A multilocus phylogeny of the non-photosynthetic parasitic plant Cistanche (Orobanchaceae) refutes current taxonomy and identifies four major morphologically distinct clades.

Phylogenetic relationships of and within non-photosynthetic parasitic lineages are notoriously poorly known, which negatively affects our understanding of parasitic plants. This is also the case for Cistanche (Orobanchaceae), an Old World genus with about two dozen species, whose relationships have not yet been addressed using molecular phylogenetic approaches. Here we infer phylogenetic relationships within the genus, employing a taxonomically and geographically broad sampling covering all previously distinguished infrageneric groups and most of the currently recognized species. A combined matrix of three plastid markers (trnL-trnF, including the trnL intron and the intergenic spacer (IGS), trnS-trnfM IGS and psbA-trnH IGS) and one nuclear marker (ITS) was analyzed using maximum parsimony, maximum likelihood and Bayesian inference. Cistanche falls into four well-supported and geographically differentiated clades: East Asian Clade, Northwest African Clade, Southwest Asian Clade and Widespread Clade. Of those, only the East Asian Clade corresponds to a previously recognized taxonomic section, whereas the others either contain members of two or three sections (Widespread Clade and Southwest Asian Clade, respectively) or have not been taxonomically recognized so far (Northwest African Clade). Whereas the Southwest Asian Clade exhibits strong phylogenetic structure among and partly within species (the East Asian Clade and the Northwest African Clade are monospecific), phylogenetic resolution within the Widespread Clade is often low and hampered by discrepancies between nuclear and plastid markers. Both molecular and morphological data indicate that species diversity in Cistanche is currently underestimated.

[Comparative studies of three Cistanche speices based on UPLC specific chromatogram and determination of main components].

Based on UPLC specific chromatogram and determination of seven main components,this study aimed at evaluating the quality of Cistanche deserticola,C. tubulosa and C. sinensis. Echinacoside,cistanoside A,verbascoside,tubuloside A,isoacteoside,2'-acetylacteoside,tubuloside B were used as reference substances. UPLC analysis was performed on a Waters ACQUITY UPLC HSS T3 column( 2. 1 mm×100 mm,1. 8 µm). The mobile phase was acetonitrile-0. 08% trifluoroacetic acid solution. The flow rate was0. 3 mL·min-1,and the injection amount was 10 µL. The column temperature was 40 ℃,and the detection wavelength was 330 nm.The UPLC specific chromatograms were processed with ChemPattern software. UPLC specific chromatograms of C. deserticola and C.tubulosa from different samples were of high similarity,but the similarities of their counterfeit C. sinensis were less than 0. 06. Both of cluster and principal component analysis can distinguish certified products and counterfeits. The content ratios of echinacoside/verbascoside and verbascoside/isoacteoside were quite different between C. deserticola and C. tubulosa,which had distinct significance.The UPLC specific chromatogram and contents of seven main components can provide a basis for quality evaluation of Cistanches Herba.

From 1H NMR-based non-targeted to LC-MS-based targeted metabolomics strategy for in-depth chemome comparisons among four Cistanche species.

The great orthogonality between 1H NMR spectroscopy and LC-MS implies that their deployments in series could offer an opportunity to gain the qualified molecular markers via comparative metabolomics, and an attempt was made here to propose an integrated strategy namely "from 1H NMR-based non-targeted to LC-MS-based targeted metabolomics". In-depth chemome comparisons of Cistanche plants, such as C. deserticola, C. salsa, C. tubulosa, and C. sinensis, that possess dramatic economic and ecological benefits for the arid regions in the northwest China attributing to their dramatic medicinal and edible values, were employed to verify the applicability. 1H NMR-based non-targeted matabolomics acted as the survey experiment to find those signals offering decisive contributions towards the species discrimination, and the signals were translated to a set of putative identities, eighteen ones in total, through matching with authentic compounds and referring to some accessible databases. Afterwards, an advanced LC-MS platform assembling reversed phase liquid chromatography, hydrophilic interaction liquid chromatography, and tailored multiple reaction monitoring, was introduced to simultaneously quantify those eighteen potential markers in a single analytical run, because those candidates exhibited great polarity span as well as wide content range. Significant species differences occurred amongst their chemome patterns. Echinacoside, acteoside, betaine, mannitol, 6-deoxycatalpol, sucrose, and 8-epi-loganic acid were disclosed as the markers enabling the discrimination of those four species. The findings offered an alternative tool to differentiate Cistanche plants. More importantly, the strategy namely "from 1H NMR-based non-targeted to LC-MS-based targeted metabolomics" facilitates the pursuit of molecular markers among analogue plants, and thereby provides a promising choice for in-depth chemome comparison.

