P2X1 Receptor-Mediated Ca2+ Influx Triggered by DA-9801 Potentiates Nerve Growth Factor-Induced Neurite Outgrowth.

Nerve growth factor (NGF)-induced neuronal regeneration has emerged as a strategy to treat neuronal degeneration-associated disorders. However, direct NGF administration is limited by the occurrence of adverse effects at high doses of NGF. Therefore, development of a therapeutic strategy to promote the NGF trophic effect is required. In view of the lack of understanding of the mechanism for potentiating the NGF effect, this study investigated molecular targets of DA-9801, a well-standardized Dioscorea rhizome extract, which has a promoting effect on NGF. An increase in intracellular calcium ion level was induced by DA-9801, and chelation of extracellular calcium ions with ethylene-bis(oxyethylenenitrilo)tetraacetic acid (EGTA) suppressed the potentiating effect of DA-9801 on NGF-induced neurite outgrowth. In addition, EGTA treatment reduced the DA-9801-induced phosphorylation of extracellular signal-regulated kinase1/2 (ERK1/2), the major mediators of neurite outgrowth. To find which calcium ion-permeable channel contributes to the calcium ion influx induced by DA-9801, we treated PC12 cells with various inhibitors of calcium ion-permeable channels. NF449, a P2X1 receptor selective antagonist, significantly abolished the potentiating effect of DA-9801 on NGF-induced neurite outgrowth and abrogated the DA-9801-induced ERK1/2 phosphorylation. In addition, transfection with siRNA of P2X1 receptor significantly reduced the DA-9801-enhanced neurite outgrowth. In conclusion, calcium ion influx through P2X1 receptor mediated the promoting effect of DA-9801 on NGF-induced neurite outgrowth via ERK1/2 phosphorylation.

Mechanical properties and in vivo healing evaluation of a novel Centella asiatica-loaded hydrocolloid wound dressing.

To develop a novel sodium alginate based Centella asiatica (CA)-loaded hydrocolloid wound dressing (HCD) providing excellent mechanical properties and improved wound healing, numerous CA-loaded HCDs were prepared with various ingredients using the hot melting method. The effect of sodium alginate, styrene-isoprene-styrene copolymer (SIS) and petroleum hydrocarbon resin (PHR) on the mechanical properties of CA-loaded HCDs was investigated. The effect of disintegrants on swelling and drug release was assessed. Moreover, the in vivo wound healing potentials of the selected CA-loaded HCD in various wound models such as abrasion, excision and infection were evaluated in comparison with the commercial product. Polyisobutylene and SIS hardly affected the mechanical properties, but PHR improved the tensile strength and elongation at break. Disintegrants such as croscarmellose sodium, sodium starch glycolate and crospovidone improved the swelling ratio of the CA-loaded HCD. Furthermore, the CA-loaded HCD without croscarmellose sodium poorly released the drug, but that with 2% croscarmellose sodium showed about 27% drug release in 24h. In particular, the CA-loaded HCD composed of CA/polyisobutylene/SIS/PHR/liquid paraffin/sodium alginate/croscarmellose sodium at the weight ratio of 1/8/25/25/12/27/2 furnished excellent mechanical properties and drug release. As compared with the commercial product, it offered improved healing effects in excision, infection and abrasion type wounds in rats. Thus, this novel CA-loaded HCD could be a potential candidate for the treatment of various wounds.

Identification of antitumor lignans from the seeds of morning glory (Pharbitis nil).

In the search for antitumor compounds from Korean natural resources, activity-guided fractionation and purification processes were used on seeds of morning glory (Pharbitis nil). Air-dried P. nil seeds were extracted with ethanol and separated into n-hexane, chloroform, ethyl acetate, and n-butanol. Four new lignans, pharbilignans A-D (1-4) were isolated from the most active ethyl acetate fraction of the ethanol extract. Their structures were characterized on the basis of spectroscopic methods, including one- and two-dimensional nuclear magnetic resonance (NMR) techniques, high resolution mass spectrometry (HRMS), and circular dichroism (CD) spectroscopy. The cytotoxic activities of the isolates (1-4) were evaluated by determining their inhibitory effects on four human tumor cell lines (A549, SK-OV-3, SK-MEL-2, and HCT15) using a sulforhodamine B (SRB) bioassay. Pharbilignan C (3) showed potent cytotoxicity against A549, SK-OV-3, SK-MEL-2, and HCT-15 cell lines with IC50 values of 1.42, 0.16, 0.20, and 0.14 µM, respectively. On the basis of the expanded understanding that inflammation is a crucial cause in tumor progress, we also evaluated anti-inflammatory activity of the isolates (1-4). Pharbilignan C (3) strongly inhibited nitric oxide (NO) production in the lipopolysaccharide (LPS)-activated BV-2 microglia cell line with an IC50 value of 12.8 µM.

