated from edible plants against H2O2-induced cell death of human brain neuroblastoma SK-N-SH cells. MAIN METHODS: Cytotoxicity was determined by MTT and lactate dehydrogenase (LDH) assays. Apoptotic cell death was monitored by annexin V-FITC/PI double staining and by TUNEL assay. The formation of reactive oxygen species (ROS), expression of antioxidant enzymes and phosphorylation of mitogen-activated protein kinase (MAPK) were determined by 2,7-dichlorofluorescein diacetate (DCF-DA) assay, RT-PCR, and western blotting, respectively. KEY FINDINGS: The ethyl acetate fractions from Cirsium setidens (CSEA) and Aster scaber (ASEA) showed neuroprotective effects in SK-N-SH cells. The phytochemicals were isolated from CSEA and ASEA and identified by spectral analyses, as ß-sitosterol, Dau, Pec, Ast, or isoquercitrin. Pretreatment with Dau, Pec, or Ast showed protective effects against H2O2-induced cell death and inhibited ROS generation by oxidative stress. HO-1 mRNA and protein levels were increased by the presence of H2O2 and were further elevated by pretreatment with Dau and Ast. Dau pretreatment resulted in further increases of H2O2-induced enhancement in levels of CAT and SOD2. Pretreatment with Dau, Pec, and Ast inhibited phosphorylation of MAPK, such as extracellular protein regulated protein kinase, p38, and c-Jun N-terminal kinase by H2O2. SIGNIFICANCE: Dau exerts its neuroprotective effects by down regulation of MAPK pathways and upregulation of the HO-1, CAT and SOD2 antioxidant genes and is associated with reduced oxidative stress in SK-N-SH cells.

Neuroprotective effects of the Phellinus linteus ethyl acetate extract against H2O2-induced apoptotic cell death of SK-N-MC cells.

Numerous studies have suggested that neuronal cells are protected against oxidative stress-induced cell damage by antioxidants, such as polyphenolic compounds. Phellinus linteus (PL) has traditionally been used to treat various symptoms in East Asian countries. In the present study, we prepared an ethyl acetate extract from the fruiting bodies of PL (PLEA) using hot water extraction, ethanol precipitation, and ethyl acetate extraction. The PLEA contained polyphenols as its major chemical component, and thus, we predicted that it may exhibit antioxidant and neuroprotective effects against oxidative stress. The results showed that the pretreatment of human brain neuroblastoma SK-N-MC cells with the PLEA (0.1-5 µg/mL) significantly and dose-dependently reduced the cytotoxicity of H2O2 and the intracellular ROS levels and enhanced the expression of HO-1 (heme oxygenase-1) and antioxidant enzymes, such as CAT (catalase), GPx-1 (glutathione peroxidase-1), and SOD-1 and -2 (superoxide dismutase-1 and -2). The PLEA also directly scavenged free radicals. PLEA pretreatment also significantly attenuated DNA fragmentation and suppressed the mRNA expression and activation of mitogen-activated protein kinases extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 kinase, which are induced by oxidative stress and lead to cell death. PLEA pretreatment inhibited the activation of the apoptosis-related proteins caspase-3 and poly (ADP-ribose) polymerase. These results demonstrate that the PLEA has neuroprotective effects against oxidative stress (H2O2)-induced neuronal cell death via its antioxidant and anti-apoptotic properties. PLEA should be investigated in an in vivo model on its potential to prevent or ameliorate neurodegenerative disease.

Immunostimulating activity of polyhydric alcohol isolated from Taxus cuspidata.

