Phylogenetic analysis of Uncaria species based on internal transcribed spacer (ITS) region and ITS2 secondary structure.

CONTEXT: The plant genus Uncaria (Rubiaceae), also known as Gouteng, is the source of an important traditional Chinese medicine. Misidentification and adulteration of Gouteng affect the safety and efficacy of the medication. Phylogenetic relationships among the species of this genus are unknown. OBJECTIVE: The present study sought to detect the phylogenetic relationships based on internal transcribed spacer (ITS) region of all 12 species of Uncaria recorded in the Flora of China. MATERIALS AND METHODS: Accession of seven species of Uncaria served as reference samples. ITS region was used for polymerase chain reaction (PCR) amplification of the reference samples representing 39 specimens. Distance analysis, species discrimination, and secondary structure of ITS2 were used to assess the ability of ITS sequence in authenticating. The phylogenetic relationships were detected using three methods: Bayesian inference (BI), maximum likelihood (ML), and neighbor joining (NJ). RESULTS: Five species of traditional Chinese medicine Gouteng were well resolved in molecular phylogenetic tree. Besides, Uncaria lancifolia Hutch. was closer to U. rhynchophylloides F.C. How and U. sessilifructus Roxb. was closer to U. laevigata Wall. within the tree. Further, we also found that ITS2 secondary structure can be a candidate tool in distinguishing two closely related species U. yunnanensis K.C.Hsia and U. lanosa Wall. For accurate identification of different species of Uncaria based on species-specific nucleotide sites, a consensus sequences database with all 12 species is established. DISCUSSIONS AND CONCLUSIONS: The results are able to discriminate Uncaria species and illustrate the phylogenetic relationships, which are essential for the investigation of adulterants and misidentifications of Uncaria.

The effects of genetic variation and environmental factors on rhynchophylline and isorhynchophylline in Uncaria macrophylla Wall. from different populations in China.

Uncaria macrophylla Wall. is an important Chinese medicinal herb. Rhynchophylline (RIN) and isorhynchophylline (IRN) are its major active compounds. We investigated the influence of genetic differentiation and environmental factors on the RIN and IRN to find the main influencing factors of their contents and lay the foundation for the following cultivation and breeding. We used inter-simple sequence repeat (ISSR) markers to investigate the genetic diversity, and high-performance liquid chromatography (HPLC) to measure the contents of RIN and IRN in 200 samples of U. macrophylla obtained from nine natural populations, and then to analyze the correlation between genetic differentiation, environmental factors of sampling sites and the contents of RIN and IRN. We found that High intra-population (80.05%) and low inter-population (19.95%) genetic diversity existed in the samples of U. macrophylla. To some extent, genetic differentiation and the contents of RIN and IRN had correlation in individual populations (such as JH, MH, XM, and ML). The RIN and IRN contents were significant negatively correlated with the precipitation in May (RIRN = -0.771, p = 0.015) and June (RRIN = -0.814, p = 0.008; RIRN = -0.921, p = 0.000), indicating that precipitation was the main affecting factor of their contents. Interestingly, the analysis results showed that the RIN content had a significant positive correlation (r = 0.585, p = 0.000) with the IRN content (they are isomers); the proportion of RIN had a significant negative correlation with the sum of the two (r = -0.390, p<0.0001), while the proportion of IRN had a significant positive correlation (r = 0.390, p<0.0001). It meant that, with the total quantity of the two compounds increased, the proportion of RIN decreased and the proportion of IRN increased, illustrating that their conversion exist some regularity. Moreover, the content ratio of RIN and IRN was significant positively correlated with the January precipitation (r = 0.716, p = 0.030), implying that January may be the key period for the mutual transformation of RIN and IRN.

Characteristic component profiling and identification of different Uncaria species based on high-performance liquid chromatography-photodiode array detection tandem ion trap and time of flight mass spectrometry coupled with rDNA ITS sequence.

Uncaria is a multi-source herb and its species identification has become a bottleneck in quality control. To study the identification method of different Uncaria species herbs through HPLC-MS coupled with rDNA Internal Transcribed Spacer (rDNA ITS) sequence, both plant morphological traits and molecular identification were used to determine the species of every collected Uncaria herb. The genetic analysis of different Uncaria species was performed using their rDNA ITS sequence as a molecular marker. Meanwhile, the phylogenetic relationships of 22 samples from six Uncaria species were divided and classified clearly. By optimizing the chromatographic conditions, a practical HPLC method to differentiate various varieties of Uncaria herbs was set up based on a set of characteristic components across each species. A high-performance liquid chromatography-photodiode array detector tandem ion trap and time of flight mass spectrometry technique combined with reference substances was utilized to derive 21 characteristic compounds containing six groups of six Uncaria species in China. Thus, this study provides a feasible method to solve the current problem of confusion in Uncaria species, and makes a significant step forward in the appropriate clinical use, in-depth research and further utilization of different Uncaria species.

