Simultaneous identification of Fritillariae cirrhosae bulbus and its common adulterants in one reaction by multiplex ligation-dependent probe amplification and high-resolution melting curve assay.

Fritillariae cirrhosae bulbus, a well-known and precious medicinal and edible herb in China, causes remarkable effects on swelling and relieving cough, with fewer side effects than other congeneric medicine. It has been subject to various cheaper congeneric adulteration because of its high price and limited production. In this paper, a rapid, high throughput, sensitive and efficient technique was described for simultaneous identification of F. cirrhosae bulbus and its common adulterants by employing multiplex ligation-dependent probe amplification coupled with high-resolution melting (MLPA-HRM) curve assay in their internal transcribed spacer 1 (ITS1) regions. This assay was highly sensitive with a detection limit of 0.19 ng genomic DNA, and highly specific with no cross-reaction with common adulterants. Mixed sample analysis showed as low as 10% adulteration can be detected from F. cirrhosae bulbus in one MLPA-HRM reaction. Overall, the method described in this paper is well suited for detecting adulteration in F. cirrhosae bulbus.

Metabolomic approach for rapid differentiation of Fritillaria bulbs by matrix-assisted laser desorption/ionization mass spectrometry and multivariate statistical analysis.

The bulbs of Fritillaria have been used for centuries as food and medicinal products in many Asian countries. Different Fritillaria species have distinct pharmacological effects despite of their similar appearances. Effective differentiation of Fritillaria species can avoid adulteration and is crucial to its clinical uses. In this paper, a hybrid method of matrix assisted laser desorption/ionization mass spectrometry and multivariate statistical analysis was developed for the rapid and reliable differentiation of Fritillaria species for the first time. Significantly different patterns for five Fritillaria species were obtained by MALDI-MS after instant sample extractions. Different groups of Fritillaria were confidently differentiated via an orthogonal partial least square model. In addition, a metabolomic taxonomy of five Fritillaria species was obtained based on MALDI-MS data.

Comparison of the abilities of universal, super, and specific DNA barcodes to discriminate among the original species of Fritillariae cirrhosae bulbus and its adulterants.

Fritillariae cirrhosae bulbus is a famous type of traditional Chinese medicine used for cough relief and eliminating phlegm. The medicine originates from dried bulbs of five species and one variety of Fritillaria. Recently, immature bulbs from other congeneric species, such as F. ussuriensis, have been sold as adulterants of Fritillariae cirrhosae bulbus in medicine markets owing to the high price and limited availability of the genuine medicine. However, it is difficult to accurately identify the bulbs from different original species of Fritillariae cirrhosae bulbus and its adulterants based on traditional methods, although such medicines have different prices and treatment efficacies. The present study adopted DNA barcoding to identify these different species and compared the discriminatory power of super, universal, and specific barcodes in Fritillaria. The results revealed that the super-barcode had strong discriminatory power (87.5%). Among universal barcodes, matK provided the best species resolution (87.5%), followed by ITS (62.5%), rbcL (62.5%), and trnH-psbA (25%). The combination of these four universal barcodes provided the highest discriminatory power (87.5%), which was equivalent to that of the super-barcode. Two plastid genes, ycf1 and psbM-psbD, had much better discriminatory power (both 87.5%) than did other plastid barcodes, and were suggested as potential specific barcodes for identifying Fritillaria species. Phylogenetic analyses indicated that F. cirrhosa was not a "good" species that was composed of multiple lineages, which might have affected the evaluation of the discriminatory ability. This study revealed that the complete plastid genome, as super barcode, was an efficient and reliable tool for identifying the original species of Fritillariae cirrhosae bulbus and its adulterants.

Combining DNA Barcoding and HPLC Fingerprints to Trace Species of an Important Traditional Chinese Medicine Fritillariae Bulbus.