Phenolic derivatives from the rhizomes of Dioscorea nipponica and their anti-neuroinflammatory and neuroprotective activities.

ETHNOPHARMACOLOGICAL RELEVANCE: Dioscorea nipponica (Dioscoreaceae) have been used as traditional medicines for diabetes, inflammatory and neurodegenerative diseases in Korea. The aim of the study was to isolate the bioactive components from the rhizomes of Dioscorea nipponica and to evaluate their anti-neuroinfalmmatory and neuroprotective activities. MATERIAL AND METHODS: The phytochemical investigation of 50% EtOH extract of Dioscorea nipponica using successive column chromatography over silica gel, Sephadex LH-20, and preparative high performance liquid chromatography (HPLC) resulted in the isolation and identification of 17 phenolic derivatives, including four new phenolic compounds (1-4). The structural elucidation of these compounds was based on spectroscopic methods, including 1D and 2D nuclear magnetic resonance (NMR) spectroscopy techniques, mass spectrometry, and optical rotation. All isolated compounds were evaluated for their effects on nerve growth factor (NGF) secretion in a C6 rat glioma cell line and nitric oxide (NO) production in lipopolysaccharide (LPS)-activated BV2 cells. The neurite outgrowth of compound 16 was further evaluated by using mouse neuroblastoma N2a cell lines. RESULTS: Three new stilbene derivatives, diosniponol C (1), D (2) and diosniposide A (3) and one new phenanthrene glycoside, diosniposide B (4), together with 13 known compounds were isolated from the rhizomes of Dioscorea nipponica. Of the tested compounds (1-17), phenanthrene, 3,7-dihydroxy-2,4,6-trimethoxy-phenanthrene (16) was the most potent NGF inducer, with 162.35±16.18% stimulation, and strongly reduced NO levels with an IC50 value of 19.56 µM in BV2 microglial cells. Also, it significantly increased neurite outgrowth in N2a cells. CONCLUSIONS: This study supports the ethnopharmacological use of Dioscorea nipponica rhizomes as traditional medicine.

Bioactive sesquiterpenes from the essential oil of Thuja orientalis.

A phytochemical investigation on the essential oil of Thuja orientalis resulted in the isolation and identification of three new sesquiterpenes, 3α-methoxy-4α-epoxythujopsane (1), Δ³,¹5-4ß-epoxythujopsene (2), and Δ³,4-thujopsen-2,15-diol (3), together with eight known sesquiterpenoids (4-11). The structures of these new compounds were elucidated based on spectroscopic data analyses including extensive 2D-NMR data and HR-ESIMS. The full assignments of ¹H and ¹³C NMR chemical shifts for thujopsadiene (4) were obtained by 2D-NMR for the first time. All compounds (1-11) showed antiproliferative activities against the SK-OV-3 and SK-MEL-2 cell lines with IC50 values of 5.85-28.64 µM. In addition, compounds 1, 3, 4, 7, 8, and 9 significantly inhibited nitric oxide production in lipopolysaccharide-activated BV-2 cells with IC50 values of 3.93-17.85 µM without cell toxicity.

Anti-neuroinflammatory diarylheptanoids from the rhizomes of Dioscorea nipponica.