A polyhydric alcohol (PAL) was isolated from Taxus cuspidata and its immunostimulatory activities were assessed. The primary monosaccharide composition of the PAL was determined to be glucose, where HPAEC analysis showed no significant amount of any other sugars. However, glycerol and xylitol were identified as the main sugar alcohols. Fourier-transform infrared (FT-IR) analysis indicated that the purified PAL is a complex glycitol, which structurally contains significant amount of hydroxyl groups. MALDI-TOF mass spectroscopy also demonstrated that PAL is a complex glycitol built in hexose polymerization. Enzyme linked immunosorbent assay showed that the PAL stimulates the release of the proinflammatory cytokines TNF-α and IL-6 in a dose-dependent manner. Furthermore, treatment of RAW 264.7 cells with PAL for 24h remarkably increased the phosphorylation levels of ERK, p38 and JNK in a dose-dependent manner, whereas the total protein levels of ERK (t-ERK), p38 (t-p38) and JNK (t-JNK) remained unchanged. These results clearly demonstrate that PAL stimulates the immune response in RAW 264.7 cells through the activation of MAPKs (ERK, p38 and JNK) signaling pathway. To the best of our knowledge, this is the first study to demonstrate the primary structure and immune-stimulating activities of PAL from the fruit of T. cuspidata.

Ginseng marc-derived low-molecular weight oligosaccharide inhibits the growth of skin melanoma cells via activation of RAW264.7 cells.

Panax ginseng C.A. Meyer has been traditionally consumed to prevent or treat various medical disorders due to its diverse health benefits. Polysaccharides isolated from Panax ginseng have been known to possess various pharmacological activities, including immune modulating, anti-diabetic, and anti-obesity properties. Despite the increasing number of reports on the bioactivities of ginseng polysaccharides, little is known regarding the medicinal potential of ginseng-derived oligosaccharides. In this study, we prepared a lower-molecular weight oligosaccharide (GOS, MW. 2.2kDa) from ginseng polysaccharides (MW. 11-605kDa) by enzymatic degradation and evaluated for its immunostimulating activities in RAW 264.7 murine macrophage cells. GOS was shown to be a glucan type oligosaccharide mainly containing glucose residues (97.48 in molar %). Treatment with GOS (100-500µg/ml) dose-dependently enhanced the production of TNF-α, IL-6, and NO in RAW 264.7 cells. Western blot analysis indicated that GOS dose-dependently induced the phosphorylation of c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), p38, and nuclear factor κB (NFκB), which are upstream signalling molecules for cytokine production. While GOS was not cytotoxic to the RAW 264.7 macrophage cells at the concentration tested (up to 1000µg/ml), when B16F10 melanoma cells were co-cultured with the GOS-activated macrophages, the cell viability of melanoma cells was dose-dependently decreased through the induction of apoptotic cell death. Taken together, these results suggested that ginseng marc-derived GOS has anti-cancer activity in vitro against melanoma cells by potentiating macrophage function.

Biodegradation-tunable mesoporous silica nanorods for controlled drug delivery.

Mesoporous silica in the forms of micro- or nanoparticles showed great potentials in the field of controlled drug delivery. However, for precision control of drug release from mesoporous silica-based delivery systems, it is critical to control the rate of biodegradation. Thus, in this study, we demonstrate a simple and robust method to fabricate "biodegradation-tunable" mesoporous silica nanorods based on capillary wetting of anodic aluminum oxide (AAO) template with an aqueous alkoxide precursor solution. The porosity and nanostructure of silica nanorods were conveniently controlled by adjusting the water/alkoxide molar ratio of precursor solutions, heat-treatment temperature, and Na addition. The porosity and biodegradation kinetics of the fabricated mesoporous nanorods were analyzed using N2 adsorption/desorption isotherm, TGA, DTA, and XRD. Finally, the performance of the mesoporous silica nanorods as drug delivery carrier was demonstrated with initial burst and subsequent "zero-order" release of anti-cancer drug, doxorubicin.

Protective Effect of Coriolus versicolor Cultivated in Citrus Extract Against Nitric Oxide-Induced Apoptosis in Human Neuroblastoma SK-N-MC Cells.