Fritillariae Bulbus is a precious Chinese herbal medicine that is grown at high elevation and used to relieve coughs, remove phlegm, and nourish the lungs. Historically, Fritillariae Bulbus has been divided into two odourless crude drugs: Fritillariae Cirrhosae Bulbus and Fritillariae Thunbergii Bulbus. However, now the Chinese Pharmacopoeia has described five Fritillariae Bulbus-the new additions include Fritillariae Pallidiflorae Bulbus, Fritillariae Ussuriensis Bulbus, and Fritillariae Hupehensis Bulbus. Because the morphology of dried Fritillariae Bulbus is similar, it is difficult to accurately identify the different types of Fritillariae Bulbus. In the current study, we develop a method combining DNA barcoding and high-performance liquid chromatography (HPLC) to help distinguish Fritillariae Cirrhosae Bulbus from other Fritillariae Bulbus and guarantee species traceability of the five types of Fritillariae Bulbus. We report on the validation of an integrated analysis method for plant species identification using DNA barcoding that is based on genetic distance, identification efficiency, inter- and intra-specific variation, calculated nearest distance, neighbour-joining tree and barcoding gap. Our results show that the DNA barcoding data successfully identified the five Fritillariae Bulbus by internal transcribed spacer region (ITS) and ITS2, with the ability to distinguish the species origin of these Fritillariae Bulbus. ITS2 can serve as a potentially useful DNA barcode for the Fritillaria species. Additionally, the effective chemical constituents are identified by HPLC combined with a chemical identification method to classify Fritillaria. The HPLC fingerprint data and HCA (hierarchical clustering analysis) show that Fritillariae Cirrhosae Bulbus is clearly different from Fritillariae Thunbergii Bulbus and Fritillariae Hupehensis Bulbus, but there is no difference between Fritillariae Cirrhosae Bulbus, Fritillariae Ussuriensis Bulbus, and Fritillariae Pallidiflorae Bulbus. These results show that DNA barcoding and HPLC fingerprinting can discriminate between the five Fritillariae Bulbus types and trace species to identify related species that are genetically similar.

Complete chloroplast genome of seven Fritillaria species, variable DNA markers identification and phylogenetic relationships within the genus.

Fritillaria spp. constitute important traditional Chinese medicinal plants. Xinjiang is one of two diversity hotspots in China in which eight Fritillaria species occur, two of which are endemic to the region. Furthermore, the phylogenetic relationships of Xinjiang Fritillaria species (including F. yuminensis) within the genus are unclear. In the present study, we sequenced the chloroplast (cp) genomes of seven Fritillaria species in Xinjiang using the Illumina HiSeq platform, with the aim of assessing the global structural patterns of the seven cp genomes and identifying highly variable cp DNA sequences. These were compared to previously sequenced Fritillaria cp genomes. Phylogenetic analysis was then used to evaluate the relationships of the Xinjiang species and assess the evolution of an undivided stigma. The seven cp genomes ranged from 151,764 to 152,112 bp, presenting a traditional quadripartite structure. The gene order and gene content of the seven cp genomes were identical. A comparison of the 13 cp genomes indicated that the structure is highly conserved. Ten highly divergent regions were identified that could be valuable in phylogenetic and population genetic studies. The phylogenetic relationships of the 13 Fritillaria species inferred from the protein-coding genes, large single-copy, small single-copy, and inverted repeat regions were identical and highly resolved. The phylogenetic relationships of the species corresponded with their geographic distribution patterns, in that the north group (consisting of eight species from Xinjiang and Heilongjiang in North China) and the south group (including six species from South China) were basically divided at 40°N. Species with an undivided stigma were not monophyletic, suggesting that this trait might have evolved several times in the genus.

Chloroplast genomic resources for phylogeny and DNA barcoding: a case study on Fritillaria.

