Selection for High Oridonin Yield in the Chinese Medicinal Plant Isodon (Lamiaceae) Using a Combined Phylogenetics and Population Genetics Approach

Oridonin is a diterpenoid with anti-cancer activity that occurs in the Chinese medicinal plant Isodon rubescens and some related species. While the bioactivity of oridonin has been well studied, the extent of natural variation in the production of this compound is poorly known. This study characterizes natural variation in oridonin production in order to guide selection of populations of Isodon with highest oridonin yield. Different populations of I. rubescens and related species were collected in China, and their offspring were grown in a greenhouse. Samples were examined for oridonin content, genotyped using 11 microsatellites, and representatives were sequenced for three phylogenetic markers (ITS, rps16, trnL-trnF). Oridonin production was mapped on a molecular phylogeny of the genus Isodon using samples from each population as well as previously published Genbank sequences. Oridonin has been reported in 12 out of 74 species of Isodon examined for diterpenoids, and the phylogeny indicates that oridonin production has arisen at least three times in the genus. Oridonin production was surprisingly consistent between wild-collected parents and greenhouse-grown offspring, despite evidence of gene flow between oridonin-producing and non-producing populations of Isodon. Additionally, microsatellite genetic distance between individuals was significantly correlated with chemical distance in both parents and offspring. Neither heritability nor correlation with genetic distance were significant when the comparison was restricted to only populations of I. rubescens, but this result should be corroborated using additional samples. Based on these results, future screening of Isodon populations for oridonin yield should initially prioritize a broad survey of all species known to produce oridonin, rather than focusing on multiple populations of one species, such as I. rubescens. Of the samples examined here, I. rubescens or I. japonicus from Henan province would provide the best source of oridonin

Comprehensive quality evaluation of Chishao by HPLC

Objective: The purpose of this paper is to comprehensively evaluate the quality of Chishao. Methods: In the experiment of this paper, the fingerprint spectrums of Chishao in all locations are established by RP-HPLC and the model of principle component analysis with the RP-HPLC peak areas is established. Results: The quality of Chishao in the northern part of China or that made of Paeonia lactiflora is better than that of these in others or that made of other species. The quality of Chishao comes from P. veitchii is in the middle class and is better than those that comes from P. obovata, P. mairei and P. anomala. The results are consistent with traditional views of the quality of this plant. These results indicates that principal component analysis (PCA) can be used as an effective and economic method to evaluate the quality of Chishao, and may be extended to other Chinese medicinal plants. Conclusions: Due to the complex basis of the efficacy of Traditional Chinese Medicine (TCM), the method such as PCA of several chemical components appears to be a more appropriate method for the quality evaluation of TCM in contrast to the determination of a single or few chemicals

Heritability of oridonin production.

<p>Heritability of oridonin production.</p

Information for <i>Isodon</i> populations collected in this study.

<p>Details include population name (species name with location code), location information, as well as the number of wild-collected parents and greenhouse-grown offspring from each population that were microsatellite genotyped and tested for oridonin content. Location codes are used throughout the text.</p

Boxplot diagram of oridonin yield in parents and offspring.

<p>Populations are indicated by a two-letter species code (en = <i>enanderianus</i>; he = <i>henryi</i>; ja = <i>japonicus</i>; lo = <i>lophanthoides</i>; ru = <i>rubescens</i>) and a two-letter location code as provided in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050753#pone-0050753-t001" target="_blank">Table 1</a>. The dashed line indicates the minimum required quantity of oridonin in the herb <i>donglingcao</i> (0.25% dry wt.), as specified in the 2010 edition of the Pharmacopoeia of the People’s Republic of China <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050753#pone.0050753-Chinese2" target="_blank">[19]</a>.</p

Presence of oridonin when mapped onto a molecular phylogeny of <i>Isodon</i>.

<p>Phylograms represent strict consensus of most parsimonious (MP) trees, using sequence data from the chloroplast (<i>rps16</i>, <i>trnL-trnF</i>; strict consensus of 140 MP trees) and nucleus (ITS; strict consensus of 390 MP trees). Presence of oridonin is indicated by dark colored branches. Taxa reported polymorphic for oridonin production (some populations produce oridonin, others do not) are shown with an asterisk. Samples newly sequenced for this study are shown with gray shading. Parsimony bootstrap (BS) values are shown above branches, Bayesian posterior probabilities (PP) are shown below branches. Support values are shown only for branches with BS>75. Outgroup taxon (<i>C. xanthanthus</i>) used for ancestral state construction not shown.</p

Chemical structure of oridonin.

<p>Chemical structure of oridonin.</p

Genetic variability of <i>Isodon</i> averaged across 11 microsatellite loci.

<p>For each population, the mean value and, in parentheses, standard error for sample size (<i>N</i>), number of alleles (<i>N_A</i>), observed heterozygosity (<i>H_O</i>), expected heterozygosity (<i>H_E</i>), and fixation index (<i>F</i>) is shown for parental microsatellite data. The maternal inbreeding coefficient (<i>F_mat</i>) and multi-locus population out-crossing rate (<i>T_m</i>) are shown based on the combined parental and offspring microsatellite data in the program MLTR.</p