Development of conventional PCR and real-time PCR assays to discriminate the origins of Chinese pepper oil and herbal materials from Zanthoxylum.

BACKGROUND: To ensure the safety, quality and therapeutic efficacy of processed foods and herbal medicines, it is important to identify and discriminate economically motivated adulterants. Zanthoxylum schinifolium is sold at a higher price than other Zanthoxylum species and is frequently adulterated with closely related Zanthoxylum species because of its high demand as a Korean food ingredient and medicinal material in markets. In addition, the pericarps of three Zanthoxylum species (Z. schinifolium, Z. bungeanum and Z. piperitum) are defined as herbal medicine Zanthoxyli Pericarpium in Korean pharmacopoeias, but not Z. piperitum in Chinese pharmacopoeias. Further confusion arises in the morphological similarity between Z. armatum (adulterant) and Z. bungeanum. Therefore, the aim of this study was to develop a sequence characterized amplified region (SCAR) marker for discrimination of four Zanthoxylum species. RESULTS: With the goal of developing rapid and reliable tools for genetic discrimination of authentic Zanthoxyli Pericarpium, we designed species-specific SCAR markers, based on ITS2 sequences, that generate amplicons of less than 200 bp. Using these markers, we established both conventional and real-time PCR assay methods capable of differentiating samples at the species level. We validated the ability of SCAR markers to authenticate edible oil and herbal medicine, and confirmed that some herbal medicines contaminated with Z. armatum are being distributed as Zanthoxyli Pericarpium in Korean and Chinese markets. CONCLUSIONS: The SCAR markers and PCR methods described represent powerful tools for protecting against adulteration and ensuring standardization of processed foods and herbal medicine. © 2018 The Authors. Journal of the Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Authentication of the Herbal Medicine Angelicae Dahuricae Radix Using an ITS Sequence-Based Multiplex SCAR Assay.

The accurate identification of plant species is of great concern for the quality control of herbal medicines. The Korean Pharmacopoeia and the Pharmacopoeia of the People's Republic of China define Angelicae Dahuricae Radix (Baek-Ji in Korean and Bai-zhi in Chinese) as the dried roots of Angelica dahurica or A. dahurica var. formosana belonging to the family Apiaceae. Discrimination among Angelica species on the basis of morphological characteristics is difficult due to their extremely polymorphic traits and controversial taxonomic history. Furthermore, dried roots processed for medicinal applications are indistinguishable using conventional methods. DNA barcoding is a useful and reliable method for the identification of species. In this study, we sequenced the internal transcribed spacer (ITS) region of nuclear ribosomal RNA genes in A. dahurica, A. dahurica var. formosana, and the related species A. anomala and A. japonica. Using these sequences, we designed species-specific primers, and developed and optimized a multiplex sequence-characterized amplified region (SCAR) assay that can simply and rapidly identify respective species, and verify the contamination of adulterant depending on the polymerase chain reaction (PCR) amplification without sequencing analysis in a single PCR reaction. This assay successfully identified commercial samples of Angelicae Dahuricae Radix collected from Korean and Chinese herbal markets, and distinguished them from adulterants. This multiplex SCAR assay shows a great potential in reducing the time and cost involved in the identification of genuine Angelicae Dahuricae Radix and adulterant contamination.

Establishment of a PCR Assay for the Detection and Discrimination of Authentic Cordyceps and Adulterant Species in Food and Herbal Medicines.

Accurate detection and differentiation of adulterants in food ingredients and herbal medicines are crucial for the safety and basic quality control of these products. Ophiocordyceps sinensis is described as the only fungal source for the authentic medicinal ingredient used in the herbal medicine "Cordyceps", and two other fungal species, Cordyceps militaris and Isaria tenuipes, are the authentic fungal sources for food ingredients in Korea. However, substitution of these three species, and adulteration of herbal material and dietary supplements originating from Cordyceps pruinosa or Isaria cicadae, seriously affects the safety and reduces the therapeutic efficacy of these products. Distinguishing between these species based on their morphological features is very difficult, especially in commercially processed products. In this study, we employed DNA barcode-based species-specific sequence characterized amplified region (SCAR) markers to discriminate authentic herbal Cordyceps medicines and Cordyceps-derived dietary supplements from related but inauthentic species. The reliable authentication tool exploited the internal transcribed spacer (ITS) region of a nuclear ribosomal RNA gene (nrDNA). We used comparative nrDNA-ITS sequence analysis of the five fungal species to design two sets of SCAR markers. Furthermore, we used a set of species-specific SCAR markers to establish a real-time polymerase chain reaction (PCR) assay for the detection of species, contamination, and degree of adulteration. We confirmed the discriminability and reproducibility of the SCAR marker analysis and the real-time PCR assay using commercially processed food ingredients and herbal medicines. The developed SCAR markers may be used to efficiently differentiate authentic material from their related adulterants on a species level. The ITS-based SCAR markers and the real-time PCR assay constitute a useful genetic tool for preventing the adulteration of Cordyceps and Cordyceps-related dietary supplements.

