Comparative Morphology of Island and Inland Agastache rugosa and Their Gastroprotective Effects in EtOH/HCl-Induced Gastric Mucosal Gastritis.

Agastache rugosa Kuntze (Lamiaceae; Labiatae), a medicinal and functional herb used to treat gastrointestinal diseases, grows well both on islands and inland areas in South Korea. Thus, we aimed to reveal the morphological and micromorphological differences between A. rugosa grown on island and inland areas and their pharmacological effects on gastritis in an animal model by combining morphological and mass spectrophotometric analyses. Morphological analysis showed that island A. rugosa had slightly smaller plants and leaves than inland plants; however, the density of all types of trichomes on the leaves, petioles, and stems of island A. rugosa was significantly higher than that of inland plants. The essential oil component analysis revealed that pulegone levels were substantially higher in island A. rugosa than in inland A. rugosa. Despite the differences between island and inland A. rugosa, treatment with both island and inland A. rugosa reduced gastric damages by more than 40% compared to the gastritis induction group. In addition, expression of inflammatory protein was reduced by about 30% by treatment of island and inland A. rugosa. The present study demonstrates quantitative differences in morphology and volatile components between island and inland plants; significant differences were not observed between the gastritis-inhibitory effects of island and inland A. rugosa, and the efficacy of island A. rugosa was found to be similar to that of A. rugosa grown in inland areas.

Anti-Periodontitis Effects of Dendropanax morbiferus H.Lév Leaf Extract on Ligature-Induced Periodontitis in Rats.

Periodontitis is caused by pathogens in the oral cavity. It is a chronic infectious disease that causes symptoms including gingival bleeding and tooth loss resulting from the destruction of periodontal tissues coupled with inflammation. Dendropanax morbiferus H.Lév (DM) is a natural product that exhibits various biological activities with few side effects. In this study, the potential of DM leaf hot-water extracts (DMWE) as a treatment for periodontitis was determined and its anti-oxidant and anti-inflammatory effects were evaluated. Compounds in DMWE were identified by high-performance liquid chromatography (HPLC) and nitric oxide (NO) and prostaglandin E2 (PGE2) production was measured in RAW 264.7 cells. We measured the gingival index and gingival sulcus depth, and micro-CT was performed in vivo using a ligature-induced periodontitis rat model, which is similar to human periodontitis. The DMWE-treated group exhibited a decrease in cytokine concentration and relieved the gingival index and gingival sulcus depth compared with the periodontitis-induced control group. In addition, micro-CT and histological analysis revealed that DMWE exhibited anti-inflammatory effects and improved alveolar bone loss in periodontitis-induced rats. These findings suggest that DMWE has excellent anti-oxidant and anti-inflammatory effects that protect and prevent periodontal tissue damage and tooth loss caused by the inflammatory response.

Floral micromorphological, palynological, and carpological characteristics of the rare endemic Korean species Peucedanum miroense (Apiaceae).

Peucedanum miroense K. Kim, H.-J. Suh & J.-H. Song, recently reported as a new species endemic to South Korea, is distributed on mountains Duta-san and Swinum-san in Gangwon Province, South Korea. Given its conspicuously limited habitat range and small populations, this species qualifies as an endangered Red List species. In this study, we undertook a detailed investigation of the floral micromorphological, palynological, and carpological characters of P. miroense based on a range of microscopic analyses. Examination of the floral micromorphology of P. miroense revealed that the surface patterns of bractlets have potential utility in taxonomy studies of the genus Peucedanum. In addition, we established that this plant is characterized a papillose conical-tabular rugose pattern of petal epidermal cells and bears sunken nectary slits on the stylopodium. Also identified were simple spherical orbicules bearing psilate surface ornamentation, as well as small tricolporate prolate to perprolate pollen grains characterized by verrucate and gemmate ornamentation. Moreover, we verified pollen wall stratification and presence of pollenkitt. The mericarps had colliculate-striate surface patterns with trichomes and eight or nine vittae, and prismatic crystals were detected in different layers of the pericarp. We believe that the findings of this study will contribute to an evaluation of the taxonomic significance of this species within the genus Peucedanum, and also provide valuable information for the conservation of this rare endemic species. RESEARCH HIGHLIGHTS: We provided the first detailed descriptions of the floral micromorphological, palynological, and carpological characters of the rare endemic Korean species Peucedanum miroense K. Kim, H.-J. Suh & J.-H. Song, based on different microscopic analyses. Our findings provide valuable information regarding the epidermal cell patterns of bractlets, petals, stylopodium, gynoecium, and anthers. We believe our study makes a significant contribution to the literature, as our findings will contribute to enhancing the taxonomical classification of species within the genus Peucedanum, and will provide a basis for the conservation of this rare endemic species. This results will be of value to researchers studying the future biology and taxonomy of species within the family Apiaceae, particularly those using different microscopic techniques, including FE-SEM, digital slide scanning, and TEM.

