Chromatographic Method for Monitoring of Pesticide Residues and Risk Assessment for Herbal Decoctions Used in Traditional Korean Medicine Clinics.

The presence of pesticide residues in herbs and the herbal products derived from them raises serious health concerns. This study was conducted to investigate the residual pesticide concentrations and assess potential human health risks from herbal medicines used in traditional Korean medicine clinics. A total of 40 samples of herbal decoctions were collected from 10 external herbal dispensaries. The pesticide residues were analyzed by the multiresidue method for 320 different pesticides using liquid chromatography tandem mass spectrometry (LC-MS/MS) and gas chromatography tandem mass spectrometry (GC-MS/MS). As a result of the monitoring, carbendazim was detected at 0.01 and 0.03 µg/g in eight samples and no pesticide was detected in the other herbal decoctions. Carbendazim was set for each individual item as less than 0.05 µg/g in Paeoniae radix, less than 0.05 µg/g in Cassiae semen, less than 2.0 µg/g in Lycii fructus, and less than 10 µg/g in Schisandrae fructus (dried). Therefore, the results of this study suggested that the detected pesticide residues in herbal decoctions could not be considered as posing a serious health risk.

Exploring the Fibrin(ogen)olytic, Anticoagulant, and Antithrombotic Activities of Natural Cysteine Protease (Ficin) with the κ-Carrageenan-Induced Rat Tail Thrombosis Model.

Although fibrinolytic enzymes and thrombolytic agents help in cardiovascular disease treatment, those currently available have several side effects. This warrants the search for safer alternatives. Several natural cysteine protease preparations are used in traditional medicine to improve platelet aggregation and thrombosis-related diseases. Hence, this study aimed to investigate the effect of ficin, a natural cysteine protease, on fibrin(ogen) and blood coagulation. The optimal pH (pH 7) and temperature (37 °C) for proteolytic activity were determined using the azocasein method. Fibrinogen action and fibrinolytic activity were measured both electrophoretically and by the fibrin plate assay. The effect of ficin on blood coagulation was studied by conventional coagulation tests: prothrombin time (PT), activated partial thromboplastin time (aPTT), blood clot lysis assay, and the κ-carrageenan thrombosis model. The Aα, Bß, and γ bands of fibrinogen are readily cleaved by ficin, and we also observed a significant increase in PT and aPTT. Further, the mean length of the infarcted regions in the tails of Sprague-Dawley rats was shorter in rats administered 10 U/mL of ficin than in control rats. These findings suggest that natural cysteine protease, ficin contains novel fibrin and fibrinogenolytic enzymes and can be used for preventing and/or treating thrombosis-associated cardiovascular disorders.

Therapeutic Effect of Rumex japonicus Houtt. on DNCB-Induced Atopic Dermatitis-Like Skin Lesions in Balb/c Mice and Human Keratinocyte HaCaT Cells.

Rumex japonicus Houtt. (RJ) is traditionally used in folk medicines to treat patients suffering from skin disease in Korea and other parts of East Asia. However, the beneficial effect of RJ extract on atopic dermatitis (AD) has not been thoroughly examined. Therefore, this study aimed to investigate the anti-inflammatory effects of RJ on AD in vitro and in vivo. Treatment with RJ inhibited the phosphorylation of mitogen-activated protein kinase (MAPK) as well as the activation of nuclear factor-kappa B (NF-κB) in tumor necrosis factor-α (TNF-α) stimulated in HaCaT cells. The five-week-old Balb/c mice were used as an AD-like mouse model by treating them with 1-chloro-2, 4-dinitrobenzene (DNCB). Topical administration of RJ to DNCB-treated mice significantly reduced clinical dermatitis severity, epidermal thickness, and decreased mast cell and eosinophil infiltration into skin and ear tissue. These results suggest that RJ inhibits the development of AD-like skin lesions by regulating the skin inflammation responses in HaCaT cells and Balb/c mice. Thus, RJ may be a potential therapeutic agent for AD.

Yin-and-yang bifurcation of opioidergic circuits for descending analgesia at the midbrain of the mouse.

