Characterization of macrophage activation after treatment with polysaccharides from ginseng according to heat processing.

The worldwide persistence of infectious diseases is a significant public health issue. Consequently, studying immunomodulatory ingredients present in natural products, such as ginseng, is important for developing new treatment options. Here, we extracted three different types of polysaccharides from white (P-WG), red (P-RG), and heat-processed (P-HPG) ginseng and analyzed their chemical properties and immunostimulatory activity against RAW 264.7 murine macrophages. Carbohydrates were the main components of all three polysaccharide types, while uronic acid and protein levels were relatively low. Chemical analysis indicated that the content of carbohydrates (total sugar) increased with processing temperature, while that of uronic acid decreased. Treatment with P-WG, P-RG or P-HPG stimulated nitric oxide (NO) production and increased tumor necrosis factor alpha (TNF-α) and interleukin (IL)-6 levels in RAW 264.7 macrophages, with P-WG showing the highest activity among the three polysaccharides. The expression of inducible NO synthase, which affects NO secretion, was highest in the macrophages treated with P-WG. Analysis of intracellular signaling pathways showed that mitogen-activated protein kinases (ERK, JNK, and p38) and NF-kB p65 were strongly phosphorylated by P-WG in macrophages but were only moderately phosphorylated by P-RG and P-HPG. Collectively, these results suggest that the polysaccharides isolated from ginseng undergo different changes in response to heat processing and display different chemical compositions and immune-enhancing activities.

Synergistic Protection by Isoquercitrin and Quercetin against Glutamate-Induced Oxidative Cell Death in HT22 Cells via Activating Nrf2 and HO-1 Signaling Pathway: Neuroprotective Principles and Mechanisms of Dendropanax morbifera Leaves.

Dendropanax morbifera leaves (DML) have long been used as traditional medicine to treat diverse symptoms in Korea. Ethyl acetate-soluble extracts of DML (DMLE) rescued HT22 mouse hippocampal neuronal cells from glutamate (Glu)-induced oxidative cell death; however, the protective compounds and mechanisms remain unknown. Here, we aimed to identify the neuroprotective ingredients and mechanisms of DMLE in the Glu-HT22 cell model. Five antioxidant compounds were isolated from DMLE and characterized as chlorogenic acid, hyperoside, isoquercitrin, quercetin, and rutin by spectroscopic methods. Isoquercitrin and quercetin significantly inhibited Glu-induced oxidative cell death by restoring intracellular reactive oxygen species (ROS) levels and mitochondrial superoxide generation, Ca2+ dysregulation, mitochondrial dysfunction, and nuclear translocation of apoptosis-inducing factor. These two compounds significantly increased the expression levels of nuclear factor erythroid-2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1) in the presence or absence of Glu treatment. Combinatorial treatment of the five compounds based on the equivalent concentrations in DMLE showed that significant protection was found only in the cells cotreated with isoquercitrin and quercetin, both of whom showed prominent synergism, as assessed by drug-drug interaction analysis. These findings suggest that isoquercitrin and quercetin are the active principles representing the protective effects of DMLE, and these effects were mediated by the Nrf2/HO-1 pathway.

Neuroprotective effects of Dendropanax morbifera leaves on glutamate-induced oxidative cell death in HT22 mouse hippocampal neuronal cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Dendropanax morbifera (DM) has long been used as a traditional herbal medicine for migraines. Glutamate toxicity and oxidative stress have emerged as the possible triggers implicated in migraine pathogenesis. AIM OF THE STUDY: We aimed to examine the neuroprotective effects of DM leaves (DML) on glutamate-induced oxidative cell death in HT22 mouse hippocampal neuronal cells. MATERIALS AND METHODS: Molecular authentication of DML was assessed using DNA barcoding analysis. Four different solvent extracts of DML were prepared and subjected to antioxidant activity and phytochemical assays. Neuroprotective effects of DML extracts were evaluated using relevant biochemical and imaging assays that measure cell viability/death, ROS generation, Ca2+ levels, mitochondrial dysfunction, and AIF nuclear translocation. RESULTS: The sequences of matK, rbcL, atpF-H, and psbK-I in DML were identical with those in voucher specimens, confirming that DML was indeed D. morbifera. The ethyl acetate extract of DML (DMLE) showed the highest flavonoid and phenolic content, and prominent DPPH/superoxide radical scavenging and reducing power activities. In the HT22 cell model, glutamate was shown to be the causative agent for apoptotic cell death via elevation of intracellular ROS and Ca2+ levels, induction of mitochondrial depolarization and membrane permeabilization, and translocation of AIF to the nucleus. Of note, N-acetyl-L-cysteine and necrostatin-1, but not z-VAD-fmk, completely prevented glutamate-induced cell death, implying that oxidative stress and AIF translocation were pivotal in glutamate cytotoxicity. DMLE significantly recovered glutamate-induced apoptotic cell death in a concentration-dependent manner. It completely inhibited intracellular/mitochondrial ROS generation, the elevation of Ca2+ levels, and mitochondrial dysfunction induced by glutamate during early exposure within 8 h. It significantly reversed subsequent AIF nuclear translocation after 12 h of treatment. Antioxidant activities of DMLE may be the protective mechanism that regulates homeostatic balance of ROS and Ca2+ as well as maintains mitochondrial function. CONCLUSIONS: DMLE shows significant neuroprotective effects against glutamate-induced oxidative neuronal cell death. Therefore, DM could be a potential therapeutic candidate for neurological disorders propagated by glutamate toxicity.