GC-MS and LC-TOF-MS profiles, toxicity, and macrophage-dependent in vitro anti-osteoporosis activity of Prunus africana (Hook f.) Kalkman Bark.

Osteoporosis affects millions of people worldwide. As such, this study assessed the macrophage-dependent in vitro anti-osteoporosis, phytochemical profile and hepatotoxicity effects in zebrafish larvae of the stem bark extracts of P. africana. Mouse bone marrow macrophages (BMM) cells were plated in 96-well plates and treated with P. africana methanolic bark extracts at concentrations of 0, 6.25, 12.5, 25, and 50 µg/ml for 24 h. The osteoclast tartrate-resistant acid phosphatase (TRAP) activity and cell viability were measured. Lipopolysaccharides (LPS) induced Nitrite (NO) and interleukin-6 (IL-6) production inhibitory effects of P. africana bark extracts (Methanolic, 150 µg/ml) and ß-sitosterol (100 µM) were conducted using RAW 264.7 cells. Additionally, inhibition of IL-1ß secretion and TRAP activity were determined for chlorogenic acid, catechin, naringenin and ß-sitosterol. For toxicity study, zebrafish larvae were exposed to different concentrations of 25, 50, 100, and 200 µg/ml P. africana methanolic, ethanolic and water bark extracts. Dimethyl sulfoxide (0.05%) was used as a negative control and tamoxifen (5 µM) and dexamethasone (40 µM or 80 µM) were positive controls. The methanolic P. africana extracts significantly inhibited (p < 0.001) TRAP activity at all concentrations and at 12.5 and 25 µg/ml, the extract exhibited significant (p < 0.05) BMM cell viability. NO production was significantly inhibited (all p < 0.0001) by the sample. IL-6 secretion was significantly inhibited by P. africana methanolic extract (p < 0.0001) and ß-sitosterol (p < 0.0001) and further, chlorogenic acid and naringenin remarkably inhibited IL-1ß production. The P. africana methanolic extract significantly inhibited RANKL-induced TRAP activity. The phytochemical study of P. africana stem bark revealed a number of chemical compounds with anti-osteoporosis activity. There was no observed hepatocyte apoptosis in the liver of zebrafish larvae. In conclusion, the stem bark of P. africana is non-toxic to the liver and its inhibition of TRAP activity makes it an important source for future anti-osteoporosis drug development.

Systematic palynology in Korean Piperales with special focus on its exine surface ornamentation and orbicule morphology.

The pollen and orbicule morphology of the Korean Piperales (Aristolochia, Asarum, Houttuynia, Piper, and Saururus) were investigated via scanning electron microscopy. Piperales pollen is a monad, its size ranging from very small to large (P = 7.78-51.4 µm, E = 6.68-43.1 µm), and having a mainly circular to sub-circular shape. The aperture type is constant in the genus [inaperturate (Aristolochia), tri to pentaporate (Asarum), and monosulcate (Houttuynia, Piper, and Saururus)]. There are four distinct types of exine ornamentation: Fossulate with perforate, microreticulate with gemmae, microperforate with granula, and microechinate. The orbicules (minute sporopollenin granules) were observed in all studied taxa and thus, may be a possible symplesiomorphic characteristic of Piperales. Further, the observed orbicule surface ornamentation was similar to pollen exine patterns, for example muri, gemmae, or granula. This resemblance between orbicule and pollen exine ornamentation may imply a similar biosynthesis pattern of sporopollenin of pollen exine and orbicules. The phenogram resulting from a cluster analysis using palynological characters was generally consistent with the known molecular phylogeny of Piperales. This initial study will help understand the palynological diversity and provide detailed information of pollen and orbicule characteristics in Piperales.