Authentication of Herbal Medicines Dipsacus asper and Phlomoides umbrosa Using DNA Barcodes, Chloroplast Genome, and Sequence Characterized Amplified Region (SCAR) Marker.

Dried roots of Dipsacus asper (Caprifoliaceae) are used as important traditional herbal medicines in Korea. However, the roots are often used as a mixture or contaminated with Dipsacus japonicus in Korean herbal markets. Furthermore, the dried roots of Phlomoides umbrosa (Lamiaceae) are used indiscriminately with those of D. asper, with the confusing Korean names of Sok-Dan and Han-Sok-Dan for D. asper and P. umbrosa, respectively. Although D. asper and P. umbrosa are important herbal medicines, the molecular marker and genomic information available for these species are limited. In this study, we analysed DNA barcodes to distinguish among D. asper, D. japonicus, and P. umbrosa and sequenced the chloroplast (CP) genomes of D. asper and D. japonicus. The CP genomes of D. asper and D. japonicus were 160,530 and 160,371 bp in length, respectively, and were highly divergent from those of the other Caprifoliaceae species. Phylogenetic analysis revealed a monophyletic group within Caprifoliaceae. We also developed a novel sequence characterised amplified region (SCAR) markers to distinguish among D. asper, D. japonicus, and P. umbrosa. Our results provide important taxonomic, phylogenetic, and evolutionary information on the Dipsacus species. The SCAR markers developed here will be useful for the authentication of herbal medicines.

Majorana-fermion origin of the planar thermal Hall effect in the Kitaev magnet α-RuCl3.

The field-induced quantum-disordered state of layered honeycomb magnet α-RuCl3 is a prime candidate for Kitaev spin liquids hosting Majorana fermions and non-Abelian anyons. Recent observations of anomalous planar thermal Hall effect demonstrate a topological edge mode, but whether it originates from Majorana fermions or bosonic magnons remains controversial. Here, we distinguish these origins from combined low-temperature measurements of high-resolution specific heat and thermal Hall conductivity with rotating magnetic fields within the honeycomb plane. A distinct closure of the low-energy bulk gap is observed for the fields in the Ru-Ru bond direction, and the gap opens rapidly when the field is tilted. Notably, this change occurs concomitantly with the sign reversal of the Hall effect. General discussions of topological bands show that this is the hallmark of an angle rotation-induced topological transition of fermions, providing conclusive evidence for the Majorana-fermion origin of the thermal Hall effect in α-RuCl3.

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.

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.

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.

Association between host wing morphology polymorphism and Wolbachia infection in Vollenhovia emeryi (Hymenoptera: Myrmicinae).

Many eusocial insects, including ants, show complex colony structures, distributions, and reproductive strategies. In the ant Vollenhovia emeryi Wheeler (Hymenoptera: Myrmicinae), queens and males are produced clonally, while sterile workers arise sexually, unlike other ant species and Hymenopteran insects in general. Furthermore, there is a wing length polymorphism in the queen caste. Despite its evolutionary remarkable traits, little is known about the population structure of this ant species, which may provide insight into its unique reproductive mode and polymorphic traits. We performed in-depth analyses of ant populations from Korea, Japan, and North America using three mitochondrial genes (COI, COII, and Cytb). The long-winged (L) morph is predominant in Korean populations, and the short-winged (S) morph is very rare. Interestingly, all L morphs were infected with Wolbachia, while all Korean S morphs lacked Wolbachia, demonstrating a association between a symbiont and a phenotypic trait. A phylogenetic analysis revealed that the S morph is derived from the L morph. We propose that the S morph is associated with potential resistance to Wolbachia infection and that Wolbachia infection does not influence clonal reproduction (as is the case in other ant species).

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.

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.

The complete chloroplast genome of Cnidium officinale Makino.

Cnidium officinale (Ligusticum officinale) is an important herbal medicine. To facilitate species identification, we determined the complete chloroplast genome of C. officinale using the Illumina MiSeq platform. The genome was 148,518 bp in length, comprising a large single copy (LSC) region of 93,977 bp, a small single copy (SSC) region of 17,607 bp, and two inverted repeat regions (IRa and IRb) of 18,467 bp each. The genome contains 113 unique genes, including 79 protein-coding genes, four ribosomal RNAs (rRNAs), and 30 transfer RNAs (tRNAs). Phylogenetic analysis revealed that C. officinale is most closely related to L. tenuissium, with high bootstrap values.

The complete chloroplast genome of Cuscuta pentagona Engelm.