Assembly of 6-aza-2-thiothymine on gold nanoparticles for selective and sensitive colorimetric detection of pencycuron in water and food samples.

A facile and novel nanosensor analytical strategy was developed for the colorimetric detection of pencycuron fungicide in rice, potato, cabbage, and water samples based on the pencycuron-induced aggregation of 6-aza-2-thiothymine-functionalized gold nanoparticles (ATT-AuNPs). The ATT-AuNPs exhibited good stability and were characterized with UV-visible spectroscopy, Fourier transform infrared (FT-IR) spectrometry, field-emission scanning electron microscopy (FE-SEM), transmission electron microscopy (TEM), dynamic light scattering (DLS), and zeta potential techniques. The addition of pencycuron facilitated strong non-covalent interactions (electrostatic, van der Waals, and H bonding) between pencycuron and ATT-AuNPs, inducing a significant red shift in the surface plasmon resonance (SPR) peak of ATT-AuNPs along with a color change from red to blue. A linear equation was established between absorption ratio (A720/A528) and pencycuron concentration (2.5-100 µM) with a correlation coefficient (R2) of 0.9915. The detection limit was calculated to be 0.42 µM, which was much lower than that of other analytical methods. The designed ATT-AuNP serves as a promising nanosensor for the rapid, simple, and selective label-free colorimetric detection of pencycuron in rice, potato, cabbage, and water samples, is highly sensitive, and does not require sophisticated instruments, tedious sample preparations, and time-consuming separation and pre-concentration procedures.

Structure analysis and antioxidant activities of an amylopectin-type polysaccharide isolated from dried fruits of Terminalia chebula.

Dried fruits of Terminalia chebula (TF) are used as herbal medicine for diverse symptoms, and their bioactivities are known to involve antioxidant activities. The aim of this study was to elucidate the structure and antioxidant effects of an active TF polysaccharide (TFP). The neutral polysaccharide (named as TFP-a) isolated by ion-exchange chromatography was a homogenous α-Glc-rich polysaccharide (over 70% α-Glc, 534.9 kDa, PDI 1.36) with a porous and flake-like morphology. Linkage and NMR data comprehensively showed that TFP-a was an amylopectin-type polysaccharide with (1→4)-α-Glc(p) backbone branched at C6/C2. Side chains were composed of (1→4)-ß-Gal(p) substituted with α-Ara(f), ß-GalUA(p), ß-GalUA(p)-Me, and α-Rham(p). In antioxidant activity assays, TFP-a exhibited potent and concentration-dependent antioxidant effects, including DPPH and superoxide radical scavenging and reducing power. We concluded that TFP-a is an amylopectin-type polysaccharide that may be used as a potential natural antioxidant.

Extracts from Dendropanax morbifera Leaves Have Modulatory Effects on Neuroinflammation in Microglia.