Plastid Phylogenomic Data Offers Novel Insights Into the Taxonomic Status of the Trichosanthes kirilowii Complex (Cucurbitaceae) in South Korea.

Trichosanthes is a genus in Cucurbitaceae comprising 90-100 species. Trichosanthes species are valuable as herbaceous medicinal ingredients. The fruits, seeds, and roots of species such as T. kirilowii and T. rosthornii are used in Korean traditional herbal medicines. T. rosthornii is only found in China, whereas in South Korea two varieties, T. kirilowii var. kirilowii and T. kirilowii var. japonica, are distributed. T. kirilowii var. kirilowii and T. kirilowii var. japonica have different fruit and leaf shapes but are recognized as belonging to the same species. Furthermore, although its members have herbal medicine applications, genomic information of the genus is still limited. The broad goals of this study were (i) to evaluate the taxonomy of Trichosanthes using plastid phylogenomic data and (ii) provide molecular markers specific for T. kirilowii var. kirilowii and T. kirilowii var. japonica, as these have differences in their pharmacological effectiveness and thus should not be confused and adulterated. Comparison of five Trichosanthes plastid genomes revealed locally divergent regions, mainly within intergenic spacer regions (trnT-UGU-trnL-UAA: marker name Tri, rrn4.5-rrn5: TRr, trnE-UUC-trnT-GGU: TRtt). Using these three markers as DNA-barcodes for important herbal medicine species in Trichosanthes, the identity of Trichosanthes material in commercial medicinal products in South Korea could be successfully determined. Phylogenetic analysis of the five Trichosanthes species revealed that the species are clustered within tribe Sicyoeae. T. kirilowii var. kirilowii and T. rosthornii formed a clade with T. kirilowii var. japonica as their sister group. As T. kirilowii in its current circumscription is paraphyletic and as the two varieties can be readily distinguished morphologically (e.g., in leaf shape), T. kirilowii var. japonica should be treated (again) as an independent species, T. japonica.

Chemical Constituents of the Egg Cases of Tenodera angustipennis (Mantidis ootheca) with Intracellular Reactive Oxygen Species Scavenging Activity.

As a traditional medicine with potential antioxidant effects, Tenodera angustipennis egg cases (Mantidis ootheca) are a potential source of new bioactive substances. Herein, three new N-acetyldopamine derivatives, namely, (+)-tenoderin A (1a), (-)-tenoderin A (1b), and tenoderin B (2), along with thirteen known compounds (3-15), were isolated from a 70% EtOH extract of T. angustipennis egg cases. Compound 1 was isolated as a racemic mixture, and two enantiomers (1a and 1b) were successfully separated by chiral-phase preparative HPLC. The chemical structures of the new compounds were established by NMR spectroscopy and high-resolution electrospray ionization mass spectrometry, and the absolute configurations of enantiomers 1a and 1b were determined by electronic circular dichroism spectroscopy. All the new compounds exhibited antioxidant activities with IC50 values of 19.45-81.98 µM, as evaluated using free-radical scavenging assays, with the highest activity observed for compound 2. In addition, compounds 1a, 1b, and 2 exhibited inhibitory activities on intracellular reactive oxygen species generation.

Three Scrophularia Species (Scrophularia buergeriana, S. koraiensis, and S. takesimensis) Inhibit RANKL-Induced Osteoclast Differentiation in Bone Marrow-Derived Macrophages.

