An Integrative Study of Scrophularia takesimensis Nakai in an Ovalbumin-Induced Murine Model of Asthma: The Effect on T Helper 2 Cell Activation.

Scrophularia have traditionally been used as herbal medicines to treat neuritis, sore throats, and laryngitis. In particular, S. takesimensis, a Korean endemic species with restricted distribution on Ulleung Island, holds significant resource and genetic value. However, its pharmacological properties have not been thoroughly evaluated. Thus, we provide detailed morphological characteristics and genomic information for S. takesimensis in this study. Moreover, its pharmacological activity was evaluated in an ovalbumin-induced asthma rat model, using extracts of S. takesimensis roots (100 or 200 mg/kg). The distinguishing features of S. takesimensis from related species include the presence or absence of stem wings, leaf shape, and habitat. The chloroplast (cp) genome of this species is 152,420 bp long and exhibits a conserved quadripartite structure. A total of 114 genes were identified, which included 80 protein-coding genes, 30 transfer RNA (tRNA) genes, and 4 ribosomal RNA (rRNA) genes. The gene order, content, and orientation of the S. takesimensis cp genome was highly conserved and consistent with the general structure observed in S. buergeriana and S. ningpoensis cp genomes. Confirming the anti-inflammatory effects of S. takesimensis extract (STE) using an established mouse model of ovalbumin-induced asthma, we observed reduced asthmatic phenotypes, including inflammatory cell infiltration, mucus production, and suppression of T helper 2 (Th2) cell. Furthermore, STE treatment reduced Th2 cell activation and differentiation. This study underscores the medicinal value of S. takesimensis. The importance of preserving S. takesimensis was revealed and crucial insights were provided for further research on its utilization as a medicinal resource.

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.

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.

Genetic and demographic signatures accompanying the evolution of the selfing syndrome in Daphne kiusiana, an evergreen shrub.

BACKGROUND AND AIMS: The evolution of mating systems from outcrossing to self-fertilization is a common transition in flowering plants. This shift is often associated with the 'selfing syndrome', which is characterized by less visible flowers with functional changes to control outcrossing. In most cases, the evolutionary history and demographic dynamics underlying the evolution of the selfing syndrome remain poorly understood. METHODS: Here, we characterize differences in the demographic genetic consequences and associated floral-specific traits between two distinct geographical groups of a wild shrub, Daphne kiusiana, endemic to East Asia; plants in the eastern region (southeastern Korea and Kyushu, Japan) exhibit smaller and fewer flowers compared to those of plants in the western region (southwestern Korea). Genetic analyses were conducted using nuclear microsatellites and chloroplast DNA (multiplexed phylogenetic marker sequencing) datasets. KEY RESULTS: A high selfing rate with significantly increased homozygosity characterized the eastern lineage, associated with lower levels of visibility and herkogamy in the floral traits. The two lineages harboured independent phylogeographical histories. In contrast to the western lineage, the eastern lineage showed a gradual reduction in the effective population size with no signs of a severe bottleneck despite its extreme range contraction during the last glacial period. CONCLUSIONS: Our results suggest that the selfing-associated morphological changes in D. kiusiana are of relatively old origin (at least 100 000 years ago) and were driven by directional selection for efficient self-pollination. We provide evidence that the evolution of the selfing syndrome in D. kiusiana is not strongly associated with a severe population bottleneck.

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.

Gekko gecko extract attenuates airway inflammation and mucus hypersecretion in a murine model of ovalbumin-induced asthma.

ETHNOPHARMACOLOGICAL RELEVANCE: Gekko gecko is used as a traditional medicine for various diseases including respiratory disorders in northeast Asian countries, mainly Korea, Japan, and China. AIM OF THE STUDY: Allergic asthma is a chronic respiratory disease caused by an inappropriate immune response. Due to the recent spread of coronavirus disease 2019, interest in the treatment of pulmonary disorders has rapidly increased. In this study, we investigated the anti-asthmatic effects of G. gecko extract (GGE) using an established mouse model of ovalbumin-induced asthma. MATERIALS AND METHODS: To evaluate the anti-asthmatic effects of GGE, we evaluated histological changes and the responses of inflammatory mediators related to allergic airway inflammation. Furthermore, we investigated the regulatory effects of GGE on type 2 helper T (Th2) cell activation. RESULTS: Administration of GGE attenuated asthmatic phenotypes, including inflammatory cell infiltration, mucus production, and expression of Th2 cytokines. Furthermore, GGE treatment reduced Th2 cell activation and differentiation. CONCLUSIONS: These results indicate that GGE alleviates allergic airway inflammation by regulating Th2 cell activation and differentiation.