Dendropanax morbifera (D. morbifera), a species endemic to Korea, is largely distributed throughout the southern part of the country. Its leaves, stems, roots, and seeds have been used as a form of alternative medicine for various diseases and neurological disorders including paralysis, stroke, and migraine. However, the molecular mechanisms that underlie the remedial effects of D. morbifera remain largely unknown. In this paper, extracts from D. morbifera leaves were prepared using ethyl acetate as a solvent (abbreviated as DMLE). The modulatory effects of DMLE on neuroinflammation were studied in a lipopolysaccharide (LPS)-stimulated BV2 murine microglial cell line. Production of pro-inflammatory cytokines, activation of mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-[Formula: see text]B), and different M1/M2 activation states of microglia were examined. DMLE treatment suppressed the production of pro-inflammatory cytokines including tumor necrosis factor-[Formula: see text] (TNF-[Formula: see text]), interleukin-6 (IL-6), and nitric oxide (NO) in LPS-stimulated BV2 cells. DMLE treatment also attenuated the activation of MAPKs and NF-[Formula: see text]B. In a novel discovery, we found that DMLE up-regulated the marker genes representing an alternative, anti-inflammatory M2 polarization, while suppressing the expression of the classical, pro-inflammatory M1 activation state genes. Here, we uncovered the cellular mechanisms underlying the beneficial effects of D. morbifera against neuroinflammation using BV2 microglia cells. These results strongly suggest that DMLE was able to counter the effects of LPS on BV2 cells via control of microglia polarization states. Additionally, study results indicated that DMLE may have therapeutic potential as a neuroinflammation-suppressing treatment for neurodegenerative diseases.

Do alumina matrix composite bearings decrease hip noises and bearing fractures at a minimum of 5 years after THA?

BACKGROUND: Ceramic-on-ceramic bearing couples are theoretically attractive in total hip arthroplasty (THA) because of low wear, but concerns regarding ceramic fracture and squeaking have arisen. Improved material properties of newer alumina matrix composite (AMC) materials, known as Delta ceramics, may reduce these risks. In addition, the use of thinner liners and larger femoral heads may be helpful clinically to lower the rate of dislocation. However, limited short-term clinical results are available and intermediate-term effects are unclear. QUESTIONS/PURPOSES: (1) What is the frequency of bearing-related complications (dissociation, fracture, and noise) with ceramic-on-ceramic AMC bearings in cementless THA? (2) What other complications arose in patients treated with these bearings? (3) What are the Harris hip scores (HHS) and survivorship free from reoperation and revision at a minimum of 5 years after cementless THA performed with AMC bearings? METHODS: Over a 9-month period in 2009, one surgeon performed 125 THAs, of which 100 (80% of the total) were performed with cementless, AMC bearings. During the period in question, the exclusion criteria for this implant were primary THAs with severe acetabular or femoral bone defect and revision THAs. Of these, 94 hips (95%) in 91 patients were available for analysis at a minimum of 5 years (range, 5-6 years), because five patients (six hips) had died. Mean age at the time of arthroplasty was 55 ± 14 years. Prostheses with an identical design and Biolox(®) Delta ceramics were used in all patients. Noise was classified into squeaking, clicking, grinding, and popping. Ceramic fracture, dislocation, and any other complications associated with the use of AMC ceramics were also investigated. Clinical evaluation included the modified HHS preoperatively and at each followup. Survivorship free from reoperation and revision was calculated using the Kaplan-Meier method. RESULTS: Of 91 patients, four developed bearing-related complications, including one with liner dissociation despite initial square seating and three with clicking. No patients had ceramic fractures. A single event of perioperative dislocation occurred in one patient and postoperative periprosthetic fracture occurred in two hips. Mean HHS improved from 56 to 93 points at the final followup (p < 0.001). Survivorship at 5 years free from reoperation and revision was 96.8% and 97.9%, respectively. CONCLUSIONS: Improved material properties combined with the possible use of larger diameter heads make AMC ceramics a promising alternative bearing option with seemingly comparable clinical outcomes reported by others with conventional ceramic bearings. Despite these encouraging results, however, meticulous technical precautions such as square seating and proper impaction in particular should be taken during liner insertion, because we did observe one liner dissociation and several patients with hip noises. LEVEL OF EVIDENCE: Level IV, therapeutic study.

Safety and efficacy evaluation of carnosine, an endogenous neuroprotective agent for ischemic stroke.