Nitric oxide (NO) is a reactive free radical and a messenger molecule in many physiological functions. However, excessive NO is believed to be a mediator of neurotoxicity. The medicinal plant Coriolus versicolor is known to possess anti-tumor and immune-potentiating activities. In this study, we investigated whether Coriolus versicolor possesses a protective effect against NO donor sodium nitroprusside (SNP)-induced apoptosis in the human neuroblastoma cell line SK-N-MC. We utilized 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometry, 4,6-diamidino-2-phenylindole (DAPI) staining, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) assay, DNA fragmentation assay, reverse transcription-polymerase chain reaction (RT-PCR), Western blot analysis, and caspase-3 enzyme activity assay in SK-N-MC cells. MTT assay showed that SNP treatment significantly reduces the viability of cells, and the viabilities of cells pre-treated with the aqueous extract of Coriolus versicolor cultivated in citrus extract (CVE(citrus)) was increased. However, aqueous extract of Coriolus versicolor cultivated in synthetic medium (CVE(synthetic)) showed no protective effect and aqueous citrus extract (CE) had a little protective effect. The cell treated with SNP exhibited several apoptotic features, while those pre-treated for 1 h with CVE(citrus) prior to SNP expose showed reduced apoptotic features. The cells pre-treated for 1 h with CVE(citrus) prior to SNP expose inhibited p53 and Bax expressions and caspase-3 enzyme activity up-regulated by SNP. We showed that CVE(citrus) exerts a protective effect against SNP-induced apoptosis in SK-N-MC cells. Our study suggests that CVE(citrus) has therapeutic value in the treatment of a variety of NO-induced brain diseases.

Estrogenic activity produced by aqueous extracts of silkworm (Bombyx mori) pupae in ovariectomized rats.

This study examined the estrogenic activity produced by aqueous extracts of silkworm (Bombyx mori) pupae in ovariectomized (OVX) rats. The components of silkworm pupae were extracted in distilled water at room temperature for 6 hours. The ovaries of six-week old female rats were then bilaterally removed. One week after OVX, the animals were treated with 200, 400 or 600 mg/kg/day of silkworm pupae extracts. The body weights of the OVX rats increased remarkably compared to the control rats, however their relative uterus weights to body weights decreased significantly. Treatment with the aqueous extracts of silkworm pupae dramatically improved the decreased uterus weights of OVX rats, with the highest increase observed in treatment with 200 mg/kg/day of the aqueous extracts. Additionally, treatment with aqueous extracts (200 mg/kg/day) of silkworm pupae significantly elevated the serum 17beta-estradiol contents of OVX rats when compared to the control animals. To examine the toxic effects of silkworm pupae on the hepatic functions of OVX rats, the levels of serum glutamate oxaloacetate transaminase (GOT) and glutamate pyruvate transaminase (GPT) were measured. The serum GOT and GPT levels did not change in response to the administration of aqueous extracts (200, 400 and 600 mg/kg/day) for 4-weeks. Taken together, these results suggest that the aqueous extracts of silkworm pupae may have estrogenic activity, which suggests that silkworm pupae may be useful in the prevention and/or treatment of menopausal disorders caused by deficiencies in female sexual hormones, including estrogen.

Moxibustion at ST36 alleviates pain in complete Freund's adjuvant-induced arthritic rats.

This study was to investigate the antinociceptive effects of moxibustion in a complete Freund's adjuvant (CFA)-induced arthritic rat model, and the effects of moxibustion on immunohistochemical changes at the spinal cord level. Moxibustion was applied to the ipsilateral (right) Zusanli (ST36) acupoint to the lesion side for 9 days to CFA-induced arthritic rats. The stepping force was measured as a behavioral test, c-Fos immunohistochemistry, NO production and nNOS Western blots were examined to evaluate antinociceptive effects. Moxibustion at ST36 significantly improved the stepping force in the affected hind limb in CFA-induced arthritis. Moreover, moxibustion at ST36 suppressed the production of NO and the protein expression of c-Fos and nNOS induced by arthritis. These results suggest that moxibustion at ST36 has a potent antinociceptive effect in an arthritic rat model, and modulates neuronal excitability and endogenous NO production by suppressing c-Fos and nNOS protein expression.