Phylogenetic position of Bupleurum sikangense inferred from the complete chloroplast genome sequence.

Bupleurum sikangense is an endemic species to China distributed in Xizang (Tibet), which has high saikosaponin content and potential medicinal value. Morphologically, it extremely resembles B. commelynoideum. In order to get a better understanding of the relationship between B. sikangense and B. commelynoideum, and on the phylogenetic status of the two species in the genus, the complete chloroplast (cp) genomes of them were sequenced. The genome organization, repeat sequences, codon usage, RNA-editing sites, and variation of their cp genomes revealed high similarity between the species. Some highly variable regions like trnK-UUU_rps16, rps16_trnQ-UUG, ndhC_trnV-UAC, petA_psbJ, accD_psaI, and petL_psbE were identified, providing potential molecular markers for differentiating the two species. Phylogenetic analysis indicated that B. commelynoideum has a closer relationship to B. chinese than that to B. sikangense. Overall, this study will not only improve our knowledge about cp genomes of these two species, and but also provide data for further research on species identification, safe medical application, conservation genetics, etc., of Bupleurum plants.

Chloroplast genomes of two Mediterranean Bupleurum species and the phylogenetic relationship inferred from combined analysis with East Asian species.

MAIN CONCLUSION: The chloroplast genomes of Mediterranean Bupleurum species are reported for the first time. Phylogenetic analysis supports the species as a basal clade of Bupleurum with divergence time at 35.40 Ma. Bupleurum is one of the most species-rich genus with high medicinal value in Apiaceae. Although infrageneric classifications of Bupleurum have been the subject of numerous studies, it still remains controversial. Chloroplast genome information will prove essential in advancing our understanding on phylogenetic study. Here we report cp genomes of two woody Bupleurum species (Bupleurum gibraltaricum and B. fruticosum) endemic to Mediterranean. The complete cp genomes of the two species were 157,303 and 157,391 bp in size, respectively. They encoded 114 unique genes including 30 tRNA genes, 4 rRNA genes and 80 protein coding genes. Genome structure, distributions of SDRs and SSRs, gene content exhibited similarities among Bupleurum species. High variable hotspots were detected in eight intergenic spacers and four genes. Most of genes were under purifying selection with two exceptions: atpF and clpP. The phylogenetic analysis based on 80 coding genes revealed that the genus was divided into 2 distinct clades corresponding to the 2 subgenera (subg. Penninervia, subg. Bupleurum) with divergence time at the end of collision of India with Eurasia. Most species diversified mainly during the later period of uplift of Qinghai-Tibetan Plateau. The cp genomes of the two Bupleurum species can be significant complementary to insights into the cp genome characteristics of this genus. The comparative chloroplast genomes and phylogenetic analysis advances our understanding of the evolution of cp genomes and phylogeny in Bupleurum.

[Comparative analysis on chemical constituents in Bupleurum chinense,B. marginatum,B. marginatum var. stenophyllum and B. smithii var. parvifolium].

UPLC-Q-TOF-MS was used to analyze the chemical differences in Bupleurum. chinense,B. marginatum,B. marginatum var. stenophyllum and B. smithii var. parvifolium. Chromatographic separation was carried out on an Acquity HSS T3 C_(18) column( 2. 1 mm ×100 mm,1. 8 µm,Waters) with the mobile phase composed of 0. 1% formic acid in water-acetonitrile in the gradient elution. A hybrid quadrupole time-of-flight tandem mass spectrometry( Q-TOF-MS~E) was used for mass spectrometric analysis. Finally,25 peaks were identified based on their exact mass data and fragmentation characteristics. B.chinense,B.marginatum,B. marginatum var. stenophyllum and B. smithii var. parvifolium were obviously clustered into 3 types through processing by principal component analysis( PCA). There was almost no difference between B. chinense and B. marginatum. However,the compounds existed in B. chinense were different from those in B. marginatum var. stenophyllum,and B. smithii var. parvifolium.

Multi-scale Gaussian/Haar wavelet strategies coupled with sub-window factor analysis for an accurate alignment in nontargeted metabolic profiling to enhance herbal origin discrimination capability.