BACKGROUND AND PURPOSE: An urgent need exists to develop therapies for stroke that have high efficacy, long therapeutic time windows, and acceptable toxicity. We undertook preclinical investigations of a novel therapeutic approach involving supplementation with carnosine, an endogenous pleiotropic dipeptide. METHODS: Efficacy and safety of carnosine treatment was evaluated in rat models of permanent or transient middle cerebral artery occlusion. Mechanistic studies used primary neuronal/astrocytic cultures and ex vivo brain homogenates. RESULTS: Intravenous treatment with carnosine exhibited robust cerebroprotection in a dose-dependent manner, with long clinically relevant therapeutic time windows of 6 hours and 9 hours in transient and permanent models, respectively. Histological outcomes and functional improvements including motor and sensory deficits were sustained on 14th day poststroke onset. In safety and tolerability assessments, carnosine did not exhibit any evidence of adverse effects or toxicity. Moreover, histological evaluation of organs, complete blood count, coagulation tests, and the serum chemistry did not reveal any abnormalities. In primary neuronal cell cultures and ex vivo brain homogenates, carnosine exhibited robust antiexcitotoxic, antioxidant, and mitochondria protecting activity. CONCLUSIONS: In both permanent and transient ischemic models, carnosine treatment exhibited significant cerebroprotection against histological and functional damage, with wide therapeutic and clinically relevant time windows. Carnosine was well tolerated and exhibited no toxicity. Mechanistic data show that it influences multiple deleterious processes. Taken together, our data suggest that this endogenous pleiotropic dipeptide is a strong candidate for further development as a stroke treatment.

Recovery of hypothalamic tuberoinfundibular dopamine neurons from acute toxicant exposure is dependent upon protein synthesis and associated with an increase in parkin and ubiquitin carboxy-terminal hydrolase-L1 expression.

Hypothalamic tuberoinfundibular dopamine (TIDA) neurons remain unaffected in Parkinson disease (PD) while there is significant degeneration of midbrain nigrostriatal dopamine (NSDA) neurons. A similar pattern of susceptibility is observed in acute and chronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse and rotenone rat models of degeneration. It is not known if the resistance of TIDA neurons is a constitutive or induced cell-autonomous phenotype for this unique subset of DA neurons. In the present study, treatment with a single injection of MPTP (20 mg/kg; s.c.) was employed to examine the response of TIDA versus NSDA neurons to acute injury. An acute single dose of MPTP caused an initial loss of DA from axon terminals of both TIDA and NSDA neurons, with recovery occurring solely in TIDA neurons by 16 h post-treatment. Initial loss of DA from axon terminals was dependent on a functional dopamine transporter (DAT) in NSDA neurons but DAT-independent in TIDA neurons. The active metabolite of MPTP, 1-methyl, 4-phenylpyradinium (MPP+), reached higher concentration and was eliminated slower in TIDA compared to NSDA neurons, which indicates that impaired toxicant bioactivation or distribution is an unlikely explanation for the observed resistance of TIDA neurons to MPTP exposure. Inhibition of protein synthesis prevented TIDA neuron recovery, suggesting that the ability to recover from injury was dependent on an induced, rather than a constitutive cellular mechanism. Further, there were no changes in total tyrosine hydroxylase (TH) expression following MPTP, indicating that up-regulation of the rate-limiting enzyme in DA synthesis does not account for TIDA neuronal recovery. Differential candidate gene expression analysis revealed a time-dependent increase in parkin and ubiquitin carboxyl-terminal hydrolase-L1 (UCH-L1) expression (mRNA and protein) in TIDA neurons during recovery from injury. Parkin expression was also found to increase with incremental doses of MPTP. The increase in parkin expression occurred specifically within TIDA neurons, suggesting that these neurons have an intrinsic ability to up-regulate parkin in response to MPTP-induced injury. These data suggest that TIDA neurons have a compensatory mechanism to deal with toxicant exposure and increased oxidative stress, and this unique TIDA neuron phenotype provides a platform for dissecting the mechanisms involved in the natural resistance of central DA neurons following toxic insult.

Anti-complementary ginsenosides isolated from processed ginseng.

As part of an ongoing search for immunomodulatory components aimed at the anti-complementary effect, ginsenosides isolated from processed ginseng were found to have inhibitory activity on complement activation through classical pathways. Activity-guided fractionation was used to isolate four ginsenosides, namely ginsenoside Rg6, F4, Rk3, and Rh4. Ginsenoside Rk3 and Rh4 had a 3 fold higher inhibition activity than rosmarinic acid which was used as a positive control while ginsenoside Rg6 and F4 showed only mild effects similar to that of the positive control. The results suggest that the activity of the corresponding ginsenosides may be increased by the glycosyl moiety at the C6 position rather than the double bond conformation at C20, and ginsenoside Rk3 and Rh4 could have a role in treating inflammatory diseases.

Pectic polysaccharides from Panax ginseng as the antirotavirus principals in ginseng.