[Study on phenotypic diversity and cluster analysis of Cistanche Herba in different populations in Xinjiang].

To intuitively understand the phenotypic diversity of intra-population and inter-population of the medicinal Cistanche Herba distributed in Xinjiang province, three species of Cistanche Herba were selected for the first time to be conducted to phenotypic observation and measurement from the morphological perspective, aiming to fill the gap in the morphological research concerning Cistanche Herba, and discuss about the relationship between the phenotypic variation and the host plants together with the geographical conditions, thus better understanding the speciation and evolutionary mechanism of Cistanche Herba and providing some scientific basis for the resource protection and germplasm breeding of Cistanche Herba. Based on sampling survey, a total of 118 well grown medicinal Cistanche samples from 17 Cistanche Herba distribution areas in Xinjiang province were selected, and various indexes were observed and measured. Besides, ANOVA and clustering analysis were conducted with 9 phenotypic quantity characters. The Cistanche Herba was plentiful in phenotypic variation. For detail, significant intra-population differences were observed in eight of the nine character indexes, and the intra-population differences were more obvious than those of inter-population. For each quantity character of the three species, the flower density possessed the maximal variable coefficient (71.1%) while the flower length was the minimum (15.9%). The phenotypic variation was also obvious among different populations. Specifically, the average variable coefficient of flower number was the maximal one (46.5%) and the flower length was the minimum one (10.0%). For different populations, the average variable coefficient of the D4 population was the maximal one (41.1%) and the S3 population was the minimum one (13.5%). According to the clustering analysis, all the samples of Cistanche Herba could be divided into three groups when the Euclidean distance was set at 15. The populations of S1, S3, D1, S2, D4, D6, D7 and D5 which distributed in the north of Xinjiang were clustered into one group, and the populations of D8, S4, D9, D2 and D3 that grown in east and central of Xinjiang were clustered into another group. The populations of C. deserticola and C. salsa could not be completely separated, but both of them were obviously differentiated from the T1, T3 and T2 populations of C. tubulosa. Besides, the C. deserticola and C. salsa displayed a patch distribution among different populations, and similar phenotypic characters were shared for each population. The research results of phenotype were consistent with that of molecular biology study of Cistanche Herba. The different phenotypic characters in different distribution areas were deduced to be arose from geographical isolation caused by mountains, which led to the specific genetic structure for each population of Cistanche Herba during the long-term adaptation and evolution. In conclusion, the current study showed the adaptation potency of Cistanche Herba exposed to different habitats.

[Identification of Chinese Traditional Medicine Cistanches Herba from Different Places by HPLC-ESI-MS and FTIR Methods].

Five samples of Cistanches Herba from different places were analyzed by HPLC-ESI-MS and FTIR methods. The effective compositions in Cistanches Herba including cistanoside A, echinacoside, acteoside , isoacteoside, 2'-actylacteoside, cistanoside C and tubluoside B were determined by HPLC-MS. The common peak ratio and variant peak ratio were calculated by FTIR spectroscopy of the five samples and the dual index sequence of common peak ratio and variant peak ratio were established. The results showed that the evaluation results of the samples by the two methods were the same. The general fake plant Cynomorii Herba could be identified by FTIR. HPLC-ESI-MS, which has high sensitivity and rapid determination procedure, can be used to evaluate quality of Cistanches Herba by quantitative analysis of the primary compositions. FTIR is a non-destructive analysis method. without complicated extraction and separation procedures to the samples. The absorption strength and the absorption shape were the synergistic effect of the functional groups and the nestification of the components in Cistanches Herba. The provided method has some advantages such as rapid analysis process, good reproducibility, non-destructive, small quantity of sample, simple treatment, good specificity, low-cost and environment-friendly. The method meets the trend of complex analysis and whole analysis for the Chinese medicines. Combination of FTIR and HPLC-ESI-MS was a good method for identification and evaluation of quality of Chinese medicines.