A Comprehensive Study of the Genus Sanguisorba (Rosaceae) Based on the Floral Micromorphology, Palynology, and Plastome Analysis.

Sanguisorba, commonly known as burnet, is a genus in the family Rosaceae native to the temperate regions of the Northern hemisphere. Five of its thirty species are distributed in Korea: Sanguisorba officinalis, S. stipulata, S. hakusanensis, S. longifolia, and S. tenuifolia. S. officinalis has been designated as a medicinal remedy in the Chinese and Korean Herbal Pharmacopeias. Despite being a valuable medicinal resource, the morphological and genomic information, as well as the genetic characteristics of Sanguisorba, are still elusive. Therefore, we carried out the first comprehensive study on the floral micromorphology, palynology, and complete chloroplast (cp) genome of the Sanguisorba species. The outer sepal waxes and hypanthium characters showed diagnostic value, despite a similar floral micromorphology across different species. All the studied Sanguisorba pollen were small to medium, oblate to prolate-spheroidal, and their exine ornamentation was microechinate. The orbicules, which are possibly synapomorphic, were consistently absent in this genus. Additionally, the cp genomes of S. officinalis, S. stipulata, and S. hakusanensis have been completely sequenced. The comparative analysis of the reported Sanguisorba cp genomes revealed local divergence regions. The nucleotide diversity of trnH-psbA and rps2-rpoC2, referred to as hotspot regions, revealed the highest pi values in six Sanguisorba. The ndhG indicated positive selection pressures as a species-specific variation in S. filiformis. The S. stipulata and S. tenuifolia species had psbK genes at the selected pressures. We developed new DNA barcodes that distinguish the typical S. officinalis and S. officinalis var. longifolia, important herbal medicinal plants, from other similar Sanguisorba species with species-specific distinctive markers. The phylogenetic trees showed the positions of the reported Sanguisorba species; S. officinalis, S. tenuifolia, and S. stipulata showed the nearest genetic distance. The results of our comprehensive study on micromorphology, pollen chemistry, cp genome analysis, and the development of species identification markers can provide valuable information for future studies on S. officinalis, including those highlighting it as an important medicinal resource.

An in vitro Propagation of Aspilia africana (Pers.) C. D. Adams, and Evaluation of Its Anatomy and Physiology of Acclimatized Plants.

Aspilia africana (Pers.) C. D. Adams is an important medicinal plant, that has been used as traditional medicine in many African countries for the treatment of various health problems, including inflammatory conditions, osteoporosis, tuberculosis, cough, measles, diabetes, diarrhea, malaria, and wounds. We developed an efficient and reproducible protocol for in vitro regeneration of A. africana from nodes. We assessed the effects of plant tissue culture media on A. africana growth, cytokinins for in vitro shoot regeneration and proliferation, and auxins for the rooting of regenerated shoots. Furthermore, chlorophyll content, photosynthetic rates, anatomy (leaves, stems, and roots), and Fourier transform near-infrared (FT-NIR) spectra (leaves, stems, and roots) of the in vitro regenerated and maternal A. africana plants were compared. Murashige and Skoog media, containing vitamins fortified with benzylaminopurine (BA, 1.0 mg/l), regenerated the highest number of shoots (13.0 ± 0.424) from A. africana nodal segments. 1-naphthaleneacetic acid (NAA, 0.1 mg/l) produced up to 13.10 ± 0.873 roots, 136.35 ± 4.316 mm length, and was the most efficient for rooting. During acclimatization, the in vitro regenerated A. africana plants had a survival rate of 95.7%, displaying normal morphology and growth features. In vitro regenerated and mother A. africana plants had similar chlorophyll contents, photosynthetic rates, stem and root anatomies, and FT-NIR spectra of the leaf, stem, and roots. The established regeneration protocol could be used for large-scale multiplication of the plant within a short time, thus substantially contributing to its rapid propagation and germplasm preservation, in addition to providing a basis for the domestication of this useful, high-value medicinal plant.