To evaluate the antidiarrheal effect of ginseng, the active principals of ginseng were studied in vitro model of rotavirus infection, the leading cause of severe diarrhea. Two pectic polysaccharides, named as GP50-dHR (56.0 kDa) and GP50-eHR (77.0 kDa), were purified from hot water extract of ginseng by bioassay-linked fractionation. Both polysaccharides rescued cell viability from rotavirus infection dose-dependently (IC50 are 15 and 10 microg/mL, respectively). Both polysaccharides had common structural features of homogalacturonan backbone with hairy regions of rhamnogalacturonan type I. Arabinose-rich side chains with abundant branch points were unique in GP50-eHR and may contribute to a greater antirotavirus effect of GP50-eHR than GP50-dHR. Because homogalacturonan itself did not show an antirotavirus effect, hairy regions might be functional sites. Of note, the antirotavirus effect of both polysaccharides resulted from inhibiting rotavirus attachment to cells. Together with a wide range of noncytotoxicity, these findings suggest that ginseng polysaccharides are viable therapeutic options for rotavirus diarrhea.

Study on the hydroxyl radical scavenging activity changes of ginseng and ginsenoside-Rb2 by heat processing.

The free radical scavenging activities of Panax ginseng C.A. MEYER are known to increase by heat processing. Phenolic acids and Maillard reaction products (MRPs) have been suggested as active free radical scavenging components from our previous research, but heat processing-induced chemical and activity changes of ginsenosides considering the Maillard reaction have not yet been fully elucidated. In this study, we investigated the hydroxyl radical (.OH) scavenging activity changes of ginsengs and ginsenoside-Rb2 (Rb2)) by heat processing using an electron spin resonance spectrometer. Especially, Rb2 was heat processed with the same amount of glycine, a frequently used amino acid in the Maillard reaction model system. As a result, the .OH scavenging activities and brown compound levels of ginseng and glycine-Rb2 mixture were increased by heat processing. However, the increase in .OH scavenging activities were not in accordance with the extents of browning. On the other hand, less-polar ginsenosides such as Rg3, Rg5, and Rk1 were generated from the glycine-Rb2 mixture by heat processing. The sugar moieties at carbon-20 of Rb2 were separated by the steaming process, less-polar ginsenosides were produced, and then the separated sugar moieties were thought to form MRPs with glycine. From the .OH scavenging activity tests of Rb2, glycine, less-polar ginsenosides, and maltol, the increase in .OH scavenging activity was thought to be more closely related to the generation of .OH scavenging ginsenosides such as 20(S)-Rg3 and Rg5 by heat processing than MRPs.

Reduction of Cisplatin-induced nephrotoxicity by ginsenosides isolated from processed ginseng in cultured renal tubular cells.

Ginsenosides, the unique constituents and secondary metabolites of the Panax species, have been known to be the pharmacologically active ingredients of ginseng. Recently, our research group has developed a new processed ginseng, called Sun ginseng (SG), which has an increased amount of the red ginseng unique ginsenosides (RGUG). In our previous studies, this new processed ginseng reduced cisplatin-induced nephrotoxicity more than white ginseng in both in vitro and in vivo systems. In this study, we isolated and characterized active principles through activity-guided fractionation. Ginsenosides Rh4 and Rk3 significantly reduced the cisplatin-induced nephrotoxicity in LLC-PK1 cells in a dose-dependent manner. The mechanisms of function and structure-activity relationships with other ginsenosides remain for further investigation.

Antioxidative activity of furanocoumarins isolated from Angelicae dahuricae.

The methylene chloride extract of the root of Angelicae dahuricae showed high protective activity against 2,2'-azobis (2-aminodinopropane) dihydrochloride (AAPH)-induced cellular damage. From this extract, 11 furanocoumarins were isolated, namely oxypeucedanin hydrate, 9-hydroxy-4-methoxypsoralen, byakangelicin, pabulenol, alloisoimperatorin, neobyakangelicol, byakangelicol, oxypeucedanin, imperatorin, phellotorin and isoimperatorin, respectively. Among these 11 furanocoumarins, 9-hydroxy-4-methoxypsoralen and alloisoimperatorin displayed potent antioxidant effects against the DPPH radical and against renal epithelial cell injury by using AAPH to generate peroxyl radicals in vitro.

Tyrosinase-inhibitory furanocoumarin from Angelica dahurica.

An extract of the roots of Angelica dahurica BENTH. et HOOK. (Umbelliferae) showed potential tyrosinase inhibition against mushroom tyrosinase. Employing a bioassay-linked HPLC method, followed by semipreparative HPLC, the active principle was isolated and characterized as 9-hydroxy-4-methoxypsoralen.