Protopine isolated from Nandina domestica induces apoptosis and autophagy in colon cancer cells by stabilizing p53.

The tumor suppressor p53 plays essential roles in cellular protection mechanisms against a variety of stress stimuli and its activation induces apoptosis or autophagy in certain cancer cells. Here, we identified protopine, an isoquinoline alkaloid isolated from Nandina domestica, as an activator of the p53 pathway from cell-based natural compound screening based on p53-responsive transcription. Protopine increased the p53-mediated transcriptional activity and promoted p53 phosphorylation at the Ser15 residue, resulting in stabilization of p53 protein. Moreover, protopine up-regulated the expression of p21WAF1/CIP1 and BAX, downstream genes of p53, and inhibited the proliferation of HCT116 colon cancer cells. Apoptosis was elicited by protopine as indicated by caspase-3/7 activation, poly ADP ribose polymerase cleavage, and increased population of Annexin V-FITC-positive cells. Furthermore, protopine induced the formation of microtubule-associated protein 1 light chain 3 (LC3) puncta and LC3-II turnover, typical biochemical markers of autophagy, in HCT116 cells. Our findings suggest that protopine exerts its antiproliferative activity by stimulating the p53 pathway and may have potential as a chemopreventive agent for human colon cancer.

Plastome phylogenomics of the early-diverging eudicot family Berberidaceae.

The relationships among the genera of the early-diverging eudicot family Berberidaceae have long been controversial. To resolve these relationships and to better understand plastome evolution within the family, we sequenced the complete plastome sequences of ten Berberidaceae genera, combined these with six existing plastomes for the family, and conducted a series of phylogenomic analyses on the resulting data set. Five of the newly sequenced plastomes were found to possess the typical angiosperm plastome complement of 79 protein-coding genes, 4 rRNA genes, and 30 tRNA genes. The infA gene was found to be pseudogenized in Bongardia, Diphylleia, Dysosma and Vancouveria; rps7 was found to be severely truncated in Diphylleia, Dysosma and Podophyllum; clpP was found to be highly divergent in Vancouveria; and a ∼19 kb inversion was detected in Bongardia. Maximum likelihood phylogenetic analyses of a 79-gene, 24-taxon data set including nearly all genera of Berberidaceae recovered four chromosome groups (x = 6, 7, 8, 10), resolved the x = 8 group as the sister to the x = 10 group, and supported the monophyly of the clade comprising x = 7, 8, 10. The generic relationships within each group were all resolved with high support. Based on gene presence within the Inverted Repeat (IR), a total of seven plastome IR types were identified within Berberidaceae. Biogeographical analysis indicated the origin and diversification of Berberidaceae has likely been strongly influenced by the distribution of its favored habitat: temperate forests.

The complete chloroplast genome of Sinopodophyllum hexandrum (Berberidaceae).

The complete chloroplast (cp) genome of the Sinopodophyllum hexandrum (Berberidaceae) was determined in this study. The circular genome is 157,940 bp in size, and comprises a pair of inverted repeat (IR) regions of 26,077 bp each, a large single-copy (LSC) region of 86,460 bp and a small single-copy (SSC) region of 19,326 bp. The GC content of the whole cp genome was 38.5%. A total of 133 genes were identified, including 88 protein-coding genes, 37 tRNA genes and eight rRNA genes. The whole cp genome consists of 114 unique genes, and 19 genes are duplicated in the IR regions. The phylogenetic analysis revealed that S. hexandrum is closely related to Nandina domestica within the family Berberidaceae.

Comparative transcriptome resources of two Dysosma species (Berberidaceae) and molecular evolution of the CYP719A gene in Podophylloideae.