Metabolic dataset can provide an overview of different herbal origin, which is conducted by some statistical procedures. Such results often deviate to a certain degree, due to peaks shifts in chromatographic signals. In order to solve this problem, an improved algorithm of combining sub-window factor analysis with the mass spectrum information is proposed. The algorithm uses a peak detection approach derived either from multi-scale Gaussian function or Haar wavelet to locate the peaks with different application scope; the candidate drift points at each peak are estimated by Fast Fourier transform cross correlation; Specifically, the best drift points at each candidate peaks are confirmed by sub-window factor analysis and mass spectrum information in nontargeted metabolic profiling. Finally, the peak regions were aligned against a reference chromatogram, and the non-peak regions were used linear interpolation. The chromatographic signals of 30 Bupleurum samples were aligned as an illustration of this algorithm, and they could be well distinguished using some statistical procedures. The result demonstrates that the presented method is stronger than other mass-spectra based algorithms, when facing the alignment of some co-eluted peaks.

Evaluation of the genetic diversity of Bupleurum using amplified fragment length polymorphism analysis.

Radix bupleuri (Chaihu), the dried root of the Bupleurum plant, is an important component of traditional Chinese medicine. In this study, we examined the genetic diversity of 11 Bupleurum strains, originating from 7 provinces in China, using amplified fragment length polymorphism analysis. A total of 274 polymorphic bands were obtained using 6 primer combinations, indicating a high level of polymorphism across all strains. An estimation of the relative relationships among strains revealed genetic distances ranging from 0.2183 to 0.7372, with an average of 0.4161. The 2 most closely related varieties were Bupleurum chinense DC. strains collected from Lushi, Henan, and Zhangjiakou, Hebei, with a genetic nearness of 0.2183. Hierarchical clustering divided the strains into 3 main groups, with B. falcatum L. from Hebei and Liaoning Provinces forming a cluster that diverged from that of B. smithii Wolff. and B. chinense DC. B. falcatum L. (Sandao chaihu), collected from Heze, Shandong, clustered independently of the other strains, suggesting that this strain may have been introduced from a different location or that it arose as a result of intraspecific variation. B. smithii Wolff. (Hei chaihu) was closely associated with B. scorzonerifolium Willd. (Nan chaihu) and B. chinense DC. (Bei chaihu), suggesting a common genetic origin.

DNA barcoding Chinese medicinal Bupleurum.

We tested 4 markers, namely nuclear internal transcribed spacer 2 (ITS2), psbA-trnH, matK, and rbcL, to evaluate these candidate DNA barcodes for distinguishing Bupleuri radix (Chaihu) from its adulterants. 51 plant samples of Bupleurum representing 19 species were collected from different areas in China. Amplification and sequencing were attempted for all the 4 candidate barcode regions, whose validity was assessed in terms of the success rate of PCR amplification and sequencing, differential intra- and inter-specific divergences, DNA barcoding gap and the ability to discriminate species. The results showed that ITS2 had the best performance in identifying Bupleurum with an identification efficiency of 73.68%, which, after combining with psbA-trnH, increased to 83.33%. We further evaluated the efficiency of ITS2 for discriminating the species of Bupleurum using a large database from GenBank, which archived data of 223 samples from 74 species, and ITS2 successfully discriminated 64.13% of the samples at the species level. In conclusion, the ITS2 can serve as a potentially useful barcode for Bupleurum species, with psbA-trnH as a supplementary locus.

[Identification of Bupleurum chinense and B. longiradiatum based on ITS2 barcode].

In this study, ITS2 barcode was used to identify Bupleurum chinense and B. longiradiatum. The ITS2 regions of 48 samples were amplified and sequenced. The sequences obtained above were aligned and the K2P distances were calculated. We used three methods, BLAST1, nearest distance and phylogenetic tree (NJ-tree), to test the identification ability. The results showed that the maximum intraspecific genetic distance of B. chinense was 0.013, and the minimum interspecific genetic distance between B. chinense and B. longiradiatum was 0.049. The NJ-tree can easily identify B. chinense and B. longiradiatum. Therefore, the ITS2 barcode is suitable to identify B. chinense and B. longiradiatum.

Antioxidant activity and total phenolic contents of three Bupleurum taxa.