In a continuing search for bioactive constituents from Dioscoreaceae medicinal plants, two new cyclic diarylheptanoids, diosniponol A (1) and B (2), together with 10 known compounds (3-12) were isolated from the rhizomes of Dioscorea nipponica. The structures of these new compounds were determined by spectroscopic analyses, including extensive two-dimensional nuclear magnetic resonance, high-resolution mass spectrometry, and optical rotation. All isolated compounds 1-12 were evaluated for their effects on nitric oxide (NO) production in murine microglia cell line BV-2. Compounds 8 and 11 showed potent inhibitory activities on NO production (IC50 13.36 and 14.36 µM, respectively) without cell toxicity in lipopolysaccharide-activated BV-2 cells.

Effects of Noscarna™ on hypertrophic scarring in the rabbit ear model: histopathological aspects.

In this study, we evaluated the effects of silicone-based gel on the healing of hypertrophic scars in the rabbit ear model. After 4-week application of silicone-based gel containing allantoin, dexpanthenol and heparin (Noscarna™) to scars in a rabbit ear model of hypertrophic scarring, significant improvements in hypertrophic scar healing and a great loss of skin pigment were observed compared to the non-treated control, base or silicone control-treated scars. Furthermore, histological analysis of Noscarna™-treated scars revealed a significant reduction in scar elevation index (SEI), anterior skin and epithelial thicknesses, inflammatory cells, vessels, collagen disorganization and fibroblasts compared to all control hypertrophic scars. Furthermore, Noscarna™ showed more favorable effects on hypertrophic scars than a commercial product, Contractubex®. Therefore, these results clearly demonstrated that the newly developed silicone-based gel, Noscarna™, could be a promising formulation as an effective therapeutic agent for hypertrophic scars.

Novel botanical drug for the treatment of diabetic neuropathy.

Diabetic neuropathy has multiple aspects of pathophysiology including metabolic derangements and alterations of microvessel with hyperglycemia. Currently, there is no effective long-term treatment based on the pathologic mechanisms. Nerve regeneration with nerve growth factor (NGF) has been on clinical trials as a treatment option. This article reviews an action of a herbal medicine, DA 9801, whose mechanism of action is related to NGF in treating diabetic neuropathy.

Two new phenolic amides from the seeds of Pharbitis nil.

Two new phenolic amides, pharnilatins A (1) and B (2), were isolated from the seeds of Pharbitis nil. These new compounds possess a p-coumaroyl unit with a structurally unique side chain, (2S,3S)-2,3-dihydroxyputrescine. The chemical structures and absolute stereochemistries of the new compounds were determined on the basis of spectroscopic analyses including 1D- and 2D-NMR experiments and chemical reactions. Compounds 1 and 2 exhibited cytotoxicity against A549, SK-OV-3, SK-MEL-2, and HCT-15 human tumor cells. However, none of the compounds inhibited nitric oxide (NO) production in lipopolysaccharide (LPS)-activated microglia cells.

New cytotoxic tetrahydroprotoberberine-aporphine dimeric and aporphine alkaloids from Corydalis turtschaninovii.

Two new tetrahydroprotoberberine-aporphine dimeric alkaloids, corydaturtschines A (1) and B (2), and a new aporphine derivative, ethyl glausuccinate (3), together with 13 known protoberberine (4-16) and nine known aporphine alkaloids (17-25), were isolated from the tubers of Corydalis turtschaninovii. The structures of these new compounds were determined through spectral analyses, including extensive 2D-NMR data. The absolute configurations of the compounds were clarified by CD spectroscopic studies. The isolated compounds were tested for their cytotoxicity against four human cancer cell lines in vitro using a sulforhodamine B bioassay.

Effects of orally administered Actinidia arguta (Hardy Kiwi) fruit extract on 2-chloro-1,3,5-trinitrobenzene-induced atopic dermatitis-like skin lesions in NC/Nga mice.