Dysosma species (Berberidaceae, Podophylloideae) are of great medicinal pharmacogenetic importance and used as model systems to study the drivers and mechanisms of species diversification of temperate plants in East Asia. Recently, we have sequenced the transcriptome of the low-elevation D. versipellis. In this study, we sequenced the transcriptome of the high-elevation D. aurantiocaulis and used comparative genomic approaches to investigate the transcriptome evolution of the two species. We retrieved 53,929 unigenes from D. aurantiocaulis by de novo transcriptome assemblies using the Illumina HiSeq 2000 platform. Comparing the transcriptomes of both species, we identified 4593 orthologs. Estimation of Ka/Ks ratios for 3126 orthologs revealed that none had a Ka/Ks significantly greater than 1, whereas 1273 (Ka/Ks < 0.5, P < 0.05) were inferred to be under purifying selection. A total of 51 primer pairs were successfully designed from 461 EST-SSRs contained in 4593 orthologs. Marker validation assay revealed that 26 (51%) and 41 (80.4%) produced clear fragments with the expected sizes in all Podophylloideae species. Specifically, 19 different sequences of CYP719A were identified from PCR-amplified genomic DNA of all 12 species of Podophylloideae using primers designed from the assembled transcripts. The data further indicated that CYP719A was likely subject to strong selective constraints maintaining only one copy per genome. In Dysosma, there was relaxed purifying selection or more positive selection for high-elevation species. Overall, this study has generated a wealth of molecular resources potentially useful for pharmacogenetic and evolutionary studies in Dysosma and allied taxa.

Flavonols, alkaloids, and antioxidant capacity of edible wild berberis species from patagonia.

There are 20 species of the Berberidaceae family described in Chile, whose fruits are edible and show high anthocyanin and hydroxycinnamic acid levels. Berberis microphylla G. Forst, commonly known as calafate, is the most extensively distributed. Flavonols and alkaloids in seed, pulp, skin, and whole calafate berry extracts and other Berberis were studied using HPLC-DAD-ESI-MS/MS and HPLC with fluorescence detector. Berry samples from different locations in Chilean Patagonia, including different phenological stages, were systematically addressed. Results were compared with other organs of the plant and with other Berberis species. Total flavonol concentration in calafate (n = 65) was 1.33 ± 0.54 µmol/g. Glycosyl metabolites of quercetin and isorhamnetin were the most abundant. Similar profiles were observed in calafate from distinct locations, but important differences were observed for the other edible Berberis species. Calafate pulp and skin have higher flavonol concentrations than seeds, and the maturation process reduced its levels. TEACCUPRAC and TEACABTS of whole calafate extracts and fractions are also explored. Finally, only berberine was detected in the fruit (0.001%), mainly in seeds. Results contribute to the promotion of this berry as a superfruit from Patagonia.

Genetic diversity and structure of Sinopodophyllum hexandrum (Royle) Ying in the Qinling Mountains, China.

Sinopodophyllum hexandrum is an important medicinal plant whose genetic diversity must be conserved because it is endangered. The Qinling Mts. are a S. hexandrum distribution area that has unique environmental features that highly affect the evolution of the species. To provide the reference data for evolutionary and conservation studies, the genetic diversity and population structure of S. hexandrum in its overall natural distribution areas in the Qinling Mts. were investigated through inter-simple sequence repeats analysis of 32 natural populations. The 11 selected primers generated a total of 135 polymorphic bands. S. hexandrum genetic diversity was low within populations (average He = 0.0621), but higher at the species level (He = 0.1434). Clear structure and high genetic differentiation among populations were detected by using the unweighted pair group method for arithmetic averages, principle coordinate analysis and Bayesian clustering. The clustering approaches supported a division of the 32 populations into three major groups, for which analysis of molecular variance confirmed significant variation (63.27%) among populations. The genetic differentiation may have been attributed to the limited gene flow (Nm = 0.3587) in the species. Isolation by distance among populations was determined by comparing genetic distance versus geographic distance by using the Mantel test. Result was insignificant (r = 0.212, P = 0.287) at 0.05, showing that their spatial pattern and geographic locations are not correlated. Given the low within-population genetic diversity, high differentiation among populations and the increasing anthropogenic pressure on the species, in situ conservation measures were recommended to preserve S. hexandrum in Qinling Mts., and other populations must be sampled to retain as much genetic diversity of the species to achieve ex situ preservation as a supplement to in situ conservation.