The genus Fritillaria comprises approximately 130 perennial herbaceous species. In the Pharmacopoeia of the People's Republic of China, the bulbs of 11 Fritillaria species are used in Chinese herbal medicines. However, the traditional methods of morphological classification cannot accurately identify closely related species of Fritillaria. Previous studies have attempted to identify these species with universal molecular markers, but insufficient phylogenetic signal was available. In this study, the complete chloroplast genomes of eight Fritillaria species were compared. The length of the eight Fritillaria chloroplast genomes ranges from 151,009 bp to 152,224 bp. A total of 136 SSR loci were identified, including 124 polymorphic SSR loci. For large repeat sequences, 108 repeat loci and four types of repeats were observed. Ten highly variable regions were identified as potential molecular markers. These SSRs, large repeat sequences and highly variable regions provide important information for the development of genetic markers and DNA fingerprints. Phylogenetic analyses showed that the topological structures of all data sets (except the IR regions) were in complete agreement and well resolved. Overall, this study provides comprehensive chloroplast genomic resources, which will be valuable for future studies of evolution and species identification in Fritillaria.

Global detection and semi-quantification of Fritillaria alkaloids in Fritillariae Ussuriensis Bulbus by a non-targeted multiple reaction monitoring approach.

Methods based on triple quadrupole tandem mass spectrometry have been widely used and reported as highly selective and sensitive methods for quantifying substances of herbal medicines. However, most of them were limited to targeted components, due to the difficulties to optimize the multiple reaction monitoring transitions without authentic standards. This study proposed a novel strategy for non-targeted optimization of multiple reaction monitoring method based on the diagnostic ion guided family classifications, tandem mass spectrometry database establishment, and transitions and collision energy screening. Applying this strategy, 59 Fritillaria alkaloids in Fritillariae Ussuriensis Bulbus have been classified, and 51 of these Fritillaria alkaloids were successfully detected by the optimal multiple reaction monitoring method. For semi-quantification, the easy-to-obtain Fritillaria alkaloids of each type, such as verticinone for cevanine type and peimisine for jervine type, were used as the reference standards to calibrate the other Fritillaria alkaloids in the same type. The method was demonstrated a good linearity (R(2) > 0.998) with satisfactory accuracy and precision, and the lower limits of quantification of verticinone and peimisine were estimated to be 0.076 and 0.216 pg, respectively. In addition, the results suggested that the proposed strategy might obtained high quality metabolomics data in discrimination of Fritillaria unibracteata and Fritillaria ussuriensis.

[Comparative Analysis of Content of Four Alkaloids in Fritillaria hupehensis and Fritillaria ebeiensis var. purpurea].

OBJECTIVE: To establish an assay method for simultaneous determination of peimine, peiminine, peimissine and hupehenine and to make a comparative analysis of the content of four alkaloids in Fritillaria hupehensis and Fritillaria ebeiensis var. purpurea for the first time. METHODS: A Unitary C18 column(250 mm x 4.6 mm, 5 µm) was chosen with acetonitrile-water (containing 0.05% diethylamine) as mobile phase in a gradient program. The column temperature was 35 degrees C and the flow-rate was 1.0 mL/min. RESULTS: There was high content of peiminine and the content of peimissine was inferior to peiminine in Fritillaria hupehensis. Relatively speaking, peimine and hupehenine were much lower than the other two ingredients. Fritillaria ebeiensis var. purpurea also contained high levels of peiminine, the minimum content of peimine and equivalent content of peimissine comparing with Fritillaria hupehensis. In addition, it didn't contain hupehenine in Fritillaria ebeiensis var. purpurea. CONCLUSION: This method is simple and fast, and it has good separation, reproducibility and reliable results. Also, it can be used as basis for the quality evaluation of Fritillaria hupehensis and Fritillaria ebeiensis var. purpurea.

[Phylogenetic analysis for Fritillaria hupehensis: evidence from ITS, rpl16 and matK sequences].

The systematic position of Fritillaria hupehensis has been in dispute. Phylogentic analyses were conducted on sequences of ITS, rpl16, matK sequences for species of F. hupehensis and allies. Lilium davidii was designed as outgroup. The analyses were performed using MP and ML methods. Conclusions could be achieved as follow. The topologies of MP and ML are consistent. The samples of F. hepehensis from different places form a supported clade with a strong bootstrap. And then form a strongly supported clade with F. anhuiensis, F. monantha. The results suggests that although F. hupehensis has a closet relation with the two ones, it exists some difference.

[Pharmacognostical study on cultivated Fritillaria taipaiensis].

