Identification of Angelica acutiloba, A. sinensis, and other Chinese medicinal Apiaceae plants by DNA barcoding.

Crude drug Angelicae acutilobae radix is one of the most important crude drugs in Japanese traditional medicine and is used mainly for the treatment of gynecological disorders. In the listing in the Japanese Pharmacopoeia XVIII, Angelicae acutilobae radix is defined as the root of Angelica acutiloba (Apiaceae), which has long been produced on an industrial scale in Japan. With the aging of farmers and depopulation of production areas, the domestic supply has recently declined and the majority of the supply is now imported from China. Due to having only slightly different morphological and chemical characteristics for the Apiaceae roots used to produce dried roots for Chinese medicines, the plant species originating the crude drug Apiaceae roots may be incorrectly identified. In particular, Angelicae sinensis radix, which is widely used in China, and Angelicae acutilobae radix are difficult to accurately identify by morphology and chemical profiles. Thus, in order to differentiate among Angelicae acutilobae radix and other radixes originated from Chinese medicinal Apiaceae plants, we established DNA markers. Using DNA sequences for the chloroplast psbA-trnH intergenic spacer and nuclear internal transcribed spacer regions, Angelicae acutilobae radix and other Chinese Apiaceae roots, including Angelicae sinensis radix, can be definitively identified.

Phylogenomic and evolutionary dynamics of inverted repeats across Angelica plastomes.

BACKGROUND: Angelica L. (family Apiaceae) is an economically important genus comprising ca. One hundred ten species. Angelica species are found on all continents of the Northern Hemisphere, and East Asia hosts the highest number of species. Morphological characters such as fruit anatomy, leaf morphology and subterranean structures of Angelica species show extreme diversity. Consequently, the taxonomic classification of Angelica species is complex and remains controversial, as the classifications proposed by previous studies based on morphological data and molecular data are highly discordant. In addition, the phylogenetic relationships of major clades in the Angelica group, particularly in the Angelica s. s. clade, remain unclear. Chloroplast (cp) genome sequences have been widely used in phylogenetic studies and for evaluating genetic diversity. RESULTS: In this study, we sequenced and assembled 28 complete cp genomes from 22 species, two varieties and two cultivars of Angelica. Combined with 36 available cp genomes in GenBank from representative clades of the subfamily Apioideae, the characteristics and evolutionary patterns of Angelica cp genomes were studied, and the phylogenetic relationships of Angelica species were resolved. The Angelica cp genomes had the typical quadripartite structure including a pair of inverted repeats (IRs: 5836-34,706 bp) separated by a large single-copy region (LSC: 76,657-103,161 bp) and a small single-copy region (SSC: 17,433-21,794 bp). Extensive expansion and contraction of the IR region were observed among cp genomes of Angelica species, and the pattern of the diversification of cp genomes showed high consistency with the phylogenetic placement of Angelica species. Species of Angelica were grouped into two major clades, with most species grouped in the Angelica group and A. omeiensis and A. sinensis grouped in the Sinodielsia with Ligusticum tenuissimum. CONCLUSIONS: Our results further demonstrate the power of plastid phylogenomics in enhancing the phylogenetic reconstructions of complex genera and provide new insights into plastome evolution across Angelica L.

Mining of simple sequence repeats (SSRs) loci and development of novel transferability-across EST-SSR markers from de novo transcriptome assembly of Angelica dahurica.

Angelica dahurica is a widely grown plant species with multiple uses, especially in the medical field. However, the frequent introduction of A. dahurica to new areas has made it difficult to distinguish between varieties. Simple sequence repeats (SSRs) detected based on transcriptome analyses are very useful for constructing genetic maps and analyzing genetic diversity. They are also relevant for the molecular marker-assisted breeding of A. dahurica. We identified 33,724 genic SSR loci based on transcriptome sequencing data. A total of 114 primer pairs were designed for the SSR loci and were tested for their specificity and diversity. Ten SSR loci in untranslated regions were ultimately selected. Subsequently, 56 A. dahurica ecotypes collected from different regions were analyzed. The SSR loci comprised 2-8 alleles, with a mean of 5.2 alleles per locus. The polymorphic information content value and Shannon's information index were 0.6274-0.2702 (average of 0.4091) and 1.3040-0.5618 (average of 0.8475), respectively. Thus, the 10 novel SSRs identified in this study were almost in accordance with Harvey-Weinberg equilibrium and will be useful for analyzing A. dahurica genetic relationships. The results of this study confirm the potential value of transcriptome databases for the development of new SSR markers.

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.

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.