Scrophulariae Radix, derived from the dried roots of Scrophularia ningpoensis Hemsl. or S. buergeriana Miq, is a traditional herbal medicine used in Asia to treat rheumatism, arthritis, and pharyngalgia. However, the effects of Scrophularia buergeriana, S. koraeinsis, and S. takesimensis on osteoclast formation and bone resorption remain unclear. In this study, we investigated the morphological characteristics and harpagoside content of S. buergeriana, S. koraiensis, and S. takesimensis, and compared the effects of ethanol extracts of these species using nuclear factor (NF)-κB ligand (RANKL)-mediated osteoclast differentiation. The harpagoside content of the three Scrophularia species was analyzed by high-performance liquid chromatography-mass spectrometry (HPLC/MS). Their therapeutic effects were evaluated by tartrate-resistant acid phosphatase (TRAP)-positive cell formation and bone resorption in bone marrow-derived macrophages (BMMs) harvested from ICR mice. We confirmed the presence of harpagoside in the Scrophularia species. The harpagoside content of S. buergeriana, S. koraiensis, and S. takesimensis was 1.94 ± 0.24 mg/g, 6.47 ± 0.02 mg/g, and 5.50 ± 0.02 mg/g, respectively. Treatment of BMMs with extracts of the three Scrophularia species inhibited TRAP-positive cell formation in a dose-dependent manner. The area of hydroxyapatite-absorbed osteoclasts was markedly decreased after treatment with the three Scrophularia species extracts. Our results indicated that the three species of the genus Scrophularia might exert preventive effects on bone disorders by inhibiting osteoclast differentiation and bone resorption, suggesting that these species may have medicinal and functional value.

Dissection for Floral Micromorphology and Plastid Genome of Valuable Medicinal Borages Arnebia and Lithospermum (Boraginaceae).

The genera Arnebia and Lithospermum (Lithospermeae-Boraginaceae) comprise 25-30 and 50-60 species, respectively. Some of them are economically valuable, as their roots frequently contain a purple-red dye used in the cosmetic industry. Furthermore, dried roots of Arnebia euchroma, A. guttata, and Lithospermum erythrorhizon, which have been designated Lithospermi Radix, are used as traditional Korean herbal medicine. This study is the first report on the floral micromorphology and complete chloroplast (cp) genome sequences of A. guttata (including A. tibetana), A. euchroma, and L. erythrorhizon. We reveal great diversity in floral epidermal cell patterns, gynoecium, and structure of trichomes. The cp genomes were 149,361-150,465 bp in length, with conserved quadripartite structures. In total, 112 genes were identified, including 78 protein-coding regions, 30 tRNA genes, and four rRNA genes. Gene order, content, and orientation were highly conserved and were consistent with the general structure of angiosperm cp genomes. Comparison of the four cp genomes revealed locally divergent regions, mainly within intergenic spacer regions (atpH-atpI, petN-psbM, rbcL-psaI, ycf4-cemA, ndhF-rpl32, and ndhC-trnV-UAC). To facilitate species identification, we developed molecular markers psaA- ycf3 (PSY), trnI-CAU- ycf2 (TCY), and ndhC-trnV-UAC (NCTV) based on divergence hotspots. High-resolution phylogenetic analysis revealed clear clustering and a close relationship of Arnebia to its Lithospermum sister group, which was supported by strong bootstrap values and posterior probabilities. Overall, gynoecium characteristics and genetic distance of cp genomes suggest that A. tibetana, might be recognized as an independent species rather than a synonym of A. guttata. The present morphological and cp genomic results provide useful information for future studies, such as taxonomic, phylogenetic, and evolutionary analysis of Boraginaceae.

In Vitro Antiosteoporosis Activity and Hepatotoxicity Evaluation in Zebrafish Larvae of Bark Extracts of Prunus jamasakura Medicinal Plant.

