Molecular identification of the traditional herbal medicines, Arisaematis Rhizoma and Pinelliae Tuber, and common adulterants via universal DNA barcode sequences.

Methods to identify Pinelliae Tuber and Arisaematis Rhizoma are required because of frequent reciprocal substitution between these two herbal medicines and the existence of several closely related plant materials. As a result of the morphological similarity of dried tubers, correct discrimination of authentic herbal medicines is difficult by conventional methods. Therefore, we analyzed DNA barcode sequences to identify each herbal medicine and the common adulterants at a species level. To verify the identity of these herbal medicines, we collected five authentic species (Pinellia ternata for Pinelliae Tuber, and Arisaema amurense, A. amurense var. serratum, A. erubescens, and A. heterophyllum for Arisaematis Rhizoma) and six common adulterant plant species. Maturase K (matK) and ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) genes were then amplified using universal primers. In comparative analyses of two DNA barcode sequences, we obtained 45 species-specific nucleotides sufficient to identify each species (except A. erubescens with matK) and 28 marker nucleotides for each species (except P. pedatisecta with rbcL). Sequence differences at corresponding positions of the two combined DNA barcodes provided genetic marker nucleotides that could be used to identify specimens of the correct species among the analyzed medicinal plants. Furthermore, we generated a phylogenetic tree showing nine distinct groups depending on the species. These results can be used to authenticate Pinelliae Tuber and Arisaematis Rhizoma from their adulterants and to identify each species. Thus, comparative analyses of plant DNA barcode sequences identified useful genetic markers for the authentication of Pinelliae Tuber and Arisaematis Rhizoma from several adulterant herbal materials.

[Analysis of genetic diversity of gemplasm of Pinellia ternata based on SRAP].

OBJECTIVE: To assess population genetic diversity of Pinellia ternata with different phenotype and from different habitat by sequence-related amplified polymorphism (SRAP) technique. METHOD: Fourteen appropriate primer pairs were selected out from a total of 80 pairs for SRAP PCR amplification. A Jaccard's genetic similarity matrix and a dendrogram were established using SPSS 16.0 software. RESULT: The 14 primer pairs could amplify 171 bands, and the percentage of polymorphic bands was 15.8%. Based on the dendrogram, P. ternata in the same habitat clustered in a clade. CONCLUSION: These results suggested that SRAP markers could be used as an effective molecular technique for the diversity study of P. ternata and the habitat was more important than the phenotype in identification of P. ternata germplasm resource.

[RAPD analysis and construction of specific DNA probes between Pinellia ternata and Typhonium flagelliforme].

OBJECTIVE: To establish a molecular marking method to identify Pinellia ternata and Typhonium flagelliforme. METHODS: Twenty-two random oligonucleotide primers were used in RAPD analysis on the genomic DNA of two types of Pinellia ternata in Sichuan and two types of Typhonium flagelliforme in Guangxi. The special fragments were sequenced, marked as probes and then conducted Southern blot. RESULTS: A great deal of special bands was found between Pinellia ternata and Typhonium flagelliforme. A Pinellia ternata specific molecule was screened. CONCLUSION: RAPD analysis and specific DNA probes show potential value in the identification of Pinellia ternata and Typhonium flagelliforme.

[Comparative study on main economic characters of different populations of Pinellia ternata in China].

OBJECTIVE: To analyze the content of guanosine, total alkaloid and individual yield of Pinellia ternata from different populations in China and evaluate its quality. METHOD: Reversed-phase high-performance liquid chromatography (HPLC) was used to determine the content of guanosine. The content of alkaloid was determined by ultra violet spectrophometry. The results were analyzed by SPSS software. RESULT: The contents of guanosine and total alkaloid in P. ternata were 0.0136% -0.0264% and 0.0155% -0.0652% respectively. Individual yield was 0.5536-2.9740 g. All of the populations could be classified into 3 types through hierarchical cluster analysis. CONCLUSION: There exist significant differences in the content of guanosine, total content of alkaloid and individual yield of P. ternata from different populations. It is suggested that breeding and selection for type II of P. ternata should be strengthened.

The complete chloroplast genome sequence of medicinal plant Pinellia ternata.