[Comparison of rattan microscopic characteristics of four Dai medicinal materials from Uncaria genus].

OBJECTIVE: To establish microscopic identification standards for four species from Uncaria genus. METHODS: The microscopic characteristics of transvers section and powder of these four species rattan were observed. RESULTS: As for these four species from Uncaria genus, the microscopic identification of rattan transvers section could be made by the existence of cork cambium and pericycle in the cortex, and the location of phloem fiber and stone cells, while the powder identification could be made according to the existence of scalariform vessel and calcium oxalate crystal, and the quantity of stone cells. CONCLUSION: The microscopic characteristics of rattan can provide information for identification of these four species from Uncaria genus.

[Molecular identification of one Uncaria plant].

OBJECTIVE: In order to identify a species of Uncaria, molecular phylogenetic analysis was carried out by using the rDNA ITS sequence as molecular marker. METHOD: Total DNA was extracted from the plant with modified CTAB method and thereby rDNA ITS regions were amplified with universally conserved primer. The rDNA ITS amplicon was characterized by cloning, sequencing, blasting in GenBank and phylogenetic analyses using PAUP by maximum parsimony (MP) criteria. RESULT: The rDNA ITS entire sequence of this species of Uncaria was 719 bp. The sequence is related to the U. sinensis available in GenBank and the similarity reaches 99.7%. CONCLUSION: Based on molecular biology methods of rDNA ITS region analysis, molecular identification is available in accurate classification on this species of Uncaria.

[Transmission spectral imaging research on folium uncariae from Guangdong].

OBJECTIVE: Build up Folium Uncariae spectral imaging characteristics of the transmission spectrum to provide a new effective method for the study of medicinal plants. METHODS: Determine five kinds of 10 populations in different parts of Folium Uncariae by electric controlled liquid crystal filter spectral imaging device. The highest spectral resolution of 0.5 nm. Spectral covergeg: 400 nm to 720 nm. Construct its fingerprint with cosine distance cluster analysis. RESULTS: Folium Uncariae spectral imaging characteristics of the transmission spectrum similarity analysis matches the systematic botany appraisal result. CONCLUSION: Spectral imaging analysis techniques can be used to construct fingerprints of traditioanal Chinese medicine, quality evaluation and study of plant phylogenetic relationships, etc., the operation method is simple, rapid.

[Molecular identification of medicinal plant genus Uncaria in Guizhou].

OBJECTIVE: To analyze rDNA ITS regions of the Medicinal Plant Genus Uncaria in Guizhou and construct their phylogenetic tree in order to supply molecular evidence of taxonomy and identification of these Medicinal Plants in genetic level. METHODS: The ITS gene fragments of the 4 Medicinal Plants were PCR amplified and sequenced. The rDNA ITS regions were analyzed by means of the software of ClustalX, BioEdit and PAUP* 4.0 beta 10. RESULTS: The entire sequences of rDNA ITS1, ITS2, and 5.8S rDNA were obtained, The Maximum-parsimony tree of four ITS regions together with those of similar sequences from GenBank were found, as Mitrayna rubrostipulata (AJ492621 ) and Mitragyna rubrostipulata (AJ605988) were designated as outgroup. CONCLUSION: The 4 medicinal plants are the 4 species in the genus Uncaria, and are mostly similar to the Uncaria rhynhcophylla.

[Recognize uncalia ganbir].

The Latin name for Jicha, which is used in bethel chewing, is Uncaria gambir. The plant belongs to the Rubiaceae family, and although its Chinese name is Jicha-Kou-tou, it is more commonly called Katsu-Jicha or Ho-Jicha. While Jicha is called Asen-yaku in Japan, the latter name dates back only to the 15th century. As it can be estimated that, in China, the use of Jicha in bethel chewing was known to occur around 200 AD, the author would like to recommend that the name Jicha be used for Asen-yaku in Chinese medical terminology, regardless of the origin of the drug Asen-yaku in Japan. This recommendation may be fortified as Prof. Sho Bai-kon, who co-edited Chugoku Honzo Zuroku (Atlas of Chinese Medicinal Herbs), a joint effort between Japan and China, has made clear that Jicha is indeed Acacia catechu.

[Survey on traditional medicinal resources of Uncaria distributed in China].

OBJECTIVE: To make clear the botanical origin of medicinal Uncaria distributed in China and the situation of its use in folk medicines. METHODS: Collecting data and making on-the-spot investigation. RESULTS: There are thirteen medicinal plants which belong to Uncaria genus, and ten of them are commercially available. A clear idea was obtained of their geographical distribution, distribution features and practical experiences in folk application. CONCLUSION: This investigation is helpful in further researching and exploring the resources of Uncaria for medical use, as well as provides a scientific basis for production, management and safe use of crude drugs.