Osteoporosis is one of the main health problems in the world today characterized by low bone mass and deterioration in bone microarchitecture. In recent years, the use of natural products approach to treat it has been in the increase. In this study, in vitro antiosteoporosis activity and hepatotoxicity of P. jamasakura bark extracts were evaluated. Methods. Mouse bone marrow macrophage (BMM) cells were incubated with tartrate-resistant acid phosphate (TRAP) buffers and p-nitrophenyl phosphate and cultured with different P. jamasakura bark extracts at concentrations of 0, 6.25, 12.5, 25, and 50 µg/ml in the presence of the receptor activator of nuclear factor kappa-Β ligand (RANKL) for 6 days. The osteoclast TRAP activity and cell viability were measured. Nitric oxide (NO) assay was conducted using murine macrophage-like RAW 264.7 cells treated with P. jamasakura ethanolic and methanolic bark extracts at concentrations of 0, 6.25, 12.5, 25, 50, 100, and 200 µg/ml. For hepatotoxicity assessment, zebrafish larvae were exposed to P. jamasakura bark extracts, 0.05% dimethyl sulfoxide as a negative control, and 5 µM tamoxifen as a positive control. The surviving larvae were anesthetized and assessed for hepatocyte apoptosis. Results. TRAP activity was significantly inhibited (p < 0.001) at all concentrations of P. jamasakura extracts compared to the control treatment. At 50 µg/ml, both ethanolic and methanolic extracts of P. jamasakura exhibited significant (p < 0.01) BMM cell viability compared to the control treatment. P. jamasakura ethanolic and methanolic extracts had significant inhibitory (p < 0.01) effects on lipopolysaccharide (LPS)-induced NO production at 200 µg/ml and exhibited significant (p < 0.01) and (p < 0.05) stimulative effects, respectively, on RAW 264.7 cell viability. No overt hepatotoxicity was observed in the liver of zebrafish larvae in any of the treatments. Conclusion. The TRAP activity of P. jamasakura bark gives a foundation for further studies to enhance future development of antiosteoporosis drug.

Taxonomic Implications of Leaf Micromorphology Using Microscopic Analysis: A Tool for Identification and Authentication of Korean Piperales.

A comparative study of the leaf micromorphology of Korean Piperales, including medicinal materials, was performed through light microscopy and scanning electron microscopy, to evaluate their taxonomic significance. Piperales possessed both amphistomatic and hypostomatic leaves. The epidermal area ranged from 38 to 5077 µm2, and the stomatal area ranged from 201 to 2129 µm2. The stomatal index on the abaxial surface was higher than that on the abaxial surface. Anomocytic stomata occurred most commonly, but actinocytic, anisocytic, tetracytic, and staurocytic stomata were also found in certain taxa. Secretory idioblasts were found on all taxa studied except Aristolochia. Three main types of trichomes were defined-(1) glandular trichome; (2) simple multicellular trichome; and (3) two-armed multicellular Y-shaped trichome. Although the quantitative data on its own had somewhat limited taxonomic value, the various qualitative characteristics (e.g., epidermal surfaces, stomata types and positions, trichome types and density, and secretory idioblast types) had great taxonomic value. These characteristics might be taxonomically relevant and useful for developing an identification key. Additionally, we evaluated and supported the previous taxonomic system of Korean Asarum, using leaf micromorphological characteristics. Finally, through the application for authentication of herbal medicine, we revealed that leaf micromorphological characteristics can be used for accurate authentication.

Rapid and Simple Species Identification of Cicada Exuviae Using COI-Based SCAR Assay.

Cicadidae periostracum (CP), the medicinal name of cicada exuviae, is well-known insect-derived traditional medicine with various pharmacological effects, e.g., anticonvulsive, anti-inflammatory, antitussive, and anticancer effects; it is also beneficial for the treatment of Parkinson's disease. For appropriate CP application, accurate species identification is essential. The Korean pharmacopoeia and the pharmacopoeia of the People's Republic of China define Cryptotympana atrata as the only authentic source of CP. Species identification of commercially distributed CP based on morphological features, however, is difficult because of the combined packaging of many cicada exuviae in markets, damage during distribution, and processing into powder form. DNA-based molecular markers are an excellent alternative to morphological detection. In this study, the mitochondrial cytochrome c oxidase subunit I sequences of C. atrata, Meimuna opalifera, Platypleura kaempferi, and Hyalessa maculaticollis were analyzed. On the basis of sequence alignments, we developed sequence-characterized amplified-region (SCAR) markers for efficient species identification. These markers successfully discriminated C. atrata from the three other cicada species, and detected the adulteration of market CP samples. This SCAR assay is a rapid, simple, cheap, reliable, and reproducible method for species identification, regardless of sample form and status, and contributes to CP quality control.

Comparative Analysis of Actaea Chloroplast Genomes and Molecular Marker Development for the Identification of Authentic Cimicifugae Rhizoma.