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.

PCR-based rapid diagnostic tools for the authentication of medicinal mistletoe species.

BACKGROUND: Taxilli Herba (TH) and Visci Herba (VH), defined as the leaves and branches of the mistletoe species Taxillus chinensis and Viscum coloratum, respectively, are popular herbal medicines in East Asia. However, commercial TH and VH products are frequently adulterated with related inauthentic mistletoe species, posing efficacy and safety concerns. Accurate species identification of herbal medicinal products is a prerequisite for quality control, but traditional morphological identification methods are hampered by difficulties in discriminating among closely related species and in identifying the source materials in processed products. PURPOSE: This study aimed to develop sequence-characterized amplified region (SCAR) markers and a multiplex-SCAR assay for rapid and accurate identification of authentic TH and VH. METHODS: The matK region was sequenced in a total of 20 samples from five mistletoe species, namely T. chinensis and V. coloratum, and three species often found in adulterated herbal medicines, T. sutchuenensis, V. articulatum, and Macrosolen tricolor. Species-specific nucleotide polymorphisms were identified and short regions (21-22 bp) containing at least two species-specific nucleotides close to the 3' end were incorporated into SCAR primers that produced uniquely sized PCR amplicons for each species. The five SCAR primer sets were also combined into a multiplex-SCAR assay. RESULTS: The SCAR primers successfully generated amplicons of the expected size for each target species even with low-DNA templates or with templates containing DNA from multiple samples. No amplification was observed in non-target species. The SCAR markers and the multiplex-SCAR assay successfully identified commercial TH and VH products that were counterfeit or adulterated in both dried and processed products. CONCLUSION: This is the first report to illustrate discrimination of genuine medicinal mistletoe species with DNA-based marker assays, enabling rapid and accurate species identification. The SCAR assays developed in this study will facilitate the standardization of commercial mistletoe products.

Role of Semisulcospira gottschei extract as medicinal food on reflux esophagitis in rats.

Gastroesophageal reflux disease (GERD) is a globally prevalent disease and results from a reflux of gastric contents into the esophagus. Existing synthetic drug-based treatments for GERD have various drawbacks including refractory symptoms, relapse, or resistance due to long-term use or may result in mucosal degeneration, polyps, and osteoporosis. Semisulcospira gottschei (SE), a freshwater snail, has been generally consumed as a food source due to its excellent flavor and nutritional value in Korea and considered to have therapeutic properties for various diseases including dyspepsia, stomachache, and hepatic diseases. The present study aims to investigate whether Semisulcospira gottschei extract (SGE) has a protective effect on reflux esophagitis-induced rat models. The anti-inflammatory effects of SGE were evaluated via NO production in LPS-induced Raw 264.7 macrophage. And the protection effects of SGE were analyzed by assessing the amelioration of mucosal damage and expression of inflammation-associated proteins in reflux esophagitis (RE) rats. Our results indicate that SGE significantly suppressed NO production in LPS-induced raw 264.7 cells without any cytotoxicity. We observed mucosal lesions and histological changes in the esophagus of RE control rats. However, SGE treatment markedly ameliorated mucosal lesion ratio indicated through histological changes. SGE administration suppressed the expression of proteins related to inflammation, such as p-NF-κB, p-IκBα, COX-2, and TNF-α, in esophageal tissue. Moreover, SGE elevated the expression of claudin-5, which is a tight junction protein, involved in barrier function of epithelium and endothelium. The results suggest that SGE is useful as a medicinal food in esophagitis and may be helpful in developing effective treatment protocols for GERD.

Notes on Allium section Rhizirideum (Amaryllidaceae) in South Korea and northeastern China: with a new species from Ulleungdo Island.

