LC-MS Metabolomics and Chemotaxonomy of Caffeine-Containing Holly ( Ilex) Species and Related Taxa in the Aquifoliaceae.

Ilex species have been consumed traditionally as medicinal teas worldwide. Though the presence of caffeine has been reported in several species, little is known about secondary-metabolite diversity within and among these taxa. Leaf samples of Ilex guayusa, Ilex paraguariensis, and Ilex vomitoria were analyzed by liquid chromatography-mass spectrometry and comparative metabolite profiling with Ilex cassine and other Ilex species to identify chemotaxonomic markers, delimit species, and provide an assessment of chemodiversity. Purine alkaloids were detected and quantified in I. guayusa, I. paraguariensis, and I. vomitoria. Reports of caffeine for I. cassine were not corroborated, suggesting that I. vomitoria was the traditional source of the Native North American tea yaupon. The tetramethyluric acid, theacrine, was detected for the first time in the genus Ilex as a low-level chemotaxonomic marker in I. vomitoria samples. Chemotaxonomy and metabolomics support a close relationship for caffeine-containing Ilex species.

Chloroplast genome structure in Ilex (Aquifoliaceae).

Aquifoliaceae is the largest family in the campanulid order Aquifoliales. It consists of a single genus, Ilex, the hollies, which is the largest woody dioecious genus in the angiosperms. Most species are in East Asia or South America. The taxonomy and evolutionary history remain unclear due to the lack of a robust species-level phylogeny. We produced the first complete chloroplast genomes in this family, including seven Ilex species, by Illumina sequencing of long-range PCR products and subsequent reference-guided de novo assembly. These genomes have a typical bicyclic structure with a conserved genome arrangement and moderate divergence. The total length is 157,741 bp and there is one large single-copy region (LSC) with 87,109 bp, one small single-copy with 18,436 bp, and a pair of inverted repeat regions (IR) with 52,196 bp. A total of 144 genes were identified, including 96 protein-coding genes, 40 tRNA and 8 rRNA. Thirty-four repetitive sequences were identified in Ilex pubescens, with lengths >14 bp and identity >90%, and 11 divergence hotspot regions that could be targeted for phylogenetic markers. This study will contribute to improved resolution of deep branches of the Ilex phylogeny and facilitate identification of Ilex species.

Molecular Evidence for the Hybrid Origin of Ilex dabieshanensis (Aquifoliaceae).

Ilex, the largest genus of dioecious woody plants, is a good study system to assess the role of hybridization in speciation and evolution. Ilex dabieshanensis, a tree endemic to Dabieshan Mountains region, was initially described as a new species. Based on morphological intermediacy and sympatric distribution with its putative parental species, I. cornuta and I. latifolia, we proposed it as a natural hybrid between them. In this study, we sequenced one chloroplast intergenic spacer (trnH-psbA) and two nuclear genes (gapC and nepGS) in I. dabieshanensis and its putative parental species to test the hybrid origin hypothesis. Our results showed that there were one to two differentially fixed sequence differences between I. cornuta and I. latifolia at the two nuclear genes. Twelve of the 14 individuals of I. dabieshanensis exhibited additivity in chromatograms on these differentially fixed sites at both nuclear genes, and the remaining two exhibited additivity in chromatograms on the fixed site at only the nepGS gene. Except one haplotype of I. cornuta at the nepGS gene, all of the haplotypes of I. cornuta at the two nuclear genes were well separated from those of I. latifolia, and most haplotypes of I. dabieshanensis were shared with those of I. cornuta and I. latifolia. Phylogenetic analysis of these haplotypes was largely consistent with haplotype network analysis. I. cornuta and I. latifolia differed by two nucleotide substitutions in the chloroplast intergenic spacer, and 12 individuals of I. dabieshanensis had the same sequences as I. latifolia, while the remaining two were identical with I. cornuta. The molecular data provide convincing evidence for the hybrid origin of I. dabieshanensis and asymmetrical direction of hybridization. One haplotype of I. cornuta at the nepGS gene was nested with those of I. latifolia, indicating introgression to I. cornuta.

The large-leaved Kudingcha (Ilex latifolia Thunb and Ilex kudingcha C.J. Tseng): a traditional Chinese tea with plentiful secondary metabolites and potential biological activities.

In China, Kudingcha has been used for almost 2,000 years as a tea to quench thirst, remove phlegm, refresh the mind, and improve eyesight. The group of large-leaved Kudingcha is coveted for its potential effects on lipid metabolism, which are attributed to the presence of characteristic ingredients. This contribution reviews studies from the past few decades regarding the plant characteristics, ethnobotanical usages, chemical constituents, and related biological activities of the large-leaved Kudingcha (Ilex latifolia Thunb and Ilex kudingcha C.J. Tseng). Triterpenoids, phenolic acids, flavonoids, and essential oils are the main metabolites in the large-leaved Kudingcha, and these ingredients protect the vascular system, regulate lipid metabolism, and have antioxidant, hypoglycemic, and anti-tumor effects. Moreover, large-leaved Kudingcha shares several properties with the popular green tea and the Yerba maté from South America.

[Studies of genetic diversity among 10 species of Ilex based on RAPD and AFLPs].