Bupleuri Radix (Bupleurum spp. root) is one of the most important crude drugs in Korea, China and Japan. We investigated the total phenolic content and antioxidant activity of three Bupleurum taxa (B. falcatum, B. falcatum 'Mishima' and B. latissimum). The highest total phenolic content was found in B. latissimum (18.6 +/- 1.7 mg/g) and the least in B. falcatum 'Mishima' (9.4 +/- 0.5 mg/g). The ethyl acetate fractions obtained from B. falcatum and B. falcatum 'Mishima' showed higher DPPH radical scavenging activity than the other fractions. In the case of B. latissimum, the DPPH radical scavenging activity of the diethyl ether fraction was higher than that of the other fractions. These results suggest that the three Bupleurum plants may be used as a food additive as a natural antioxidant.

Phylogeographic analysis of a temperate-deciduous forest restricted plant (Bupleurum longiradiatum Turcz.) reveals two refuge areas in China with subsequent refugial isolation promoting speciation.

This study investigates the influence of climate-induced oscillations and complicated geological conditions on the evolutionary processes responsible for species formation in presently fragmented temperate forest habitats, located in continental East Asia. In addition to this, we also investigate the heavily debated issue of whether temperate forests migrated southwards during such glacial periods or, alternatively, whether there existed refugia within north China, enabling localized survival of temperate forests within this region. In order to achieve these, we surveyed the phylogeography of Bupleurum longiradiatum Turcz. (a herbaceous plant solely confined to temperate forests) constructed from sequence variation in three chloroplast (cp) DNA fragments: trnL-trnF, psbA-trnH and rps16. Our analyses show high genetic diversity within species (h(T)=0.948) and pronounced genetic differentiation among groups (yellow and purple flowers) with a significant phylogeographical pattern (N(ST)>G(ST), P<0.05). Forty-three haplotypes were identified and clustered into two lineages (the purple-flowered lineage and the yellow-flowered lineage). Two corresponding refuge areas, one in Qinling and its adjacent regions and one in the Changbai Mountains/eastern China, were revealed across the entire distribution ranges of Bupleurum longiradiatum. These results provide evidence for the hypothesis that independent refugia were maintained across the range of temperate forests in northern China during the last glacial maximum or earlier cold periods. Bupleurum longiradiatum var. porphyranthum formed a single taxon based on molecular data. This specific formation process suggests that the historical vicariance factors, i.e. climate-induced eco-geographic isolation through the biotic displacement of temperate-deciduous forest habitats, enhanced the divergence of the yellow and purple flower lineages at different spatial-temporal scales and over glacial and interglacial periods. Additionally, geological conditions that restricted gene flow might also be responsible for the observed high genetic and geographic differentiation. A nested clade analysis (NCA) revealed that allopatric fragmentation was a major factor responsible for the phylogeographic pattern observed, and also supported a role for historical vicariance factors. Our results therefore support the inference that Quaternary refugial isolation promoted allopatric speciation of temperate plants in East Asia. This may help to explain the existence of high diversity and endemism of plant species in East Asia.

[Investigation on bupleurum resources in northwest area of Hubei Province].

OBJECTIVE: To investigate the species and the distribution of Bupleurum genus in northwest of Hubei province and provide basis for the standardization planting of Bupleurum in the region. METHODS: Investigated and collected specimens on the spot, com- pared them with literatures and specimen collected before. RESULTS: The main species of Bupleurum plants was B. marginatum, only very few of them was B. scorzonerifolium, no B. chinense was found in northwest of Hubei province. CONCLUSION: B. marginatum is the mainstream species of Bupleurum plants and the roots of B. marginatum are the actual source of commodity with the name of "Beichaihu (the roots of B. chinense)" in northwest of Hubei province.

[Advances in studies on classification of Bupleurum].

This article introduced the herbal medicine studies on Bupleurum in recent years, focused on the classification just like morphological and chemical classification, microscopic characteristics and molecular biology classification for Bupleurum. Identification combined with a variety of classification is the most effective method for Bupleurum. Due to the short supply of Bupleurum in the current market the current Bupleurum classification studies should focuses on combining pharmacological research to expand the Bupleurum's herbal sources.

[Clustering analysis of karyotype resemblance-near coefficient for 6 Bupleurum species].