Allium section Rhizirideum is reviewed for South Korea and neighboring northeastern China based on critical observation of wild populations and herbarium materials. Species delimitations are re-evaluated on the basis of morphological and somatic chromosome numbers, resulting in the recognition of five species. Allium dumebuchum from Ulleungdo Island, South Korea, is described as a new species. This species is most similar to A. senescens due to its habits, but is clearly distinguished particularly by its rhomboid scapes in cross-secion, light purple perianth color, entire and narrowly triangular inner filaments, and flowering season from late September. One previously recognized species is placed into synonymy: A. pseudosenescens (under A. senescens). Photographs and a key to species of Allium section Rhizirideum in South Korea and northeastern China are provided in addition to information on nomenclatural types, synonymies, chromosome numbers, distribution, and specimens examined.

Indirect in vitro Regeneration of the Medicinal Plant, Aspilia africana, and Histological Assessment at Different Developmental Stages.

The medicinal plant, Aspilia africana, has been traditionally used in several African countries to treat many diseases such as tuberculosis, cough, inflammation, malaria, osteoporosis, and diabetes. In this study, we developed a protocol for in vitro propagation of A. africana using indirect shoot organogenesis from leaf and root explants of in vitro-grown seedlings and assessed the tissues at different developmental stages. The highest callus induction (91.9 ± 2.96%) from leaf explants was in the Murashige and Skoog (MS) medium augmented with 1.0 mg/L 6-Benzylaminopurine (BAP) and 1.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) while from root explants, the highest callus induction (92.6 ± 2.80%) was in the same plant tissue culture medium augmented with 0.5 mg/L BAP and 1.0 mg/L 2,4-D. The best shoot regeneration capacity from leaf-derived calli (i.e., 80.0 ± 6.23% regeneration percentage and 12.0 ± 6.23 shoots per callus) was obtained in medium augmented with 1.0 mg/L BAP and 0.05 mg/L α-Naphthaleneacetic acid (NAA); the best regeneration capacity for root-derived calli (i.e., 86.7 ± 6.24% shoot regeneration percentage and 14.7 ± 1.11 shoots per callus) was obtained in the MS medium augmented with 1.0 mg/L BAP, 0.05 mg/L NAA, and 0.1 mg/L Thidiazuron (TDZ). Regenerated plantlets developed a robust root system in 1/2 MS medium augmented with 0.1 mg/L NAA and had a survival rate of 93.6% at acclimatization. The in vitro regenerated stem tissue was fully differentiated, while the young leaf tissue consisted of largely unorganized and poorly differentiated cells with large intercellular airspaces typical of in vitro leaf tissues. Our study established a protocol for the indirect regeneration of A. africana and offers a basis for its domestication, large-scale multiplication, and germplasm preservation. To the best of our knowledge, this is the first study to develop an indirect regeneration protocol for A. africana and conduct anatomical assessment through the different stages of development from callus to a fully developed plantlet.

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.

A Micropropagation Protocol for the Endangered Medicinal Tree Prunus africana (Hook f.) Kalkman: Genetic Fidelity and Physiological Parameter Assessment.

Prunus africana is an endangered medicinal plant and hence new propagation methods are urgently required to increase its populations. Unfortunately, propagation through seeds is challenging due to its long flowering cycle and recalcitrant seeds. We developed a protocol for micropropagation using nodal segment explants. A woody plant medium supplemented with vitamins, 15 g L-1 sucrose, and 1.0 mg L-1 6-benzylaminopurine (BAP) supported the optimum rate (100%) of axillary shoot initiation. Supplementation with 15 g L-1 sucrose and 1.5 mg L-1 indole-3-acetic acid (IAA) provided the optimum rate (75%) of root initiation. Rooted plantlets were successfully planted in sterilized horticultural soil containing perlite (2:1 v/v) and the survival rate was 98% following acclimatization. The photosynthetic rate assessed using FlourPen FP110 series showed that the ratio of variable fluorescence to maximum fluorescence mean value for in vitro regenerated P. africana (0.830 ± 0.0008) was similar to that of the maternal P. africana plant (0.825 ± 0.005), indicating similarity in their photosynthetic performance; a pivotal process for growth and development. The Fourier transform near-IR (FT-NIR) spectrometer analysis of the in vitro regenerated and the maternal P. africana plant samples exhibited homogeneity in the absorbance peaks at 8,273, 6,344, and 4,938-4,500 cm-1 associated with lipids, starch, and proteins. The genetic fidelity of regenerated plants was confirmed using the randomly amplified polymorphic DNA (RAPD) technique. Our protocol is suitable for use in large-scale P. africana to meet the increasing demands for it in the global market.