Rapid Authentication of the Herbal Medicine Plant Species Aralia continentalis Kitag. and Angelica biserrata C.Q. Yuan and R.H. Shan Using ITS2 Sequences and Multiplex-SCAR Markers.

Accurate identification of the plant species that are present in herbal medicines is important for quality control. Although the dried roots of Aralia continentalis (Araliae Continentalis Radix) and Angelica biserrata (Angelicae Pubescentis Radix) are used in the same traditional medicine, namely Dok-Hwal in Korean and Du-Huo in Chinese, the medicines are described differently in the national pharmacopeia. Further confusion arises from the distribution of dried Levisticum officinale and Heracleum moellendorffii roots as the same medicine. Medicinal ingredients from all four plants are morphologically similar, and discrimination is difficult using conventional methods. Molecular identification methods offer rapidity and accuracy. The internal transcribed spacer 2 (ITS2) region of the nuclear ribosomal RNA gene (rDNA) was sequenced in all four plant species, and the sequences were used to design species-specific primers. Primers for each species were then combined to allow sample analysis in a single PCR reaction. Commercial herbal medicine samples were obtained from Korea and China and analyzed using the multiplex assay. The assay successfully identified authentic medicines and also identified inauthentic or adulterated samples. The multiplex assay will be a useful tool for identification of authentic Araliae Continentalis Radix and/or Angelicae Pubescentis Radix preparations in Korea and China.

Identification of species and materia medica within Angelica L. (Umbelliferae) based on phylogeny inferred from DNA barcodes.

DNA barcodes have been increasingly used in authentication of medicinal plants, while their wide application in materia medica is limited in their accuracy due to incomplete sampling of species and absence of identification for materia medica. In this study, 95 leaf accessions of 23 species (including one variety) and materia medica of three Pharmacopoeia-recorded species of Angelica in China were collected to evaluate the effectiveness of four DNA barcodes (rbcL, matK, trnH-psbA and ITS). Our results showed that ITS provided the best discriminatory power by resolving 17 species as monophyletic lineages without shared alleles and exhibited the largest barcoding gap among the four single barcodes. The phylogenetic analysis of ITS showed that Levisticum officinale and Angelica sinensis were sister taxa, which indicates that L. officinale should be considered as a species of Angelica. The combination of ITS + rbcL + matK + trnH-psbA performed slight better discriminatory power than ITS, recovering 23 species without shared alleles and 19 species as monophyletic clades in ML tree. Authentication of materia medica using ITS revealed that the decoction pieces of A. sinensis and A. biserrata were partially adulterated with those of L. officinale, and the temperature around 80 °C processing A. dahurica decoction pieces obviously reduced the efficiency of PCR and sequencing. The examination of two cultivated varieties of A. dahurica from different localities indicated that the four DNA barcodes are inefficient for discriminating geographical authenticity of conspecific materia medica. This study provides an empirical paradigm in identification of medicinal plants and their materia medica using DNA barcodes.

[Qualitative and quantitative research on sulfur fumigation of Angelicae Dahuricae Radix (Baizhi) by near-infrared spectroscopy].

The contents of coumarins in the sulfur fumigated Angelicae Dahuricae Radix (Baizhi, ADR) were reduced significantly. To achieve the quality control of ADR, the qualitative identification of sulfur fumigated ADR and quantitative model of imperatorin content should be established. The near-infrared (NIR) spectrograms of non-sulfur and sulfur fumigated ADR were collected by NIR diffuse reflectance spectroscopy technology and pretreated by the method of first derivative derivation and vector normalization. The Ward's Algorithm method was used for the cluster analysis. The non-sulfur and sulfur fumigated ADR can be quickly identified in the range of 8,806. 0-3 811.0 cm(-1) based on the cluster analysis. The NIR quantitative model of imperatorin was established by the contents of imperatorin determined by HPLC in combination with partial least squares regression analysis. According to the calibration model established in this study, correlation coefficients (R2), the root-mean-square error of cross-validation (RMSECV), and the root-mean-square error of prediction (RMSEP) for imperatorin were 0.982 8, 0.006 8, 0.011 8, respectively. The quantitative model of imperatorin can be applied to determine the content of imperatorin in ADR accurately.

Authentication of Angelica anomala Avé-Lall cultivars through DNA barcodes.