[Rapid Identification of Cistanche via Fluorescence Spectrum Imaging Technology Combined with Principal Components Analysis and Fisher Distinction].

In order to explore rapid reliable Hebra cistanche detection methods, identification of 3 different sources of Hebra cistanche: cistanche deserticola, cistanche tubulosa, sand rossia is studied via fluorescent spectral imaging technology combined with pattern recognition. It is found in experiment that cistanche samples have obvious fluorescence properties. Forty fluorescence spectral images of 3 different sources of Hebra cistanche samples are collected through fluorescent spectral imaging system. After carrying on denoising and binarization processing to these images, the spectral curves of each sample was drawn according to the spectral cube. The obtained spectra data in the 450 - 680 nm wavelength range is regarded as the study object of discriminant analysis. Then, principal component analysis (PCA) is applied to reduce the dimension of spectroscopic data of the three kinds of cistanche and fisher distinction is used in combination to classify them; During the experiment were compared the effects of three methods of data preprocessing on the model: multiplicative scatter correction (MSC), standard normal variable correction (SNV) and first-order differential (FD) and then according to the cumulative contribution rate of the principal component and the effect of number of factors on the discriminant model to optimize the number of principal components factor. The results showed that. identification of the best after the first derivative pretreatment then the first four principal components is extracted to carry on fisher discriminant, discriminant model of 3 different sources of Hebra cistanche is set up through PCA combined with fisher discriminant the precision of original discrimination is 100%, recognition rate of the cross validation is 95%. It was thus shown that the fluorescent spectral imaging technology combined with principal components analysis and fisher distinction can be used for the identification study of 3 different sources of Hebra cistanche and has the advantages of easy operation, speediness, reliability.

[Study on identification of cistanche hebra and its adulterants by PCR amplification of specific alleles based on ITS sequences].

To explore the new method of discriminating Cistanche deserticola, Cynomorium songaricum and Orobanche pycnostachya by using PCR amplification of specific alleles. 30 samples of the different C. deserticola, 21 samples of C. songaricum and O. pycnostachya were collected. The total DNA of the samples were extracted, the ITS sequences from C. deserticola, C. songaricum and O. pycnostachya were amplified by PCR and sequenced unidirectionally. These sequences were aligned by using ClustulW. Specific primer was designed according to the ITS sequences of specific alleles, and PCR reaction system was optimized. Additionally, compare with the identification of specific PCR method and DNA sequence analysis method. The result showed that the 331 bp identification band for C. deserticola and the adulterants not amplified bands by a single PCR reaction, which showed good identification ability to the three species. PCR amplification of specific alleles can be used to identify C. deserticola, C. songaricum and O. pycnostachya successfully.

[Rapid and undamaged determination of C. deserticola by Fourier-transform infrared spectroscopy and clustering analysis].

Cistanche deserticola, an endemic species in China, has been one of the grade II national key conservation rare and endangered plants. The spectra of cultivated and wild C. deserticola samples were determined by Fourier transform infrared (FTIR) spectrometry. Based on the fingerprint infrared spectrum from 450 to 2 000 cm(-1), C. deserticola samples were rapidly classfied and closely studied by using the method of clustering analysis. Results showed that although there were tiny differences between the spectra of different origin, including the wild and cultivated C. deserticola samples, these samples could be successfully classified by soft independent modeling of class analogy (SIMCA). Recognition rate and rejection rate of different C. deserticola samples were up to 90%. When testing with the blind sample which the authors picked out from the chosen samples, the accuracy of clustering reaches up to 95%. On the whole, combined with clustering analysis, FTIR provides a effective way to evaluate the origin of the Chinese medicines rapidly and undamagedly.

[Studies on seed quality evaluation technique and grading rules of Cistanche deserticola].