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.

An Integrated Approach for Efficient and Accurate Medicinal Cuscutae Semen Identification.

To guarantee the safety and efficacy of herbal medicines, accurate identification and quality evaluation are crucial. The ripe dried seeds of Cuscuta australis R.Br. and C. chinensis Lam. are known as Cuscutae Semen (CS) and are widely consumed in Northeast Asia; however, the seeds of other species can be misidentified as CS owing to morphological similarities, leading to misuse. In this report, we propose a multilateral strategy combining microscopic techniques with statistical analysis and DNA barcoding using a genus-specific primer to facilitate the identification and authentication of CS. Morphology-based identification using microscopy revealed that the useful diagnostic characteristics included general shape, embryo exudation, hairiness, and testa ornamentation, which were used to develop an effective identification key. In addition, we conducted DNA barcoding-based identification to ensure accurate authentication. A novel DNA barcode primer was produced from the chloroplast rbcL gene by comparative analysis using Cuscuta chloroplast genome sequences, which allowed four Cuscuta species and adulterants to be discriminated completely. Therefore, this investigation overcame the limitations of universal DNA barcodes for Cuscuta species with high variability. We believe that this integrated approach will enable CS to be differentiated from other species, thereby improving its quality control and product safety in medicinal markets.

Accurate and Rapid Identification of Longan Arillus and Litchi Semen by a Multiplex PCR Assay.

Dimocarpus longan, Litchi chinensis, and Nephelium lappaceum are commercially valuable subtropical and tropical fruits of the Sapindaceae family. Arillus and seeds of the three species have very similar morphologies; however, the arillus of D. longan is used as the herbal medicine Longan Arillus and seeds of L. chinensis are used as Litchi Semen in Korean and Chinese pharmacopoeias. The adulteration of herbal medicines with inauthentic species, including the use of Aril and seed fractions acquired from a single species for two herbal medicines (e.g., Longan Arillus and Litchi Semen), is often driven by economic motives. DNA markers are a tool for the detection of adulterants in commercial products. To establish rapid and reliable assays for the genetic identification of authentic Longan Arillus and Litchi Semen, we developed DNA markers with high specificity and sensitivity based on internal transcribed spacer (ITS) sequences. The newly developed DNA markers and multiplex PCR assay may contribute to efforts to protect against adulteration, quality control, and the standardization of herbal medicines.

Establishment of conventional PCR and real-time PCR assays for accurate, rapid and quantitative authentication of four mistletoe species.

Adulterants in processed food and herbal medicines reduce their safety, quality control, or pharmacological efficacy. Four mistletoe species, including Viscum coloratum, inhabit Korea. Leaves and branches of V. coloratum, defined as edible or medicinal mistletoe species in Korean, are used to prepare Korean herbal medicines as well as leached tea. However, other mistletoe species including Taxillus sutchuenensis var. duclouxii, Korthalsella japonica, and Loranthus tanakae are frequently distributed as authentic V. coloratum in Korean markets because of similarities in the branches morphology and Korean names of these species with V. coloratum. Although herbal medicines and food products prepared from the other mistletoe species are inauthentic, they are sold at high prices because of the rarity of these species. Thus, it is important to distinguish between authentic and inauthentic adulterant mistletoe species. In this study, we developed species-specific primer, based on matK sequences, suitable for both conventional PCR and real time PCR (qPCR) assay. These assays allowed rapid discrimination among all four mistletoe species. Moreover, qPCR assay enabled the detection of trace amounts of adulterant mistletoe species in V. coloratum samples. Furthermore, we used these assays to monitor commercial mistletoe products distributed in Korean markets. Our data suggest that these methods would serve as a reliable genetic tool to prevent adulteration and standardize the quality of commercial V. coloratum products.