OBJECTIVE: To study on the pharmacognostical characteristics of cultivated Fritillaria taipaiensis for providing basis for further development and research. METHODS: Botanical, macroscopic and microscopic identifications, and determination of the content of extract, total saponins and total alkaloids were carried out. RESULTS: Because of various growing years, cultivated Fritillaria taipaiensis had diffferent properties,in addition to tip slightly resembling songbei's tip "embracing the moon", there were greatly different characteristics in the rest of specifications comparing with the traditional Fritillaria cirrhosa. Some were shallow conical or cylindrical, some had slightly rough surface,and some bases were constricted, bitter in taste. There were great differences in its extract and total alkaloids con-tent,and no obvious differences in the content of total saponins. CONCLUSION: The experimental results show that the extract,total saponins and total alkaloids content are not positively correlated or relevant with the current classification of Fritillariae Cirrhosae Bulbus. To consider the medicinal appearance diameter and length, the grade classification should be based on different application requirements, and combined with the evaluation of active ingredients.

Evolutionary relationships in the medicinally important genus Fritillaria L. (Liliaceae).

Fritillaria (Liliaceae) is a genus of approximately 140 species of bulbous perennial plants that includes taxa of both horticultural and medicinal importance. As well as being commercially valuable, Fritillaria species have attracted attention because of their exceptionally large genome sizes, with all values recorded to date in excess of 30Gb. Despite such interest in the genus, phylogenetic relationships between the majority of species have remained untested. Here we present the first phylogenetic reconstruction of relationships to encompass most of the currently recognised species diversity in the genus. Three regions of the plastid genome were sequenced in 117 individuals of Fritillaria, representing 92 species (c. 66% of the genus) and in representatives of nine other genera of Liliaceae. Eleven low-copy nuclear gene regions were also screened in selected species for their potential utility. Phylogenetic analysis of a combined plastid dataset using maximum parsimony and Bayesian inference provided support for the monophyly of the majority of currently recognised subgenera. However, subgenus Fritillaria, which is by far the largest of the subgenera and includes the most important species used in traditional Chinese medicine, is found to be polyphyletic. Moreover, several taxa that were represented by multiple individuals show evidence of species non-monophyly. The Japanese endemic subgenus Japonica, which contains the species with the largest recorded genome size for any diploid plant, is resolved as sister to the predominantly Middle Eastern and Central Asian subgenus Rhinopetalum. Whilst relationships between most of the major Fritillaria lineages can now be resolved, our results also highlight the need for data from additional independently evolving loci; an endeavour that may be particularly challenging in light of the huge nuclear genomes found in these plants.

Authentication of Bulbus Fritillariae Cirrhosae by RAPD-derived DNA markers.

Bulbus Fritillariae is the most commonly used antitussive herb in China. Eleven species of Fritillaria are recorded as Bulbus Fritillariae in the Chinese Pharmacopoeia. Bulbus Fritillariae Cirrhosae is a group of six Fritillaria species with higher efficiency and lower toxicity derived mainly from wild sources. Because of their higher market price, five other Fritillaria species are often sold deceptively as Bulbus Fritillariae Cirrhosae in the herbal market. To ensure the efficacy and safety of medicinal herbs, the authentication of botanical resources is the first step in quality control. Here, a DNA based identification method was developed to authenticate the commercial sources of Bulbus Fritillariae Cirrhosae. A putative DNA marker (0.65 kb) specific for Bulbus Fritillariae Cirrhosae was identified using the Random Amplified Polymorphic DNA (RAPD) technique. A DNA marker representing a Sequence Characterized Amplified Region (SCAR) was developed from a RAPD amplicon. The SCAR marker was successfully applied to differentiate Bulbus Fritillariae Cirrhosae from different species of Fritillaria. Additionally, the SCAR marker was also useful in identifying the commercial samples of Bulbus Fritillariae Cirrhosae. Our results indicated that the RAPD-SCAR method was rapid, accurate and applicable in identifying Bulbus Fritillariae Cirrhosae at the DNA level.

Phytochemical and biological research of Fritillaria medicine resources.