OBJECTIVE: To establish a seed quality evaluation technique system and quality grading rules for Cistanche deserticola. METHODS: We used stereomicroscope, microphotography and image analysis system, tetrazolium method to measure, analyze and evaluate the size, thousand kernel weight, plumpness, embryo rate and viability of Cistanche deserticola seed. Then we used SPSS 11.0 statistical analysis software to analyze the seed viability and related indexes of another 55 batches. RESULTS: Seed size was significantly correlated with thousand kernel weight, but wasn't correlated with seed viability. However, grain plumpness was negatively correlated with seed viability. Seed quality was not determined by seed size but by seed viability and grain plumpness. CONCLUSION: Seed viability, thousand kernel weight and grain plumpness are significant indicators of seed quality and they can be took in the granding rules of Cistanche deserticola seed.

[Study on conditions of seed germination of Cistanche].

OBJECTIVE: To study the effect of fluridone concentration, stimulating period, temperature and salt on the seed germination of three species of Cistanche. METHOD: The seeds were cultured in Petri dish, and the germination percentage was counted. RESULT: The highest germination percentage was observed in Cistanche tubulosa, C. deserticola, C. sala seeds pre-treated by 0.1 mg x L(-1) fluridone for 24-29 h. The optimal temperature for the seeds germination of three species of Cistanche was at 20-30 degrees C, and the seeds did not germinate at sub-or supraoptimal temperatures (5 and 35 degrees C). The salt tolerance of C. sala seeds was strong, and the critical value of NaCl concentration was 0.04 mol x L(-1). By contrast, C. tubulosa and C. deserticola seeds were more sensitive to the salt stress, the critical value of NaCl concentration was 0.02 mol x L(-1). CONCLUSION: The optimal germination condition and the method of testing germination percentage of three species of Cistanche seeds are as follow: the seeds are pre-treated by 0.1 mg x L(-1) fluridone for 24 h and then cultured at 20-30 degrees C in salt solution which concentration is lower than 0.02 mol x L(-1).

[Microscopic identification of commercial Herba Cistanches by digital imaging technique].

OBJECTIVE: To identify the commercial drugs of Herba Cistanches collected from 10 different areas. METHOD: Descriptions identification and digital imaging technique. RESULT: The original plants of the commercial drugs were three species, Cistanche deserticola Y. C. Ma, C. salsa (C. A. Mey) Benth et Hook. f. and C. tubelosa (Schrenk.) R. Wight. CONCLUSION: The confusion of Herba Cistanches in markets is exist. The main current species was C. deserticola, C. salsa (each 2/5) and C. tubelosa (1/5). The descriptions of C. deserticola and C. salsa are similar and not easy to distinguish. But the digital photographs offered by the paper visually reflected the microscopic differences of two species.

[Study on genetic diversity of herba Cistanches by RAPD].

OBJECTIVE: To determine the genetic diversity of Cistanche species. METHOD: Two populations of Cistanche deserticola and four populations of C. tubulosa were analyzed by random amplified polymorphic DNA (RAPD) markers. RESULT: A total of 76 and 87 loci were amplified using 10 random primers each other. The average percentage of polymorphic loci of C. deserticola was 47.37%. The PPL were 39.47% and 35.53% for two populations. Average Nei's gene diversity was 0.1358, Shannon' s genetic diversity was 0.2072, and Gst was 0.2546. The average PPL of C. tubulosa was 27.59%. It was 19.54% to 25.29% in different populations and Andi'er population had the highest. Average Nei's gene diversity was 0.0823, and Shannon' s genetic diversity was 0.125 8, Cst was 0.175 5. CONCLUSION: The diversity of Cistanche deserticola is higher than that of C. tubulosa, but both has differentiation among populations, C. deserticola has already separated itself into two different ecotypes.

[Morphotype diversity of Cistanche deserticola].

OBJECTIVE: To provide theoretical basis for selecting high quality seeds by studying the modality diversity of Cistanche deserticola. METHOD: Four populations were collected in the field and its biodiversity was studied by comparative morphoaanatory to identify its mutation of nutrition organ and reproduction organ in laboratory and herbarium. RESULT AND CONCLUSION: There are several types of C. deserticola that come from different types, which results in the difference in pharmacody and effect of medicine.

[Identification and survey of commercial crude drug of Herba Cistanchis].

This paper deals with the morphological identification, TLC analysis and survey of commercial crude drug of Herba Cistanchis (Genus Cistinche). This results provide authentic methods for the identification of Herba Cistanchis.