In the descending analgesia pathway, opioids are known to disinhibit the projections from the periaqueductal gray (PAG) to the rostral ventromedial medulla (RVM), leading to suppression of pain signals at the spinal cord level. The locus coeruleus (LC) has been proposed to engage in the descending pathway through noradrenergic inputs to the spinal cord. Nevertheless, how the LC is integrated in the descending analgesia circuit has remained unknown. Here, we show that the opioidergic analgesia pathway is bifurcated in structure and function at the PAG. A knockout as well as a PAG-specific knockdown of phospholipase C ß4 (PLCß4), a signaling molecule for G protein-coupled receptors, enhanced swim stress-induced and morphine-induced analgesia in mice. Immunostaining after simultaneous retrograde labeling from the RVM and the LC revealed two mutually exclusive neuronal populations at the PAG, each projecting either to the LC or the RVM, with PLCß4 expression only in the PAG-LC projecting cells that provide a direct synaptic input to LC-spinal cord (SC) projection neurons. The PAG-LC projection neurons in wild-type mice turned quiescent in response to opiates, but remained active in the PLCß4 mutant, suggesting a possibility that an increased adrenergic function induced by the persistent PAG-LC activity underlies the enhanced opioid analgesia in the mutant. Indeed, the enhanced analgesia in the mutant was reversed by blocking α2-noradrenergic receptors. These findings indicate that opioids suppress descending analgesia through the PAG-LC pathway, while enhancing it through the PAG-RVM pathway, i.e., two distinct pathways with opposing effects on opioid analgesia. These results point to a therapeutic target in pain control.

Cyperus Rotundus L. extract suppresses RANKL-induced osteoclastogenesis through NFATc1/c-fos downregulation and prevent bone loss in OVX-induced osteoporosis rat.

ETHNOPHARMACOLOGICAL RELEVANCE: Cyperus Rotundus L. (CyR) has been widely used for the treatment of gynecologic disorder. Recent studies have reported that CyR can prevent the formation of cystic follicles and ovarian malfunction. However, the effects of CyR on osteoclastogenesis and postmenopausal osteoporosis remain unknown. AIM OF THE STUDY: This study was aimed to investigate the preventive effects of CyR on RANKL-induced osteoclast formation and ovariectomy (OVX)-induced bone loss. MATERIALS AND METHODS: In this in vitro study, we investigate the anti-osteoporotic effect of CyR on receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclastogenesis, the formation of tartrate-resistant acid phosphatase (TRAP) multinucleated cells, pit formation, transcription factors such as NFATc1 and c-Fos, and mRNA expression of osteoclast-associated genes were investigated. Forty 12-weeks female Sprague-Dawley rats for in vivo effect of CyR were used and OVX rat model was determined. The rats were randomly assigned into sham group and four OVX groups, i.e. OVX with D.W; OVX with estradiol (E2, 100µg/kg/day), OVX with CyR-L (16mg/kg/day), OVX with CyR-H (160mg/kg/day). The treatment lasted for 8weeks. RESULTS: CyR inhibited osteoclast differentiation and pit formation in the RANKL-induced osteoclastogenesis of RAW 264.7 cells. Reverse transcription polymerase chain reaction analysis also showed that CyR reduced the mRNA expression of osteoclast-associated genes such as carbonic anhydrase II, TRAP, RANK, cathepsin K, matrix metalloproteinase 9, nuclear factor of activated T cells cytoplasmic 1 (NFATc1), and c-Fos. In addition, CyR decreased protein levels of NFATc1 and c-Fos. CyR inhibited trabecular bone loss in the femur caused by OVX. CONCLUSION: The results of this study indicate that CyR inhibits the RANKL-induced osteoclast differentiation in RAW 264.7 cells and trabecular bone loss in OVX rats.

Porcine placenta hydrolysates enhance osteoblast differentiation through their antioxidant activity and effects on ER stress.