The genus Fritillaria is a botanical source for various pharmaceutically active components, which have been commonly used in traditional Chinese medicine for thousands of years. Increasing interest in Fritillaria medicinal resources has led to additional discoveries of steroidal alkaloids, saponins, terpenoids, glycosides and many other compounds in various Fritillaria species, and to investigations on their chemotaxonomy, molecular phylogeny and pharmacology. In continuation of studies on Fritillaria pharmacophylogeny, the phytochemistry, chemotaxonomy, molecular biology and phylogeny of Fritillaria and their relevance to drug efficacy is reviewed. Literature searching is used to characterize the global scientific effort in the flexible technologies being applied. The interrelationship within Chinese Bei Mu species and between Chinese species, and species distributed outside of China, is clarified by the molecular phylogenetic inferences based on nuclear and chloroplast DNA sequences. The incongruence between chemotaxonomy and molecular phylogeny is revealed and discussed. It is essential to study more species for both the sustainable utilization of Fritillaria medicinal resources and for finding novel compounds with potential clinical utility. Systems biology and omics technologies will play an increasingly important role in future pharmaceutical research involving the bioactive compounds of Fritillaria.

[Fingerprint analysis of Fritillaria thunbergii using rapid resolution liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry].

A fingerprint method for quality assessment of Fritillaria thunbergii was developed by rapid resolution liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry (RRLC-Q-TOF-MS). The separation was performed using Agilent Eclipse Plus C18 column (2.1 mm x 100 mm, 1.8 microm) by gradient elution with acetonitrile and 0.1% formic acid aqueous solution (containing 10 mmol x L(-1) ammonium formate) as the mobile phase. Q-TOF-MS was used to obtain the accurate mass for precursor and product ions. Under this chromatographic and MS condition, 12 batches of F. thunbergii and its adulterants (F. hupehensis and F. pallidiflora) were analyzed by RRLC-Q-TOF-MS. Fifteen steroidal alkaloids were identified from F. thunbergii, F. hupehensis and F. pallidiflora and nine were assigned as the common characteristic peaks for F. thunbergii. The RRLC-Q-TOF-MS fingerprint of F. thunbergii was different significantly with those of F. hupehensis and F. pallidiflora. The quality of 12 batches of F. thunbergii samples were finally evaluated by hierarchical clustering analysis (HCA) and principle component analysis (PCA). This convenient and high specific method could be used to identify and evaluate the quality of the F. thunbergii.

[Genetic diversity of Fritillaria from Sichuan province based on ISSR].

OBJECTIVE: To provide more proofs for expounding the genetic relationships among the (varietal) species in genus Fritillaria from Sichuan province. METHOD: The ISSR marker technique was used to study relationships and genetic polymorphism of nineteen populations in ten species and one varietal species of genus Fritillaria. Genetic similarities were calculated by using NTSYS software and the dendrogram was constructed by using UPGMA method. RESULT: Eleven primers were selected from 35 ISSR primers, and 179 DNA fragments were amplified from 19 populations. Of which, 179 fragments were polymorphic (percentage of polymorphic bands was 86.8%). The genetic similarity among all accessions ranged from 0.569 to 0.855. Clustering analysis showed that the 19 populations of Fritillaria could be distinctively classified into 4 groups. F. cirrhosa, F. przewalskii, F. cirrhosa var. logirnectarea and F. dajitensis were in the first group; The second group was the cluster of F. cirrhosa and F. mellea (wild and cultivated species); The third group was F. sulcisquamosa, F. thunbergii, wabunesis and F. delavayi; F. hupehensis alone formed the fourth group. CONCLUSION: ISSR marker technique is suitable for the genetic diversity of Fritillaria from Sichuan province. Interspecific identifications among the four original species of Bulhus Fritillariae Cirrhosae recorded by pharmacopoeia of China, and between them and the other species of genus Fritillaria from Sichuan province could not be gained by using ISSR markers technique. In addition, the cluster result of genus Fritillaria had some relationships with the geographical distribution.

Sensitive determination of verticine and verticinone in Bulbus Fritillariae by ionic liquid assisted capillary electrophoresis-electrochemiluminescence system.