Angelica anomala Avé-Lall (Chuanbaizhi in Chinese) is an important medicinal plant which can be used in traditional Chinese medicines; however, there are no authentic and universal methods to differentiate this Sichuan famous-region drug of A. anomala from a large number of non-famous-region and false drugs. It has been demonstrated that DNA barcoding is a molecular diagnostic method for species identification, which uses a single standardized DNA fragment. In this study, we tested five DNA barcoding candidates (matK, ITS, ITS2, rbcL, and psbA-trnH), and we found that ITS was the best candidate to authenticate the famous-region drug of A. anomala. Moreover, through comparative analysis of these five DNA barcodes between A. anomala and Angelica dahurica, we found that ITS had the most and ITS2 had more variable regions, but the psbA-trnH, rbcL, and matK regions were identical. Hence, we suggest ITS as the DNA barcoding to identify A. anomala and A. dahurica. Moreover, we are determined to adopt the A. anomala as the accurate Latin name of Chuanbaizhi.

Chemometric classification of morphologically similar Umbelliferae medicinal herbs by DART-TOF-MS fingerprint.

INTRODUCTION: It needs many years of special training to gain expertise on the organoleptic classification of botanical raw materials and, even for those experts, discrimination among Umbelliferae medicinal herbs remains an intricate challenge due to their morphological similarity. OBJECTIVE: To develop a new chemometric classification method using a direct analysis in real time-time of flight-mass spectrometry (DART-TOF-MS) fingerprinting for Umbelliferae medicinal herbs and to provide a platform for its application to the discrimination of other herbal medicines. METHODOLOGY: Angelica tenuissima, Angelica gigas, Angelica dahurica and Cnidium officinale were chosen for this study and ten samples of each species were purchased from various Korean markets. DART-TOF-MS was employed on powdered raw materials to obtain a chemical fingerprint of each sample and the orthogonal partial-least squares method in discriminant analysis (OPLS-DA) was used for multivariate analysis. RESULTS: All samples of collected species were successfully discriminated from each other according to their characteristic DART-TOF-MS fingerprint. Decursin (or decursinol angelate) and byakangelicol were identified as marker molecules for Angelica gigas and A. dahurica, respectively. Using the OPLS method for discriminant analysis, Angelica tenuissima and Cnidium officinale were clearly separated into two groups. Angelica tenuissima was characterised by the presence of ligustilide and unidentified molecular ions of m/z 239 and 283, while senkyunolide A together with signals with m/z 387 and 389 were the marker compounds for Cnidium officinale. CONCLUSION: Elaborating with chemoinformatics, DART-TOF-MS fingerprinting with chemoinformatic tools results in a powerful method for the classification of morphologically similar Umbelliferae medicinal herbs and quality control of medicinal herbal products, including the extracts of these crude drugs.

Metabolite profiling of Angelica gigas from different geographical origins using 1H NMR and UPLC-MS analyses.

Angelica gigas obtained from different geographical regions was characterized using (1)H nuclear magnetic resonance (NMR) spectroscopy and ultraperformance liquid chromatography-mass spectrometry (UPLC-MS) followed by multivariate data analyses. Principal component analysis (PCA) and orthogonal partial least-squares-discriminant analysis (OPLS-DA) score plots from (1)H NMR and UPLC-MS data sets showed a clear distinction among A. gigas from three different regions in Korea. The major metabolites that contributed to the discrimination factor were primary metabolites including acetate, choline, citrate, 1,3-dimethylurate, fumarate, glucose, histamine, lactose, malate, N-acetylglutamate, succinate, and valine and secondary metabolites including decursin, decursinol, nodakenin, marmesin, 7-hydroxy-6-(2R-hydroxy-3-methylbut-3-ethyl)coumarin in A. gigas roots. The results demonstrate that (1)H NMR and UPLC-MS-based metabolic profiling coupled with chemometric analysis can be used to discriminate the geographical origins of various herbal medicines and to identify primary and secondary metabolites responsible for discrimination.

[Quality control and discrimination of angelica different processed products based on HPLC fingerprints combined chemometrics methods].

OBJECTIVE: To establish a chemical fingerprint method for reorganizing and validating angelica different processed products. METHOD: A high-performance liquid chromatographic method was developed to establish the fingerprint. Principal component analysis, hierarchical cluster analysis and discriminate analysis were applied to study HPLC finger printing and chemical pattern reorganization. RESULT: There were difference of characteristic peaks and its relative peak area of HPLC fingerprints between different processed products. Fish's discriminate functions were generated by using six selected predictor variables, the tested samples of different processed products were classified with 100% accuracy, and discriminate analysis plots for the five groups were well-resolved. CONCLUSION: The developed HPLC finger print, combined with chemometrics, can accurately identify and validate angelica different processed products, the research provide theoretical basis for the processing mechanism and quality assess of angelica different processed products.