OBJECTIVE: To explore the genetic evolutionary distance between plants by using karyotype parameters identification of medicinal plants. METHOD: The cluster analysis of karyotype resemblance-near coefficient and evolutionary distance was used for 6 Bupleurum species. RESULT: The results showed that there were the biggest karyotype resemblance-near coefficient (0.9920) and the smallest evolutionary distance (D(e) = 0.0080) between B. scorzonerifolium and B. chinense, indicating the closest relationship, and the minimum karyotype resemblance-near coefficient (0.4794) and the maximum evolutionary distance (D(e) = 0.7352) between B. smityii and B. falcatum, indicating the most distant relationship. CONCLUSION: Karyotype was an important parameter for identification of medicinal plants because karyotype was stabilized for species. The genetic distance between in 6 species of Bupleurum species was obtained by karyotype clustering analysis of karyotype resemblance-near coefficient. There was the bigger evolutionary distance between the species which had different chromosome number.

Species discrimination of Radix Bupleuri through the simultaneous determination of ten saikosaponins by high performance liquid chromatography with evaporative light scattering detection and electrospray ionization mass spectrometry.

A simple, rapid and robust high performance liquid chromatography-evaporative light scattering detection (HPLC-ELSD) method was established for the species discrimination and quality evaluation of Radix Bupleuri through the simultaneous determination of ten saikosaponins, namely saikosaponin-a, -b(1), -b(2), -b(3), -b(4), -c, -d, -g, -h, and -i. These compounds were chromatographed on an Ascentis(®) Express C18 column with a gradient elution of acetonitrile and water containing 0.1% acetic acid at a flow rate of 1.0 mL/min. Saikosaponins were monitored by ELSD, which was operated at a 50°C drift tube temperature and 3.0 bar nebulizer gas (N(2)) pressure. The developed method was validated with respect to linearity, intra- and inter-day accuracy and precision, limit of quantification (LOQ), recovery, robustness and stability, thereby showing good precision and accuracy, with intra- and inter-assay coefficients of variation less than 15% at all concentrations. Furthermore, a high performance liquid chromatography-electrospray ionization mass spectrometry (HPLC-ESI-MS) method was developed to certify the existence of ten saikosaponins, as well as to confirm the reliability of ELSD. The extraction conditions of saikosaponins from Radix Bupleuri were also optimized by investigating the effect of extraction methods (sonication, reflux and maceration) and various solvents on the extraction efficiencies for saikosaponins. Sonication with 70% methanol for 40 min was found to be simple and effective for extraction of major saikosaponins. This analytical method was applied to determine saikosaponin profiles in 20 real samples consisting of four Bupleurum species, namely B. falcatum, B. chinense, B. sibiricum and the poisonous B. longiradiatum. It was found that three major saikosaponin-a, -c and -d were the major constituents in B. falcatum, B. chinense, and B. longiradiatum, while one major saikosaponin (saikosaponin-c) was not identified from B. sibiricum. In addition, no saikosaponin-b(3) was detected in B. longiradiatum samples, indicating that the toxic B. longiradiatum may be tentatively distinguished from officially listed Bupleurum species (B. falcatum and B. chinense) based on their saikosaponin profiles. Overall the simultaneous determination of ten saikosaponins in Radix Bupleuri was shown to be a promising tool to adopt for the discrimination and quality control of closely related Bupleurum species.

[Comparison on HPLC analysis of aerial part of Bupleurums from different germplasm resources].

OBJECTIVE: To investigate ingredient differences of the aerial part of Bupleurums from different germplasm resources by HPLC. METHOD: Twenty-one different germplasm resources were sown and cultured at the same conditions and the aerial parts of the biennial plants were analyzed by HPLC. RESULT: The chromatograms were established and the data were analyzed by Hierarchical Cluster Analysis to compare the similarity of the samples. The results showed that ingredients from various germplasm resources were significantly different, which indicated the germplasm diversity of Bupleurum. CONCLUSION: The method can be used for comparison of the ingredient differences and quality control, the results provide important references for classification of Bupleurum species.

Identification of crude drugs from Chinese medicinal plants of the genus Bupleurum using ribosomal DNA ITS sequences.

To determine the variation of rDNA ITS sequences in medicinal plants of the genus Bupleurum and identify corresponding DNA molecular markers associated with medicinally important members, ITS regions were amplified, sequenced, and analyzed by DNAssist version 2.2. We found that the homologous alignment of ITS sequences between members of the genus Bupleurum and associated out-groups was lower than 75%, while within-group alignment was greater than 87%. The conclusion can be drawn that ITS sequences can be used as reliable molecular markers for the identification of Radix Bupleuri.