[Studies on main interspecific association of rare and endangered medicinal plant Sinopodophyllum hexandrum community in Kangding Zheduo mountain of Sichuan province].

Based on the 2 x 2 contingency table, by using multi-species relevance (variance ratio, VR), chi2-test, Ochiai index, Dice index, Jaccard index, t-test of v/x and F-test of Morisita, s index, the interspecific relationships and the spatial distribution pattern between 20 dominants in Kangding Zheduo Mountain of Sichuan province were studied. The results indicated that the interspecific association between dominants of Sinopodophyllum hexandrum community in this area did not show significant association, which suggested that the S. hexandrum community was in mature stage, and showed stronger independency, among total 190 pairs in 20 dominant species, 2 species pairs exhibited extremely significantly positive association, 12 species pairs showed significantly positive association, 6 species pairs exhibited significantly negative association and there were no pairs showed extremely significantly negative association. S. hexandrum in community did not show significant association, which indicates they are independent in community, the spatial distribution pattern of S. hexandrum is characterized by random distribution.

[Genetic diversity and genetic structure of endangered wild Sinopodophyllum emodi by start codon targeted polymorphism].

OBJECTIVE: Revealed the genetic diversity level and genetic structure characteristics in Sinopodophyllum emodi, a rare and endangered species in China. METHOD: We detected the genetic polymorphism within and among six wild populations (45 individuals) by the approach of Start Codon Targeted (SCoT) Polymorphism. The associated genetic parameters were calculated by POP-GENE1.31 and the relationship was constructed based on UPGMA method. RESULT: A total of 350 bands were scored by 27 primers and 284 bands of them were polymorphic. The average polymorphic bands of each primer were 10.52. At species level, there was a high level of genetic diversity among six populations (PPB = 79.27%, N(e) = 1.332 7, H = 0.210 9 and H(sp) = 0.328 6). At population level, the genetic diversity level was low (PPB = 10.48% (4.00% -23.71%), N(e) = 1.048 7 (1.020 7-1.103 7), H = 0.029 7 (0.012 9-0.063 1), H(pop) = 0.046 2 (0.019 9-0.098 6). The Nei's coefficient of genetic differentiation was 0.841 1, which was consistent with the Shannon's coefficient of genetic differentiation (0.849 4). Two calculated methods all showed that most of the genetic variation existed among populations. The gene flow (N(m) = 0.094 4) was less among populations, indicating that the degree of genetic differentiation was higher. Genetic similarity coefficient were changed from 0.570 8 to 0.978 7. By clustering analysis, the tested populations were divided into two classes and had a tendency that the same geographical origin or material of similar habitats clustered into one group. CONCLUSION: The genetic diversity of samples of S. emodi is high,which laid a certain foundation for effective protection and improvement of germplasm resources.

[Identification on three kinds of Diphylleia sinensis (I)].

OBJECTIVE: In order to provide reference for the development of quality standards and distinction of authenticity, some sources of Diphylleia sinensis Li. in the market were identified by sampling. METHODS: To identify the traits and microscopic features of six batches of Diphylleia sinensis Li. in the market. RESULTS: They were the dried rhizome of Diphylleia sinensis Li., Dysosma versipellis (Hance) M. Cheng and D. pleiantha (Hance) Woods. CONCLUSION: The differences in traits and microscopic features of them are obvious.

[Identification on three kinds of Diphylleia sinensis (II)].

OBJECTIVE: In order to provide reference for the development of quality standards and distinction of authenticity, some sources of Diphylleia sinensis Li. in the maket were identified by sampling. METHODS: The TLC of six batches of Diphylleia sinensis Li. in the market were identified. RESULTS: They were the dried rhizome of Diphylleia sinensis Li., Dysosma versipellis (Hance.) M. Cheng and D. pleiantha (Hance) Woods. According to the pharmaceutical characteristics, retrieval tables of identification on plants, traits, microscopic and TLC were established. CONCLUSION: It is simple and reliable to use TLC and retrieval table to identify three sources of Diphylleia sinensis Li.

Phylogeny of flowering plants by the chloroplast genome sequences: in search of a "lucky gene".