Atopic dermatitis (AD) is characterized by highly pruritic, chronic, relapsing inflammatory skin lesions. Furthermore, therapeutic choices are limited, especially in long-standing cases, despite its increasing prevalence. This study was performed to examine the clinical efficacy and the therapeutic mechanism underlying the effects of Actinidia arguta (hardy kiwi) fruit extract in an animal model of AD. To examine the effects of A. arguta extract on AD, 2-chloro-1,3,5-trinitrobenzene-treated NC/Nga mice were orally administered A. arguta extract (100 mg/kg/day), tacrolimus (1 mg/kg/day), or dexamethasone (3 mg/kg/day) for 8 weeks. Skin severity scores, epidermal thickening, mast cell infiltration and degranulation, total serum immunoglobulin (Ig) isotypes (IgE, IgG(1)), and cytokine (interleukin [IL]-4 and interferon [IFN]-gamma) and Toll-like receptor (TLR) (TLR-2, TLR-4, and TLR-9) expressions were examined in each of the study groups. Orally administered A. arguta extract significantly reduced clinical dermatitis severity, epidermal thickness, mast cell dermal infiltration and degranulation, and total levels of serum IgE and IgG(1). Furthermore, this suppression of total serum IgE and IgG(1) levels was accompanied by a decrease in IL-4 and an increase in IFN-gamma expression in skin and splenocytes. Interestingly, TLR-9 expression was increased by oral A. arguta extract. This study confirms that A. arguta extract has potential as a dietary therapeutic agent for the treatment of AD. Furthermore, our findings suggest that its clinical efficacy and mode of action against AD are associated with the modulation of biphasic T-helper (Th) 1/Th2 cytokines, with the inhibition of Th2-mediated IgE overproduction, and possibly with the up-regulation of TLR-9.

Monitoring of peptide acylation inside degrading PLGA microspheres by capillary electrophoresis and MALDI-TOF mass spectrometry.

The purpose of this research was to assess the acylation reactions of peptides, salmon calcitonin (sCT), human parathyroid hormone 1-34 (hPTH1-34) and leuprolide, in poly(lactic-co-glycolic acid) (PLGA) microspheres. Capillary electrophoresis (CE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) were used for determining and monitoring peptide acylation and quantitating acylation products in the degrading PLGA microspheres. In the degrading PLGA microspheres of sCT and hPTH1-34, the acylation products were observed and determined to be adducts with glycolic acid units from degradable PLGA polymer by MALDI-TOF MS. In the microsphere of leuprolide, however, the acylation product was not observed even after 28 days of incubation at the release medium, which represents the different stabilities among peptides according to the primary structure. As the leuprolide contains tyrosine and serine having hydroxyl group of nucleophilic amino acids, the acylation reaction of peptide is shown to be mainly due to the primary amino groups of N-terminus or lysine residue. The complementary use of CE and MALDI-TOF MS will be useful for searching the counter measures as well as determining the peptide acylation in the manufactured formulations on the market.

Inhibitory effects of the standardized extract (DA-9601) of Artemisia asiatica Nakai on phorbol ester-induced ornithine decarboxylase activity, papilloma formation, cyclooxygenase-2 expression, inducible nitric oxide synthase expression and nuclear transcription factor kappa B activation in mouse skin.

Artemisia asiatica Nakai has been used in traditional Asian medicine for the treatment of inflammatory and other disorders. Previous studies have revealed that the formulated ethanol extract (DA-9601) of A. asiatica has pronounced antioxidative and antiinflammatory activities and exhibits cytoprotective effects against experimentally induced gastrointestinal, hepatic and pancreatic damage. In the present study, we assessed the inhibitory effect of DA-9601 on tumor promotion, which is closely linked to inflammatory tissue damage. As an initial approach to evaluating the possible antitumor-promoting potential of DA-9601, its effects on TPA-induced ear edema were examined in female ICR mice. Pretreatment of the inner surface of the mouse ear with DA-9601 30 min prior to topical application of TPA inhibited ear edema at 5 hr. TPA-stimulated expression of epidermal COX-2 and iNOS was also mitigated by topical application of the same extract. Moreover, DA-9601 abrogated the TPA-mediated activation of NF-kappa B/Rel and AP-1 in mouse epidermis. Suppression of epidermal NF-kappa B by DA-9601 appeared to be mediated in part through inhibition of I kappa B alpha degradation, thereby blocking the nuclear translocation of p65, the functional subunit of NF-kappa B. DA-9601 also significantly suppressed TPA-induced ODC activity and papilloma formation in mouse skin. Taken together, these findings suggest that DA-9601 derived from A. asiatica possesses potential chemopreventive activities.