BACKGROUND: Osteoporosis is a disease characterized by decreased bone strength, decreased bone mass, and bone deterioration. Oxidative damage is an important contributor to functional changes in the development of osteoporosis. Here we found that porcine placenta hydrolysates (PPHs) protect MC3T3-E1 osteoblastic cells against hydrogen peroxide (H2O2)-induced oxidative damage. METHODS: In vitro cell viability was determined using trypan blue dye exclusion. ER stress and apoptosis were evaluated using immunoblotting and a commercially available caspase kit. ALP, osteocalcin, Runx2, and osterix expression levels were evaluated by RT-PCR using isolated RNA. ROS, NADPH oxidase, and SOD activity levels were also measured. RESULTS: We investigated the mechanisms underlying PPH-mediated inhibition of H2O2-induced ER stress and ROS production. PPHs also regulated osteoblast differentiation via the upregulation of alkaline phosphatase (ALP) expression in MC3T3-E1 osteoblastic cells. Also, treatment with PPHs enhanced the transcription of osteocalcin, Runx2, and osterix. These effects were all associated with the antioxidant actions of PPHs. Moreover, PPHs reversed the decrease in SOD activity, decreased ROS release, and inhibited NADPH oxidase activity in H2O2-treated MC3T3-E1 osteoblastic cells. CONCLUSIONS: PPHs protect cells against H2O2-induced cell damage when ER stress is involved. In addition, PPHs enhance osteoblast differentiation. This enhancement likely explains the regulatory effect of PPHs on bone metabolism disturbances, i.e. PPHs control ER stress and the related ROS production in osteoblasts.

Porcine placenta hydrolysates regulate calcium disturbance in MC3T3-E1 osteoblastic cells.

BACKGROUND: In bone metabolism, Ca(2+) disturbance and oxidative damage are the main biochemical factors related to pathology. Osteoblasts are bone-forming cells that also control bone endocrinology. Endocrine hormones and proteins are matured, folded, and secreted in the endoplasmic reticulum (ER). ER stress has emerged as a new pathological mechanism to explain bone disturbance. Here we studied the role of porcine placenta hydrolysates (PPHs) in the regulation of ER stress. METHODS: Cell viability was determined in vitro using trypan blue dye exclusion. ER stress and apoptosis were evaluated using immunoblotting and a caspase kit. The fluorescent Ca(2+)-binding dye Fura-2/AM was used to measure changes in intracellular Ca(2+) ([Ca(2+)]i). ROS levels, NADPH oxidase activity, and superoxide dismutase (SOD) activity were also measured. RESULTS: PPHs protected MC3T3-E1 osteoblastic cells against thapsigargin (Tg)-induced ER stress. Moreover, PPHs regulated caspase-12 and -3 activities, thereby protecting against cell death, and also regulated Tg-induced Ca(2+) release. The Ca(2+) chelator BAPT/AM also regulated caspase-12 and -3 activities and prevented Ca(2) stress-induced cell death. In the presence of PPHs or BAPTA/AM, Ca(2+)-related ROS were also regulated, as demonstrated by alterations in NADPH oxidase and SOD activity. CONCLUSIONS: PPHs appear to regulate bone metabolism disturbance by controlling Ca(2+) concentrations, and thus ER stress and ROS, in osteoblasts cultured in vitro.

Commercial Motion Sensor Based Low-Cost and Convenient Interactive Treadmill.

Interactive treadmills were developed to improve the simulation of overground walking when compared to conventional treadmills. However, currently available interactive treadmills are expensive and inconvenient, which limits their use. We propose a low-cost and convenient version of the interactive treadmill that does not require expensive equipment and a complicated setup. As a substitute for high-cost sensors, such as motion capture systems, a low-cost motion sensor was used to recognize the subject's intention for speed changing. Moreover, the sensor enables the subject to make a convenient and safe stop using gesture recognition. For further cost reduction, the novel interactive treadmill was based on an inexpensive treadmill platform and a novel high-level speed control scheme was applied to maximize performance for simulating overground walking. Pilot tests with ten healthy subjects were conducted and results demonstrated that the proposed treadmill achieves similar performance to a typical, costly, interactive treadmill that contains a motion capture system and an instrumented treadmill, while providing a convenient and safe method for stopping.

Low-cost implementation of a self-paced treadmill by using a commercial depth sensor.

A self-paced treadmill that can simulate overground walking has the potential to improve the effectiveness of treadmill training for gait rehabilitation. We have implemented a self-paced treadmill without the need for expensive equipment such as a motion capture system and an instrumented treadmill. For this, an inexpensive depth sensor, ASUS XtionTM, substitutes for the motion capture system, and a low-cost commercial treadmill is considered as the platform of the self-paced treadmill. The proposed self-paced treadmill is also convenient because the depth sensor does not require markers placed on user's body. Through pilot tests with two healthy subjects, it is quantitatively and qualitatively verified that the proposed self-paced treadmill achieves similar performance as one which utilizes a commercial motion capture system (VICON) as well as an instrumented treadmill.