Rapid authentication of Bupleurum species using an array of immobilized sequence-specific oligonucleotide probes.

Standardization and modernization of Chinese medicinal herbs are limited partially by misidentification of processed materials. Our goal was to develop an efficient method for verification of Chinese medicinal herbs, based on the variable sites of the rDNA internal transcribed spacer (ITS) region. We analyzed sequence differences in ITS of three Bupleurum species, B. kaoi Liu Chao et Chuang, B. falcatum L. and B. chinense DC., and developed a rapid detection method using a sequence-specific oligonucleotide probe (SSOP) array. The SSOP array, composed of poly-T tailed sequence-specific oligonucleotides, was hybridized to the digoxigenin (DIG)-labeled target ITS DNA of the Bupleurum species. The detected signals corresponded precisely to the specific sequences. This array provides a reliable and economical method for authenticating a large number of Chinese medicinal herbs. The short duration of the procedure (within 30 h) makes it an especially useful tool in verifying processed plant material.

ITS sequence analysis used for molecular identification of the Bupleurum species from northwestern China.

Radix Bupleuri (Chaihu), a famous Traditional Chinese Medicine, is derived from the dried roots of Bupleurum chinense DC. and B. scorzonerifolium Willd. But nowadays, other nine species and varieties from genus Bupleurum are also used as Radix Bupleuri in northwestern China. The authentic identification of dried roots of B. chinense and B. scorzonerifolium, however, is difficult to just base on the appearance and morphology. A molecular genetic method was developed to help discriminate the original species of Radix Bupleuri. The ITS sequences ( approximately 600 bp) were amplified by the PCR technique from genomic DNA isolated from all the collected samples. According to analyzing the information given by ITS sequences, the conclusion can be made that ITS sequence could serve as markers for authentication of Radix Bupleuri.

[ITS sequence of 9 Bupleurum species and its application in identification of Chaihu (Radix Buplurei)].

OBJECTIVE: The study the patterns of rDNA ITS sequence variation of 9 medicinal species of genus Bupleurum in China to find the DNA molecular markers for identification of these crude drugs. METHOD: The ITS regions of these species were amplified by PCR and sequenced, and their sequences were analyzed by DNAssist Version 2.2. RESULTS: Homologous alignment indicated that the consanguinity of the ITS sequences between genus Bupleurum and outgroups was lower than 75%, and that within genus Bupleurum was higher than 88%. For the same species, the consanguinity was higher than 99%. CONCLUSION: The ITS sequences may serve as reliable molecular markers for identification of Radix Bupleuri.

[Studies on fingerprints of Bupleurum chinense from Hebei Province with HPLC-UV].

OBJECTIVE: To establish a HPLC fingerprint analysis method for identification of Bupleurum chinense of Hebei Province and compare the fingerprints of Radix Bupleuri collected from different habitats so as to establish a sensitive and specific method for controlling the quality of Radix Bupleuri. METHODS: The HPLC-UV fingerprints of Radix Bupleuri from different habitats were obtained from Waters 1525 instruments. The HPLC separation was performed on a C18 analytical column gradient eluted with a mixture consisting of acetonitrile and water at a flow rate of 1.0 ml/min with UV detector at 203 nm. The temperature of column was 30 degrees C. RESULTS: The mutual mode of HPLC-UV fingerprints was set up, and the similar degrees to the crude drugs of different habitats were compared. CONCLUSION: It is simple and quick to differ Bupleurum chinense from different habitats with the method that can be used as a quality control item for Radix Bupleuri.

[ITS sequence identification of Radix Bupleuri].

OBJECTIVE: To provide the basis of molecular authentication of Radix Bupleuri by the comparison of the internal transcribed spacer (ITS) sequences of five kinds of Radix Bupleuri in common use. METHOD: Firstly, total DNA of five kinds of Radix Bupleuri was extracted. Secondly, the ITS sequence was amplified by PCR with universal primer of ITS and PCR product was directly sequenced after purification. RESULT: The length of ITS1 and ITS2 sequence was 214-220 bp and 230-231 bp respectively. There were distinct variation sites between the ITS sequences of the five kinds of Radix Bupleuri. CONCLUSION: ITS sequence may be the evidence for the molecular authentication of Radix Bupleuri.