[Studies on the chemical constituents in roots of Angelica tianmuensis and A. megaphylla].

OBJECTIVE: To study the chemical constituents in roots of Angelica tianmuensis and A. megaphylla. METHODS: Compounds were isolated by column chromatography with silica gel, their structures were identified by spectral analysis. RESULTS: Another three compounds (ligustilone, 5-methoxyhamaudol, cimifugin) were obtained from the roots of Angelica tianmuensis, and another two compounds (ligustilone, angelol) were obtained from the roots of A. megaphylla. CONCLUSION: All the compounds are isolated in these two plants for the first time, and ligustilone is first found from Angelica L..

Non-targeted metabolite fingerprinting of oriental folk medicine Angelica acutiloba roots by ultra performance liquid chromatography time-of-flight mass spectrometry.

The potential of ultra-performance (UP)LC-TOF-MS based metabolite fingerprinting was explored in the attempt to establish a standard methodology for the quality control of dried angelica roots (Angelica acutiloba) in commercial markets. Accurate mass chromatographic fingerprints of positive and negative ion modes were collected simultaneously at a high-throughput manner with high resolution and sensitivity, where analysis of hydrophobic, low molecular weight compounds, which includes secondary metabolites, could be achieved. The comparison of various metabolite profiles was performed through the use of chemometric technique, in which distinct partitioning of root samples was effectively achieved by principal component analysis. The discrimination was illustrated to have been subjective to cultivation area and was reported to be an important influential factor for quality determination. Further insights to the chemical constituents in relation to quality were attained where some ion markers significantly linked to dissociation of angelica roots were tentatively identified as some secondary metabolites. Reliable classification models by partial least square discriminant analysis gave good capability in categorizing test set samples. Overall, through the utilization of UPLC-TOF-MS, analysis could be attained with great sufficiency and accuracy for angelica root discrimination.

Molecular systematics of Angelica and allied genera (Apiaceae) from the Hengduan Mountains of China based on nrDNA ITS sequences: phylogenetic affinities and biogeographic implications.

Maximum parsimony, maximum likelihood, and Bayesian analyses of nuclear ribosomal DNA internal transcribed spacer sequences were used to infer the phylogenetic affinities and historical biogeography of Angelica and its allies (Apiaceae tribe Selineae), with emphasis on those species of Angelica and Peucedanum endemic to the Hengduan Mountains of south-central China. Results of these analyses corroborate a monophyletic Angelica (Angelica sensu stricto) upon the inclusion of Coelopleurum, Czernaevia, and one of two examined species of Ostericum, but with the exclusion of several species previously attributable to Angelica. Angelica oncosepala and A. likiangensis arise within the genus Heracleum in tribe Tordylieae; the former is recognized under its original name, Heracleum oncosepalum. Angelica sinensis, A. tianmuensis and A. paeoniifolia arise within the Sinodielsia clade of previous circumscription, closely related to Levisticum officinale. Angelica anomala is a sister group to Ostericum grosseserratum in the previously delimited Acronema clade. Angelica apaensis and A. decursiva, taxa whose phylogenetic affinities have previously been controversial, are confirmed within Angelica. Northeast Asia (including Japan, northeast China, Korea and adjacent areas of Russia), Western Europe, and North America are inferred to be ancestral areas of Angelica based on optimal solutions of a dispersal-vicariance analysis, with the Hengduan Mountains likely providing a refugium for Angelica during the latter part of the Tertiary.

Quality evaluation of Angelica acutiloba Kitagawa roots by 1H NMR-based metabolic fingerprinting.

The prices of Angelica acutiloba Kitagawa (yamato-toki) and A. acutiloba Kitagawa var. sukiyamae Hikino (hokkai-toki) are now mainly determined according to the sensory quality determined by experts in addition to the physical properties. This method provides a low reliability result for differentiating and qualifying their qualities. In addition, the quality in terms of pharmacological efficiency is not taken into account for consideration in the ordinary sensory method. A combination of a 1H NMR technique and a multivariate analysis was preliminarily applied for the quality evaluation of both toki roots with regard to their geographical and variety differences. A broad range of metabolites was detected by a single-run 1H NMR spectrometry. Partial least-squares discrimination analysis (PLS-DA), a pattern recognition method, was applied to the 1H NMR spectra of aqueous extracts of toki samples having different sensory qualities. The PLS-DA result showed a clear clustering corresponding to the cultivation area between toki samples cultivated in Hokkaido (Japan) and those cultivated in the southern part of China and the Nara prefecture (Japan), while there was no separation corresponding to the toki's variety and sensory qualities, indicating the inconsistency of the sensory evaluation result. The chemical metabolites contributing to the discrimination of toki samples in relation to pharmacological and sensory properties were reported for the first time. A reliable multivariate calibration model used to predict the sensory quality was successfully carried out by PLS regression.