Effects of Xanthium stramarium and Psoralea corylifolia Extracts Combined with UVA1 Irradiation on the Cell Proliferation and TGF-ß1 Expression of Keloid Fibroblasts.

BACKGROUND: Xanthium stramarium (XAS) and Psoralea corylifolia (PSC), phototoxic oriental medicinal plants, has been used in traditional medicines in Asian countries. OBJECTIVE: The effects of highly purified XAS or PSC extract combined with ultraviolet A1 (UVA1) irradiation on cell proliferation and transforming growth factor-beta1 (TGF-ß1) expression of the keloid fibroblast were being investigated to define potential therapeutic uses for keloid treatments. METHODS: The keloid fibroblasts were treated with XAS or PSC alone or in the combination with UVA1 irradiation. The cell viability, apoptosis, and expression of TGF-ß1 and collagen I were investigated. RESULTS: XAS and PSC in combination with UVA1 irradiation suppressed cell proliferation and induced apoptosis of keloid fibroblasts. Furthermore, the XAS and PSC in combination with UVA1 irradiation inhibited TGF-ß1 expression and collagen synthesis in keloid fibroblasts. CONCLUSION: These findings may open up the possibility of clinically used XAS or PSC in combination with UVA1 irradiation for keloid treatments.

Schizonepeta tenuifolia inhibits the development of atopic dermatitis in mice.

Historically, Schizonepeta tenuifolia (ST) has been used for the treatment of skin disorders, such as allergic dermatitis, eczema, and inflammatory diseases. In this study, we examined whether ST inhibited 2,4-dinitrochlorobenzene (DNCB)-induced atopic dermatitis (AD) in BALB/c mice. In histopathological analyses of the epidermis and dermis, skin thickness was significantly increased in DNCB-induced mice as compared with normal group. Treatment with ST inhibited this inflammatory change and markedly suppressed the secretion of immunoglobulin E, tumor necrosis factor α, and interleukin 6 levels in the serum of DNCB-induced mice. In addition, ST treatment significantly restored the upregulation of proinflammatory factors, such as nuclear factor (NF)-κB and mitogen-activated protein kinase expression. Taken together, due to its ability to suppress inflammatory factors and upregulate proinflammatory factors, ST may be useful as a therapeutic treatment for AD. ST extract application decreased both epidermis and dermis thickness in DNCB-induced mice. In serum, ST reduced immunoglobulin E, tumor necrosis factor, and interleukin 6 level. In addition, ST suppressed NF-κB activation as well as the mitogen-activated protein kinase activities.

Improvement of modified charcoal-cefoperazone-deoxycholate agar by supplementation with a high concentration of polymyxin B for detection of Campylobacter jejuni and C. coli in chicken carcass rinses.

Modified charcoal-cefoperazone-deoxycholate agar (mCCDA) was improved by supplementation with a high concentration of polymyxin B. The ability of the supplemented medium to isolate Campylobacter jejuni and C. coli from chicken carcass rinses was compared to that of Campy-Cefex agar and mCCDA. Modification of mCCDA with increased polymyxin B yielded a significantly (P < 0.05) higher isolation rate and greater selectivity than those achieved using Campy-Cefex agar and mCCDA.

Fritillaria ussuriensis extract inhibits the production of inflammatory cytokine and MAPKs in mast cells.

Fritillaria ussuriensis (FU, derived from the bulbs of various species of the genus Fritillaria, including Fritillaria thunbergii Miq.) is used in herbal medicine to treat conditions such as eczema, skin burns, and frostbite. In this study, we investigated the mechanism of the anti-allergy effect of FU. FU extract (80 mg/kg), orally administered to Sprague-Dawley (SD) rats, significantly inhibited the passive cutaneous anaphylaxis (PCA) reaction. It inhibited the compound 48/80-induced release of histamine from rat peritoneal mast cells in a concentration-dependent manner. Significant inhibitory effects of the FU extract on IL-6, IL-8, and TNF-α (1, 10, and 100 µg/mL) were observed in HMC-1 cells. Treatment with FU attenuated PMA plus A23187-induced phosphorylation of all three MAPKs, especially at concentrations of 10 and 100 µg/mL. Further, it (80 mg/kg) led to significant inhibition of mast-cell accumulation in ear tissue at the chronic phase. These results indicate that it inhibits allergic reactions.