Actaea (Ranunculaceae; syn. Cimicifuga) is a controversial and complex genus. Dried rhizomes of Actaea species are used as Korean traditional herbal medicine. Although Actaea species are valuable, given their taxonomic classification and medicinal properties, sequence information of Actaea species is limited. In this study, we determined the complete chloroplast (cp) genome sequences of three Actaea species, including A. simplex, A. dahurica, and A. biternata. The cp genomes of these species varied in length from 159,523 to 159,789 bp and contained 112 unique functional genes, including 78 protein-coding genes, 30 transfer RNA genes, and 4 ribosomal RNA genes. Gene order, orientation, and content were well conserved in the three cp genomes. Comparative sequence analysis revealed the presence of hotspots, including ndhC-trnV-UAC, in Actaea cp genomes. High-resolution phylogenetic relationships were established among Actaea species based on cp genome sequences. Actaea species were clustered into each Actaea section, consistent with the Angiosperm Phylogeny Group (APG) IV system of classification. We also developed a novel indel marker, based on copy number variation of tandem repeats, to facilitate the authentication of the herbal medicine Cimicifugae Rhizoma. The availability Actaea cp genomes will provide abundant information for the taxonomic and phylogenetic analyses of Actaea species, and the Actaea (ACT) indel marker will be useful for the authentication of the herbal medicine.

Scrophularia koraiensis Nakai Attenuates Allergic Airway Inflammation via Suppression of NF-κB and Enhancement of Nrf2/HO-1 Signaling.

Scrophularia koraiensis Nakai (Scrophulariaceae) is a medicinal herb that grows in Korea and which has been widely used to treat fever, edema, neuritis and laryngitis. Hence, we evaluated the anti-inflammatory and antioxidant effects of the ethanol extract (SKE) of S. koraiensis Nakai in an ovalbumin (OVA)-induced mouse model. We injected 20 µg of OVA with 2 mg of aluminum on day 0 and day 14 to induce allergic airway inflammation in six-week-old BALB/c mice, and mice were challenged with 1% OVA by nebulization for 1 h on days 21, 22, and 23. SKE was orally administered at 20 mg/kg and 40 mg/kg from day 18 to 23, and its effects were compared with those of montelukast treatment. SKE significantly reduced proinflammatory cytokines, inflammatory cell counts, immunoglobulin-E, and airway hyperresponsiveness during the OVA-induced allergic airway inflammation model; it also reduced airway inflammation and mucus production. In addition, SKE reduced the OVA-induced nuclear factor kappa B (NF-κB) phosphorylation in lung tissues while enhancing nuclear factor erythroid-derived 2-related factor (Nrf-2) and heme oxygenase-1 (HO-1) expression. In conclusion, SKE showed the protective effects on OVA-induced allergic airway inflammation via the suppression of NF-κB phosphorylation and the enhancement of the Nrf2/HO-1 signaling pathway. These results indicate that SKE is a potential therapeutic agent for allergic airway inflammation.

Mantidis Oötheca (mantis egg case) original species identification via morphological analysis and DNA barcoding.

ETHNOPHARMACOLOGICAL RELEVANCE: Mantidis Oötheca (mantis egg case; sangpiaoxiao) is a medicine from an insect source, which has been widely used in Asian countries. However, misidentification due to a lack of information given variations in the medicinal portion of the ootheca and morphological similarities of the ootheca as an egg chamber. AIM OF THE STUDY: Thus, this study aims to provide the first comprehensive data for discriminating authentic of Mantidis Oötheca. Here, we provide detailed ootheca morphology and their molecular information to accurately identify Mantidis Oötheca. MATERIALS AND METHODS: Oothecae of Tenodera angustipennis (Saussure, 1869), Tenodera sinensis (Saussure, 1871), Hierodula patellifera Serville, 1839, and Hierodula sp. were used in the comparative morphological, principal component analysis, and DNA barcoding. RESULTS: The morphological analyses revealed that the emergence area, outline, angle of distal end, width of air-filled layer, and weight are useful diagnostic characters. Using these quantitative and qualitative characteristics, we developed the effective identification key. Furthermore, our CO1 sequences from all individuals were monophyletic with high bootstrap values at genus and species levels. Moreover, morphological identification using our developed key among all studied individuals agreed with molecular identification results using CO1 barcoding data. CONCLUSIONS: These multilateral approaches, including morphological, statistical, and DNA barcoding methods are highly reliable identification tools. Moreover, our diagnostic key characteristics and molecular barcoding should aid in the accurate identification, authentication, and quality control of Mantidis Oötheca medicinal materials.