The techniques of random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphisms (AFLPs) were used to amplify the genomic DNA of 10 species of Ilex, so as to reveal the polymorphism in different species of Ilex. In RAPD analysis, a total of 301 lines were amplified with 11 kinds of primers selected from 100 kinds of 10 bp random primers, and the polymorphism was 98.63%. In AFLPs analysis,a huge diversity was showed by 3 primers combinations carried out on these species. Both RAPD and AFLPs analysis were clustered by UPGMA and the results showed that the genetic distances were the closest between I. purpurea and I. vomitoria Ait, I. Litseaefolia and I. zhejiangensis, I. hylonoma var. glabra and I. triflora var. kanehirai.

Molecular evidence of host-associated genetic divergence in the holly leafminer Phytomyza glabricola (Diptera: Agromyzidae): apparent discordance among marker systems.

Host races play a central part in understanding the role of host plant mediated divergence and speciation of phytophagous insects. Of greatest interest are host-associated populations that have recently diverged; however, finding genetic evidence for very recent divergences is difficult because initially only a few loci are expected to evolve diagnostic differences. The holly leafminer Phytomyza glabricola feeds on two hollies, Ilex glabra and I. coriacea, that are broadly sympatric throughout most of their ranges. The leafminer is often present on both host plants and exhibits a dramatic life history difference on the two hosts, suggesting that host races may be present. We collected 1393 bp of mitochondrial cytochrome oxidase I (COI) sequence and amplified fragment length polymorphism (AFLP) data (45 polymorphic bands) from sympatric populations of flies reared from the two hosts. Phylogenetic and frequency analysis of mitochondrial COI sequence data uncovered considerable variation but no structuring by the host plant, and only limited differentiation among geographical locations. In contrast, analysis of AFLP frequency data found a significant effect with host plant, and a much smaller effect with geographical location. Likewise, neighbour-joining analysis of AFLP data resulted in clustering by host plant. The AFLP data indicate that P. glabricola is most likely comprised of two host races. Because there were no fixed differences in mitochondrial or AFLP data, this host-associated divergence is likely to have occurred very recently. P. glabricola therefore provides a new sympatric system for exploring the role of geography and ecological specialization in the speciation of phytophagous insects.

Classification of Ilex species based on metabolomic fingerprinting using nuclear magnetic resonance and multivariate data analysis.

The metabolomic analysis of 11 Ilex species, I. argentina, I. brasiliensis, I. brevicuspis, I. dumosavar. dumosa, I. dumosa var. guaranina, I. integerrima, I. microdonta, I. paraguariensis var. paraguariensis, I. pseudobuxus, I. taubertiana, and I. theezans, was carried out by NMR spectroscopy and multivariate data analysis. The analysis using principal component analysis and classification of the (1)H NMR spectra showed a clear discrimination of those samples based on the metabolites present in the organic and aqueous fractions. The major metabolites that contribute to the discrimination are arbutin, caffeine, phenylpropanoids, and theobromine. Among those metabolites, arbutin, which has not been reported yet as a constituent of Ilex species, was found to be a biomarker for I. argentina,I. brasiliensis, I. brevicuspis, I. integerrima, I. microdonta, I. pseudobuxus, I. taubertiana, and I. theezans. This reliable method based on the determination of a large number of metabolites makes the chemotaxonomical analysis of Ilex species possible.

[Pharmacological studies of trilex on treatment of pharyngitis].

Trilex consisted of three species: Ilex latifolia, Ilex asprella and Ilex rotunda. Trilex had the antipyretic effects, decreased the rat body temperature about 0.8 degree C on yeast induced hyperthermia. Trilex could obviously enhance the threshold of hot plate induced pain in mice. The maximum threshold increased 43.8%. Trilex could also inhibit acetic acid induced inflammatory ooze in mice abdominal cavity. The inhibitory rate was 34.8%. These results showed that trilex had antipyretic, analgesic and anti-inflammatory effects. It could be used to treat acute and chromic pharyngitis.

[Electron microscope observation on effect of kudingcha inspissation tea on small intestine villus in the adiposity rats].

OBJECTIVE: Experimental study on pharmacological action of Guang Dong kudingcha inspissation tea on small intestine villus in the adiposity rats (nutrition obesity). METHODS: By using electron microscope method, check on small intestine villus of 60 experiment rats of just wean and count and analyse and conclude. RESULTS: Under the scan electron imcroscope, the surface configuration on small intestine villus of model group and various kudingcha dosage groups is similar to the blank (P > 0.05), but fenfluramine group appear constriction on top end of small intestine villus. CONCLUSION: Compring with fenfluramine, Guang Dong kudingcha inspissation tea has not effect on configuration of small intestine of adiposity rats (nutrition obesity), but has more strong modulation function on fat tissue lipocyte hypertrophy and quantitative.

[Chemical pattern recognition of traditional Chinese medicine kudingcha (I)].

In this paper, the non-linear mapping method of pattern recognition was adopted to classify 78 samples of traditional Chinese medicine Kudingcha, with macro and trace elements as classified characteristic features. Ilex cornuta Lindl., Ilex latifolia Thunb. and Ligustrum lucidum Ait. were identified accurately. The results agree with those from pharmacognosy. This paper provides a new method for identification of traditional Chinese medicine.

[Chemical pattern recognition of traditional Chinese medicine kudingcha (II)].

In this paper, the HPLC data from 78 samples of Kudingcha were treated with back propagation algorithm of artifical neural network pattern recognition, and the computer-aided classification of Ilex cornuta Lindl., Ilex latifolia Thunb. and Ligustrum lucidum Ait. was accomplished. This paper provides a scientific, advanced and feasible method for identification of traditional Chinese medicine.