Tooth discoloration of immature permanent incisor associated with triple antibiotic therapy: a case report.

INTRODUCTION: A triple antibiotic mixture of ciprofloxacin, metronidazole, and minocycline was used as an intracanal medicament in an attempt to disinfect the root canal system for revascularization of a tooth with a necrotic pulp. However, discoloration developed after applying the triple antibiotic mixture. METHODS: Six weeks after a triple antibiotic paste had been applied to the root canal of tooth #8 of a 7-year-old girl, the tooth showed a dark discoloration. An in vitro experiment with human extracted teeth was performed to determine which of the 3 antibiotics caused the tooth discoloration. Another experiment was then carried out to examine whether a currently used dentin bonding agent would prevent or reduce such discoloration. The degree of discoloration was assessed by using a colorimeter. RESULTS: Among the components of the triple antibiotic paste, only minocycline caused the tooth discoloration. Moreover, the dentin bonding agent reduced the intensity of the discoloration but did not prevent it. CONCLUSIONS: The possible esthetic problems with the tooth color should be considered when using minocycline as a canal medication.

Therapeutic effect of rokitamycin in vitro and on experimental meningoencephalitis due to Naegleria fowleri.

Inhalation of freshwater containing the free-living amoeba Naegleria fowleri leads to a potentially fatal infection known as primary amoebic meningoencephalitis (PAME). Amphotericin B is the only agent with clinical efficacy in the treatment of PAME in humans, however this drug is often associated with adverse effects on the kidney and other organs. In an attempt to select other useful therapeutic agents for treating PAME, the amoebicidal activities of antibacterial agents including clarithromycin, erythromycin, hygromycin B, neomycin, rokitamycin, roxithromycin and zeocin were examined. Results showed that the growth of amoeba was effectively inhibited by treatment with hygromycin B, rokitamycin and roxithromycin. Notably, when N. fowleri trophozoites were treated with rokitamycin, the minimal inhibitory concentration was 6.25 microg/mL on Day 2. In the treatment of experimental meningoencephalitis due to N. fowleri, survival rates of mice treated with roxithromycin and rokitamycin were 25% and 80%, respectively, over 1 month. The mean time to death for roxithromycin and rokitamycin treatment was 16.2 days and 16.8 days, respectively, compared with 11.2 days for control mice. Finally, rokitamycin showed both in vitro and in vivo therapeutic efficacy against N. fowleri and may be a candidate drug for the treatment of PAME.

Susceptibility of cholangiocarcinoma cells to parthenolide-induced apoptosis.

Cholangiocarcinomas are intrahepatic bile duct carcinomas that are known to have a poor prognosis. Sesquiterpene lactone parthenolide, which is the principal active component in medicinal plants, has been used to treat tumors. Parthenolide effectively induced apoptosis in all four cholangiocarcinoma cell lines in a dose-dependent manner. However, the sarcomatous SCK cells were more sensitive to parthenolide than the other adenomatous cholangiocarcinoma cells. Therefore, this study investigated whether or not the expression of p53, the Fas/Fas ligand (FasL), Bcl-2/Bcl-X(L) determines the enhanced drug susceptibility of SCK cells. The results showed that Bcl-2 family molecules, such as Bid, Bak, and Bax, are involved in the parthenolide-induced apoptosis and that the defective expression of Bcl-X(L) might contribute to the higher parthenolide sensitivity in the SCK cells than in the other adenomatous cholangiocarcinoma cells. SCK cells, which stably express Bcl-X(L), were resistant to parthenolide, whereas Bcl-X(L)-positive Choi-CK cells transfected with the antisense Bcl-X(L) showed a higher parthenolide sensitivity than the vector control cells. Molecular dissection revealed that Bcl-X(L) inhibited the translocation of Bax to the mitochondria, decreased the generation of intracellular reactive oxygen species, reduced the mitochondrial transmembrane potential (deltapsi(m)), decreased the release of cytochrome c, decreased the cleavage of poly(ADP-ribose) polymerase, and eventually inhibited apoptotic cell death. These results suggest that parthenolide effectively induces oxidative stress-mediated apoptosis, and that the susceptibility to parthenolide in cholangiocarcinoma cells might be modulated by Bcl-X(L) expression in association with Bax translocation to the mitochondria.