Pinellia ternata is an important medicinal plant used in the treatment of cough, to dispel phlegm, to calm vomiting and to terminate early pregnancy, as an anti-ulcer and anti-tumor medicine. In this study, we found that the complete chloroplast genome of Pinellia ternata was 164 013 bp in length, containing a pair of inverted repeats of 25 625 bp separated by a large single-copy region and a small single-copy region of 89 783 bp and 22 980 bp, respectively. The chloroplast genome encodes 132 predicted functional genes, including 87 protein-coding genes, eight ribosomal RNA genes, and 37 transfer RNA genes. The chloroplast DNA is GC-rich (36.7%). The phylogenetic analysis showed a strong sister relationship with Colocasia esculenta, which also strongly supports the position of Pinellia ternata. The complete chloroplast genome sequence of Pinellia ternata reported here has the potential to advance population and phylogenetic studies of this medicinal plant.

[Study on growth rhythm in populations of Pinellia ternata (Thunb.) Breit].

OBJECTIVE: To study the genetic diversity of Pinellia ternata. the growth rhythm. METHOD: Sixteen populations of P. ternata. originated mostly from middle and lower reaches of Yangtze River valley were collected and cultivated in Weigang Experimental Farm of Nanjing Agricultural University with same cultural conditions, whose differences in growth rhythm have been observed and compared continuously for two years. RESULT AND CONCLUSION: There are obvious differences in sprouting, lamina spreading, bolting and "sprout tumble", Three times of "sprouting" and so-called "sprout tumble" and two fast-growing periods for plants in most of the populations in spring and autumn respectively were observed within a year, including mass bolting in May. But the starting time and duration are very different from population to population, with flowering being prior to sprouting in the population originated from Jurong county.

[Contents of total flavonoids in Rhizoma Arisaematis].

OBJECTIVE: Comparing the contents of total flavonoides of Rhizoma Arisaematis, which collected in different time, regions, different varieties and processed. METHOD: Determining the contents by ultraviolet spectro-photometry. RESULT AND CONCLUSION: The contents were found in the following sequence: 1. the end of July, the begin of July, August, September; 2. Beijing, Shanxi, Sichuan, Anhui; 3. Arisaema erubenscens, A. heterophyllum, A. amurense; 4. unprocessed product, processed product.

Authentication of Pinellia ternata and its adulterants based on PCR with specific primers.

Tubers of Pinellia ternata are one of the well known traditional Chinese medicines. According to the Chinese Pharmacopoeia, the remedy is commonly used as an antitussive and expectorant. The shapes of young tubers from species of P. ternata are similar to those of P. pedatisecta and Arisaema heterophyllum, but different in medicinal properties. In order to provide molecular evidence for genuine origin identification of P. ternata species, the mannose-binding lectin sequences of P. ternata and its adulterants P. pedatisecta and A. heterophyllum were cloned using genomic walker technology. Based on the sequence analyses, we designed a pair of species-specific primers to authenticate P. ternata. For PCR-selective restriction (PCR-SR), we identified two distinctive sites which can be recognized by the restriction endonucleases BAMHI and NCOI in the open reading frame sequences of P. ternata, P. pedatisecta and A. heterophyllum. Our results indicate that the methods of PCR and PCR-SR are effective, accurate and applicable for identification of the bulbs of P. ternata.

Determination of the site of origin of Pinellia ternata roots based on RAPD analysis and PCR-RFLP.

Analyses of randomly amplified polymorphic DNA (RAPD) and PCR-restriction fragment length polymorphism (PCR-RFLP) were performed in an effort to distinguish between two different origins of Pinellia ternata. To determine whether the origin of Pinellia ternata is in China or Korea. RAPD analysis was carried out using ten 20-mer random primers. Although the coefficients of similarity between the DNA of Chinese and Korean accessions of Pinellia ternata were high, distinguishable band patterns were observed in the reaction performed using primer numbers 3, 6 and 10. Primer 3 produced one (410 bp) and primer 6 produced four (410 bp, 350 bp, 300 bp, 250 bp) Chinese Pinellia-specific fragments. Primer 10 produced one (900 bp) Korean Pinellia rhizome-specific fragment. In addition, using PCR-RFLP analysis, different fingerprints were obtained from Korean and Chinese Pinellia ternata respectively. These results suggest that the analyses with RAPD and PCR-RFLP can be used to authenticate the relevant Chinese and Korean herbal medicines.