Scrophularia buergeriana attenuates allergic inflammation by reducing NF-κB activation.

BACKGROUND: Scrophularia buergeriana Miq. (Scrophulariaceae) (SB) has been used as an oriental medicine for the treatment of inflammatory diseases, such as neuritis and pharyngolaryngitis. PURPOSE: We explored the therapeutic effects of S. buergeriana ethanol extract (SBE) on airway inflammation in ovalbumin (OVA)-induced asthmatic mice and lipopolysaccharide (LPS)-stimulated RAW264.7 cells. METHODS: Mice were intraperitoneally injected with OVA on days 0 and 14 to elevate the immune response. On days 21 to 23, the mice were challenged with OVA solution and SBE (20 and 40 mg/kg) was administered daily by oral gavage from days 18 to 23. RAW264.7 cells were pretreated with SBE 1 h before LPS stimulation. RESULTS: SBE administration effectively suppressed inflammatory cell infiltration, the expression of interleukin (IL)-5, IL-13, and IL-17, immunoglobulin E, and airway hyperresponsiveness in an OVA-induced allergic asthma model. A reduction in histological alterations, including airway inflammation and mucus hypersecretion, was observed. These effects of SBE were accompanied by a decrease in matrix metalloproteinase-9 (MMP-9) expression and nuclear factor kappa B (NF-κB) phosphorylation. These responses were observed in LPS-stimulated RAW264.7 cells. SBE treatment reduced the mRNA expression of tumor necrosis factor (TNF)-α, IL-6, and MMP-9, and NF-κB phosphorylation, in LPS-stimulated RAW264.7 cells. CONCLUSION: Our results indicated that SBE effectively attenuated airway inflammation in an OVA-induced allergic asthma model. These properties of SBE were thought to be involved in the suppression of NF-κB phosphorylation, suggesting that the material has the potential to regulate the development of allergic asthma.

Comparative Morphological, Ultrastructural, and Molecular Studies of Four Cicadinae Species Using Exuvial Legs.

Previous studies have suggested that exuviae can be used for the identification of cicada species, but the precise characteristics that differ among species have not been determined. Thus, we performed the first comparative analyses of the leg morphology, ultrastructure, and mitochondrial DNA sequences of exuviae of four dominant cicada species in Korea, Hyalessa maculaticollis (Motschulsky, 1866), Meimuna opalifera (Walker, 1850), Platypleura kaempferi (Fabricius, 1794) and Cryptotympana atrata (Fabricius, 1775), the source of Cicadidae Periostracum, a well-known traditional medicine. A morphological analysis revealed that the profemur length, femoral tooth angle, and distance between the intermediate and last tooth of the femoral comb are useful characteristics for identification. We also evaluated the usefulness of the size, degree of reflex, and number of spines on the mid-legs and hind legs as diagnostic features. An ultrastructural study showed that Meimuna opalifera has a unique surface pattern on the legs. The sequences obtained using exuviae were identical to previously obtained sequences for adult tissues. Moreover, in a phylogenetic analysis using CO1 sequences, each species formed a monophyletic cluster with high bootstrap support. Accordingly, multiple methodological approaches using exuviae might provide highly reliable identification tools. The integrative data provide useful characteristics for the exuviae-based identification of closely related species and for further taxonomic and systematic studies of Cicadinae.

Cuscuta Species Identification Based on the Morphology of Reproductive Organs and Complete Chloroplast Genome Sequences.