Simultaneous determination of eight coumarins in Angelica gigas and in various other Angelica species by high performance liquid chromatography and comparative micro-morphology study of Angelica species.

This article outlines the development of a simple and specific analytical method for the quantitative determination of eight coumarin constituents from the methanolic extract of the roots of Angelica gigas. The coumarin constituents present in the roots of Angelica gigas were separated using an acetonitrile (0.1 % acetic acid)-water (0.1 % acetic acid)-reagent alcohol gradient solvent system at a flow rate of 1.0 mL per minute. The HPLC separation was performed using a Phenomenex C12 reversed phase column with detection at 328 nm. The method was successfully used to study the percentage compositions of eight coumarins present in various Angelica species procured from S. Korea. Additionally, this manuscript provides a detailed microscopic account for the authentication of Angelica gigas and compared it with other Angelica species. The microscopic evaluation of plant tissue samples plays a vital role in botanical authentication. The combination of these two methodologies will provide additional tools for the evaluation of various Angelica species.

Development of gas chromatographic/mass spectrometry-pattern recognition method for the quality control of Korean Angelica.

This paper describes gas chromatographic/mass spectrometry (GC/MS)-pattern recognition methods for the quality control of Korean Angelica. A total of 57 Angelicae radix samples, including Angelica gigas (Korean origin), A. sinensis (Chinese origin) and A. acutiloba (Japanese origin), were analyzed by GC/MS, with a principal component analysis (PCA) subsequently applied to 10 common peaks selected from each chromatogram. As a result, the samples were clustered according to their origins on the PC score plot. The loading plot revealed that decursin and decursinol angelate were the most contributive principles distinguishing Korean samples from Chinese and Japanese samples, In addition, a discriminant model was developed for classification of the Angelicae radix, using a discriminant analysis (DA), and validated with a training set (three from A. gigas, four from A. sinensis, and three from A. acutiloba). All samples tested were successfully classified according to their species origin.

[A new molecular identification method: anchored primer amplification polymorphism DNA].

To build up a stable and easy doing method for molecular identification in traditional Chinese medicine, on basis of RAPD, the new method mainly changed the primer length and PCR annealing temperature. Panax ginseng, Panax quinquefolius and its nine adulterants were used to establish the method and test it using MARMS primers published in 2004. The new method also used to authenticate Chinese Materia Medica of Tian-hua-fen (Radix Trichosanthes) and Bai-zhi (Radix Angelica). Primer Pg-q36F obtained polymorphic bands of P. Ginseng, P. quinquefolius and its adulterants. The identification result is identical to that published before and more stable. Primer TkS1-64F obtained polymorphic bands of Tian-hua-fen and its nine adulterants. Primer AfS1-100F obtained polymorphic bands of Bai-zhi and its three adulterants. The method has good stability and reproducibility and can easily identify authertic medicines from their adulterants. It was a potential molecular method to identify other Chinese Materia Medica. The method was named as anchored primer amplification polymorphism DNA (APAPD).

Optimized conditions for the extraction of secondary volatile metabolites in Angelica roots by accelerated solvent extraction.

Accelerated solvent extraction (ASE) of three common Angelica species found in Asia: Angelica sinensis (Oliv.) Diels from China, Angelica acutiloba (Sieb. et Zucc.) Kitagawa from Japan, and Angelica gigas Nakai from Korea was investigated. Preliminary experiments, including the selection of the solvent, extraction time, pressure, static cycle and time were investigated to optimize experimental parameters. Kováts indices and mass spectra were used to identify the components in the various fractions. These were then confirmed using gas chromatography-mass spectrometry (GC-MS). A total of 18 compounds were identified, with qualitative differences and similarities observed among the cultivars. From the 18 compounds found in the ASE extract of danggui cultivars, the major components were decursin, decursinol angelate (A. gigas); butylidene dihydrophthalide, 4-hydroxy-4-methyl-2-pentanone (A. sinensis); and 9,12-octadecanoic acid in Angelica acutiloba. The optimum ASE operating conditions were n-hexane as extraction solvent, extraction temperature and pressure of 80 degrees C and 1500 atm, respectively, static cycle of 2 min, and static time of 10 min. Under these conditions, the percentages of main analytes were increased.