CE/Ru(bpy)(3)(2+) electrochemiluminescence (ECL) system with the assistance of ionic liquids (ILs) was successfully established for sensitive determination of verticine and verticinone in Bulbus Fritillariae for the first time. Migration behavior of alkaloid largely relies on the hydrogen bonding interactions between alkyl imidazolium cations in ILs and the alkaloids. Running buffer containing 40 mmol/L 1-butyl-3-methylimidazolium tetrafluoroborate (BMImBF(4)) IL-8 mmol/L phosphate resulted in significant changes in separation selectivity for alkaloids with similar structures. The highest sensitivity of the detection was obtained by maintaining the detection potential at 1.2V. Under the optimized conditions, relative standard derivations of the ECL intensity and the migration time were 3.27 and 2.84% for verticine and 4.42 and 1.69% for verticinone, respectively. The standard curves were linear between 1x10(-8) and 1x10(-6) mol/L for verticine and between 5x10(-8) and 1x10(-6) mol/L for verticinone, respectively. Detection limits of 1.25x10(-10) mol/L for verticine and 1x10(-10) mol/L for verticinone were obtained (S/N=3). Developed method was successfully applied to determine the amounts of alkaloids in Bulbus Fritillariae.

[Progress of taxonomic study on Fritillaria (Liliaceae) medicinal plant].

In this review, we described the taxonomic study of the Fritillaria medicinal plant in the recent years. The taxonomic study of the Fritillaria medicinal plant was carried out from three main aspects: the traditional morphological character, the characteristic constituents of the plant and genotyping and species identification of Fritillaria by DNA chips. By comparison, we concluded that the DNA chip technology can provide a rapid, high throughput for genotyping and quality assurance of the plant species verification. It is the most prosperous method of species identification of the plant.

[Identification of Chinese medicine material of Fritillaria by themogravimetric/diffrential thermal analysis].

OBJECTIVE: To provide a quick and simple method to identify different Chinese medicine material of Fritillaria. METHOD: The thermograms and differential thermograms of nine Fritillaria powders were obtained by thermal analyzer. RESULT: By analyzing the thermograms of nine Fritillaria powders, we concluded that the thermal stability of nine Fritillaria powders was much different each other due to the different geography origin. The thermal stability of F. hupehensis was highest among nine Fritillaria, while F. ussurensis was the lowest. The different Fritillaria showed their own DTA spectra respectively. CONCLUSION: According to the differences in the thermal properties of nine Fritillaria powders, the origins of Fritillaria could be easily identfied.

[Study on the application of chemometrics methods in chemical characteristic fingerprinting of Fritillaria].

A method for determination of eleven samples in Fritillaria by HPLC with UV detection at 215 nm was described. The column was used a XDP-C18, 4.6 x 250 mm, 5 microm, and the mobile phase was methanol-water (volume: volume = 70: 30, to be added 7.5 mmol/L SDS, pH 4.5 +/- 0.1). Important parameters with chromatograms of eleven samples were obtained and crusted. The results indicated that it is similar between clustering analysis and taxonomy of Fritillaria. Identification of Fritillaria and establishment of the chemical characteristic fingerprinting can used the methods.

[Determination of peimine and peiminine in Fritillaria thunbergii by HPLC-ELSD].

AIM: To determine peimine and peiminine in Fritillaria drug simultaneously by RP-HPLC-ELSD. METHODS: HPLC was carried out with a Waters Alliance, Model 2690, equipped with XTerra RP18 column (150 mm x 3.9 mm ID, 5 microm) and evaporated light scattering detector. The mobile phase (acetonitrile-10 mmol.L(-1) NH4HCO3 adjusted to pH 10.10 by ammonia solution) was eluted in gradient mode. RESULTS: The recoveries of peimine and peiminine were 98.96% (n = 4), with RSD 1.01% and 98.40% (n = 4), with RSD 2.63%, respectively. CONCLUSION: The method is simple, sensitive and reliable. It can be used for quantitative determination of Fritillaria drug.