The genus Cuscuta (Convolvulaceae) comprises well-known parasitic plants. Cuscuta species are scientifically valuable, as their life style causes extensive crop damage. Furthermore, dried seeds of C. chinensis are used as a Korean traditional herbal medicine. Despite the importance of Cuscuta species, it is difficult to distinguish these plants by the naked eye. Moreover, plastid sequence information available for Cuscuta species is limited. In this study, we distinguished between C. chinensis and C. japonica using morphological characterisation of reproductive organs and molecular characterisation of chloroplast genomes. The differences in morphological characteristics of reproductive organs such as style, stigma, infrastaminal scale, seed shape and testa ornamentation were useful for distinguishing between C. japonica and C. chinensis. Analysis of chloroplast genomes revealed drastic differences in chloroplast genome length and gene order between the two species. Although both species showed numerous gene losses and genomic rearrangements, chloroplast genomes showed highly similar structure within subgenera. Phylogenetic analysis of Cuscuta chloroplast genomes revealed paraphyletic groups within subgenera Monogynella and Grammica, which is consistent with the APG IV system of classification. Our results provide useful information for the taxonomic, phylogenetic and evolutionary analysis of Cuscuta and accurate identification of herbal medicine.

Sequencing and Comparative Analysis of the Chloroplast Genome of Angelica polymorpha and the Development of a Novel Indel Marker for Species Identification.

The genus Angelica (Apiaceae) comprises valuable herbal medicines. In this study, we determined the complete chloroplast (CP) genome sequence of A. polymorpha and compared it with that of Ligusticum officinale (GenBank accession no. NC039760). The CP genomes of A. polymorpha and L. officinale were 148,430 and 147,127 bp in length, respectively, with 37.6% GC content. Both CP genomes harbored 113 unique functional genes, including 79 protein-coding, four rRNA, and 30 tRNA genes. Comparative analysis of the two CP genomes revealed conserved genome structure, gene content, and gene order. However, highly variable regions, sufficient to distinguish between A. polymorpha and L. officinale, were identified in hypothetical chloroplast open reading frame1 (ycf1) and ycf2 genic regions. Nucleotide diversity (Pi) analysis indicated that ycf4⁻chloroplast envelope membrane protein (cemA) intergenic region was highly variable between the two species. Phylogenetic analysis revealed that A. polymorpha and L. officinale were well clustered at family Apiaceae. The ycf4-cemA intergenic region in A. polymorpha carried a 418 bp deletion compared with L. officinale. This region was used for the development of a novel indel marker, LYCE, which successfully discriminated between A. polymorpha and L. officinale accessions. Our results provide important taxonomic and phylogenetic information on herbal medicines and facilitate their authentication using the indel marker.

Inhibition of Wnt/ß-catenin signaling mediates ursolic acid-induced apoptosis in PC-3 prostate cancer cells.

BACKGROUND: Ursolic acid, a pentacyclic triterpenoid, is known to exert antitumor activity in breast, lung, liver and colon cancers. Nonetheless, the underlying mechanism of ursolic acid in prostate cancer cells still remains unclear. To investigate the antitumor mechanism, the apoptotic mechanism of ursolic acid via Wnt/ß-catenin signaling was examined in PC-3 prostate cancer cells. METHODS: Cytotoxicity assay, flow cytometry, immunofluorescence assay and western blotting were performed. RESULTS: Ursolic acid showed cytotoxicity against PC-3, LNCaP and DU145 prostate cancer cells with IC50 of 35 µM, 47 µM and 80 µM, respectively. Also, ursolic acid significantly increased the number of ethidium homodimer stained cells and apoptotic bodies, and dose-dependently enhanced the sub-G1 apoptotic accumulation in PC-3 cells. Consistently, western blotting revealed that ursolic acid effectively cleaved poly (ADP-ribose) polymerase (PARP), activated caspase-9 and -3, suppressed the expression of survival proteins such as Bcl-XL, Bcl-2 and Mcl-1, and upregulated the expression of Bax in PC-3 cells. Interestingly, ursolic acid suppressed the expression of Wnt5α/ß and ß-catenin, and enhanced the phosphorylation of glycogen synthase kinase 3 ß (GSK3ß). Furthermore, the GSK3ß inhibitor SB216763 or Wnt3a-conditioned medium (Wnt3a-CM) reversed the cleavages of caspase-3 and PARP induced by ursolic acid in PC-3 cells. CONCLUSIONS: Our findings suggest that ursolic acid induces apoptosis via inhibition of the Wnt5/ß-catenin pathway and activation of caspase in PC-3 prostate cancer cells. These results support scientific evidence that medicinal plants containing ursolic acid can be applied to cancer prevention and treatment as a complement and alternative medicine (CAM) agent.