Cuscuta pentagona is a parasitic plant whose seeds are often mixed with the seeds of medicinal Cuscuta species. To facilitate the identification of C. pentagona seeds, we generated the complete chloroplast genome sequence of C. pentagona Engelm. using the Illumina MiSeq platform. The complete chloroplast genome is 86,380 bp long, with a 50,958 bp LSC region, a 7022 bp SSC region, and two inverted repeat (IRa and IRb) regions comprising 14,200 bp. The chloroplast genome consists of 85 unique genes, 57 protein-coding genes, four ribosomal RNA (rRNA) genes, and 24 transfer RNA (tRNA) genes. Two gene families, NADH oxidoreductases and RNA polymerase-related genes, are absent in this genome. Phylogenetic analysis revealed that C. pentagona is closely related to C. reflexa and C. exaltata, with strong support values.

Complete chloroplast genome of Sanguisorba × tenuifolia Fisch. ex Link.

Sanguisorba × tenuifolia Fisch. ex Link is an important herbal medicine. To facilitate species identification, we sequenced its complete chloroplast genome using the Illumina MiSeq platform. The data show that the chloroplast genome of S. × tenuifolia is 155,403 bp in size, comprising an 85,525 bp large single-copy (LSC) region, a 18,726 bp small single-copy (SSC) region, and two inverted repeats (IR) regions, IRa and IRb (each 25,576 bp). The genome contains 112 unique genes, including 79 protein-coding genes, four ribosomal RNAs genes, and 30 transfer RNAs genes. Phylogenetic analysis revealed that S. × tenuifolia is most closely related to Hagenia abyssinica.

Complete chloroplast genome of Actaea heracleifolia (Kom.) J. Compton.

Dried rhizomes of Actaea heracleifolia, used as a traditional Korean herbal medicine, are frequently adulterated with other plant species. For accurate species identification, we sequenced the complete chloroplast genome of A. heracleifolia using Illumina MiSeq. A. heracleifolia harbours a 159,578 bp chloroplast genome comprising a large single-copy region (88,770 bp), small single-copy region (18,070 bp) and two inverted repeat (IR) regions (IRa and IRb; each 26,519 bp). The chloroplast genome contains 112 unique genes, including 78 protein-coding genes, 4 ribosomal RNA genes, and 30 transfer RNA genes. Phylogenetic analysis revealed that A. heracleifolia was closely related to Gymnaconitum gymnandrum.

The Complete Chloroplast Genomes of Six Ipomoea Species and Indel Marker Development for the Discrimination of Authentic Pharbitidis Semen (Seeds of I. nil or I. purpurea).

Ipomoea L. is the largest genus within the Convolvulaceae and contains 600-700 species. Ipomoea species (morning glories) are economically valuable as horticultural species and scientifically valuable as ecological model plants to investigate mating systems, molecular evolution, and both plant-herbivore and plant-parasite interactions. Furthermore, the dried seeds of I. nil or I. purpurea are used in Korean traditional herbal medicines. In this study, chloroplast (cp) genomes were sequenced from six Ipomoea species, namely, I. nil and I. purpurea and, for the first time, I. triloba, I. lacunosa, I. hederacea, and I. hederacea var. integriuscula. The cp genomes were 161,354-161,750 bp in length and exhibited conserved quadripartite structures. In total, 112 genes were identified, including 78 protein-coding regions, 30 transfer RNA genes, and 4 ribosomal RNA genes. The gene order, content, and orientation of the six Ipomoea cp genomes were highly conserved and were consistent with the general structure of angiosperm cp genomes. Comparison of the six Ipomoea cp genomes revealed locally divergent regions, mainly within intergenic spacer regions (petN-psbM, trnI-CAU-ycf2, ndhH-ndhF, psbC-trnS, and ccsA-ndhD). In addition, the protein-coding genes accD, cemA, and ycf2 exhibited high sequence variability and were under positive selection (Ka/Ks > 1), indicating adaptive evolution to the environment within the Ipomoea genus. Phylogenetic analysis of the six Ipomoea species revealed that these species clustered according to the APG IV system. In particular, I. nil and I. hederacea had monophyletic positions, with I. purpurea as a sister. I. triloba and I. lacunosa in the section Batatas and I. hederacea and I. hederacea var. integriuscula in the section Quamoclit were supported in this study with strong bootstrap values and posterior probabilities. We uncovered high-resolution phylogenetic relationships between Ipomoeeae. Finally, indel markers (IPOTY and IPOYCF) were developed for the discrimination of the important herbal medicine species I. nil and I. purpurea. The cp genomes and analyses in this study provide useful information for taxonomic, phylogenetic, and evolutionary analysis of the Ipomoea genome, and the indel markers will be useful for authentication of herbal medicines.