One of the most complicated remaining problems of molecular-phylogenetic analysis is choosing an appropriate genome region. In an ideal case, such a region should have two specific properties: (i) results of analysis using this region should be similar to the results of multigene analysis using the maximal number of regions; (ii) this region should be arranged compactly and be significantly shorter than the multigene set. The second condition is necessary to facilitate sequencing and extension of taxons under analysis, the number of which is also crucial for molecular phylogenetic analysis. Such regions have been revealed for some groups of animals and have been designated as "lucky genes". We have carried out a computational experiment on analysis of 41 complete chloroplast genomes of flowering plants aimed at searching for a "lucky gene" for reconstruction of their phylogeny. It is shown that the phylogenetic tree inferred from a combination of translated nucleotide sequences of genes encoding subunits of plastid RNA polymerase is closest to the tree constructed using all protein coding sites of the chloroplast genome. The only node for which a contradiction is observed is unstable according to the different type analyses. For all the other genes or their combinations, the coincidence is significantly worse. The RNA polymerase genes are compactly arranged in the genome and are fourfold shorter than the total length of protein coding genes used for phylogenetic analysis. The combination of all necessary features makes this group of genes main candidates for the role of "lucky gene" in studying phylogeny of flowering plants.

AFLP analysis of genetic diversity of the endangered species Sinopodophyllum hexandrum in the Tibetan region of Sichuan Province, China.

Amplified fragment length polymorphism (AFLP) markers were used to estimate the genetic diversity of seven wild populations of Sinopodophyllum hexandrum (Royle) Ying from the Tibetan region of Sichuan Province, China. Six primer combinations generated a total of 428 discernible DNA fragments, of which 111 were polymorphic. The percentage of polymorphic bands (PPB) was 25.93 at the species level, and PPB within population ranged from 4.91 to 12.38%. Genetic diversity (H(E)) within populations varied from 0.01 to 0.04, averaging 0.05 at the species level. As revealed by the results of AMOVA analysis, 58.8% of the genetic differentiation occurred between populations, and 41.2% within populations. The genetic differentiation was, perhaps, due to the limited gene flow (Nm = 0.43) of the species. The correlation coefficient (r) between genetic and geographical distance using Mantel's test for all populations was 0.698 (P = 0.014). The UPGMA cluster analysis revealed a similar result in that the genetic distances among the populations show, to a certain extent, a spatial pattern corresponding to their geographic locations. On the basis of the genetic and ecological information, we propose some appropriate strategies for conserving the endangered S. hexandrum in this region.

Molecular identification of Sinopodophyllum hexandrum and Dysosma species using cpDNA sequences and PCR-RFLP markers.

Rhizomes of Sinopodophyllum hexandrum and Dysosma species, which have long been used in the traditional Chinese herbal medicine, have similar morphology and chemical composition. However, the podophyllotoxin content is higher in the rhizomes of S. hexandrum than in those of Dysosma species. The PCR-amplified fragments of trnT -trnL showed length variation between S. hexandrum and Dysosma species, and sequence comparison indicated that the length variation resulted from differential indels. There were species-specific PCR-RFLP markers of the chloroplast trnD -trnT region. Our results suggest that both chloroplast intergenic regions can be used for the identification of S. hexandrum and Dysosma Rhizoma medicines on the market.

[Novel application of FTIR in medical herb chemotaxonomy].

Typical medical plants in Araliaceae, Campanulaceae, Magnoliaceae, Lauraceae, Leguminosae, Berberidaceae, Pteridophyta, etc. were studied with FTIR for the first time, and the similarities and differences within each familiar were also pointed out. Furthermore the differences in spectra of samples from different parts or collected at different time on the same plant were also discussed. The characteristic radicals of the mainly effective components in plants were identified, and the primary peaks were deciphered. It was considered that FTIR could become a rapid, reliable, impersonal and effective method in chemotaxonomy as a supplement of morphologic plant taxonomy.

[The descriptions and microscopy identification of three allied species of Dysosma versipellis].

The descriptions, histological and microscopical characteristics of three allied species of Dysosma versipellis were described.