Isodon Species Research Articles

Three new species of Isodon (Nepetoideae, Lamiaceae) from China.

Three new species of Isodon (Lamiaceae) from China are described and illustrated, based on both morphological evidence and our recent phylogenomic studies of the genus. Isodonattenuatus, a herbaceous new species known only from the Fanjing Mountain, is shown to be sister to I.villosus, but they can be easily distinguished by leaf and inflorescence indumentum, calyx teeth shape and corolla tube morphology. Isodongongshanensis, a herbaceous new species collected from the Hengduan Mountains in southwest China, represents a distinct lineage within the genus. Isodonsukungii, a shrubby new species also endemic to the Hengduan Mountains, was previously misidentified as I.tenuifolius, but they are phylogenetically distantly related and differ in lamina size and margin, inflorescence type and corolla length and shape.

Comparative analysis of medicinal plant Isodon rubescens and its common adulterants based on chloroplast genome sequencing.

Isodon rubescens (Hemsley) H. Hara is the source of Donglingcao under the monograph Rabdosiae Rubescentis Herba in Chinese Pharmacopoeia. In the local marketplace, this medicine can be accidentally contaminated, deliberately substituted, or mixed with other related species. The contaminants of herbal products are a threat to consumer safety. Due to the scarcity of genetic information on Isodon plants, more molecular markers are needed to avoid misidentification. In the present study, the complete chloroplast (cp) genome of seven species of Isodon was sequenced, de novo assembled and characterized. The cp genomes of these species universally exhibited a conserved quadripartite structure, i.e., two inverted repeats (IRs) containing most of the ribosomal RNA genes and two unique regions (large single copy and small single copy). Moreover, the genome structure, codon usage, and repeat sequences were highly conserved and showed similarities among the seven species. Five highly variable regions (trnS-GCU-trnT-CGU, atpH-atpI, trnE-UUC-trnT-GGU, ndhC-trnM-CAU, and rps15-ycf1) might be potential molecular markers for identifying I. rubescens and its contaminants. These findings provide valuable information for further species identification, evolution, and phylogenetic research of Isodon.

Characterization of the complete chloroplast genome sequence of Isodon japonicus (N. Burman) H. Hara (Lamiaceae)

Isodon japonicus (N. Burman) H. Hara (family: Lamiaceae), is a traditional herbal plant from Henan Province, China. In this study, we sequenced the complete chloroplast genome of I. japonicus sampled from the Funiu Mountains, Henan Province, China. The total length of the chloroplast genome is 152,298 bp, and has a typical quadripartite structure, including a large single-copy region (LSC) of 83,184 bp, a small single-copy region (SSC) of 17,806 bp and a pair of inverted repeats of 25,654 bp. The chloroplast genome of I. japonicus consists of 133 genes, including 88 protein-coding genes, eight rRNA genes, and 37 tRNA genes. There are 34 tandem repeats and 50 simple sequence repeats (SSRs) in the chloroplast genome. The total guanine-cytosine (GC) content of the whole chloroplast genome is 37.6%. Phylogenetic analysis indicated that I. japonicus forms a clade with six other Isodon species and is closely associated with I. rubescens.

Comparative analysis of chloroplast genomes reveals phylogenetic relationships and intraspecific variation in the medicinal plant Isodon rubescens.

Isodon rubescens (Hemsley) H. Hara (Lamiaceae) is a traditional Chinese medicine plant that has been used to treat various human diseases and conditions such as inflammation, respiratory and gastrointestinal bacterial infections, and malignant tumors. However, the contents of the main active components of I. rubescens from different origins differ significantly, which greatly affected its quality. Therefore, a molecular method to identify and classify I. rubescens is needed. Here, we report the DNA sequence of the chloroplast genome of I. rubescens collected from Lushan, Henan province. The genome is 152,642 bp in length and has a conserved structure that includes a pair of IR regions (25,726 bp), a LSC region (83,527 bp) and a SSC region (17,663 bp). The chloroplast genome contains 113 unique genes, four rRNA genes, 30 tRNA genes, and 79 protein-coding genes, 23 of which contain introns. The protein-coding genes account for a total of 24,412 codons, and most of them are A/T biased usage. We identified 32 simple sequence repeats (SSRs) and 48 long repeats. Furthermore, we developed valuable chloroplast molecular resources by comparing chloroplast genomes from three Isodon species, and both mVISTA and DnaSP analyses showed that rps16-trnQ, trnS-trnG, and ndhC-trnM are candidate regions that will allow the identification of intraspecific differences within I. rubescens. Also 14 candidate fragments can be used to identify interspecific differences between species in Isodon. A phylogenetic analysis of the complete chloroplast genomes of 24 species in subfamily Nepetoideae was performed using the maximum likelihood method, and shows that I. rubescens clustered closer to I. serra than I. lophanthoides. Interestingly, our analysis showed that I. rubescens (MW018469.1) from Xianyang, Shaanxi Province (IR-X), is closer to I. serra than to the other two I. rubescens accessions. These results strongly indicate that intraspecific diversity is present in I. rubescens. Therefore, our results provide further insight into the phylogenetic relationships and interspecific diversity of species in the genus Isodon.

Discovery of ent-kaurane diterpenoids, characteristic metabolites of Isodon species, from an endophytic fungal strain Geopyxis sp. XY93 inhabiting Isodon parvifolia

Isogeopyxins A–C (1–3), three new diterpenoids with ent-kaurane, ent-pimarane, and ent-abietane scaffolds, respectively, along with six known ent-kauranoids, were isolated from the fermentation culture of Geopyxis sp. XY93 inhabiting the leaves of Isodon parvifolia. Their structures were elucidated by interpretation of spectroscopic data, and single crystal X-ray diffraction. It marks the first time that ent-kauranoids, characteristic metabolites of Isodon species, have been isolated from an associated endophytic fungus.

High-content screening of diterpenoids from Isodon species as autophagy modulators and the functional study of their antiviral activities.

Autophagy is a conserved lysosomal degradation process, and abnormal autophagy has been associated with various pathological processes, e.g., neurodegeneration, cancer, and pathogen infection. Small chemical modulators of autophagy show the potential to treat autophagy-associated diseases. Diterpenoids, nature products found in various plants, exhibit a wide range of bioactivity, and we have recently isolated and characterized over 150 diterpenoids from Isodon species distributed in China. Here, we applied a high-content fluorescence imaging-based assay to assess these diterpenoids' ability to affect autophagic flux in HeLa cells. We found that enanderinanin J, an ent-kauranoid dimer, is an autophagy inhibitor, manifested by its ability to increase lysosomal pH and inhibit the fusion between autophagosomes and lysosomes. Autophagy has been shown to be either positively or negatively involved in the life cycle of Zika virus (ZIKV), Japanese encephalitis virus (JEV), Dengue virus (DENV), and enterovirus-A71 (EV-A71). We found that enanderinanin J significantly inhibited the infection of ZIKV, DENV, JEV, or EV-A71. Interestingly, although ATG5 knockdown inhibited ZIKV or JEV infection, enanderinanin J further inhibited the infection of ZIKV or JEV in ATG5-knockdown cells. Taken together, our data indicate that enanderinanin J inhibits autophagosome-lysosome fusion and is a potential antiviral agent.

Molecular signature of nrDNA-ITS marker in Isodon rugosus (Lamiaceae)

Molecular features of nrDNA-ITS sequences in medicinal plant Isodon rugosus were assessed with universal ITS-1 and ITS-2 primers. ITS sequences of 46 Isodon species were defined for their respective ITS-1, 5.8S and ITS-2 regions through in-silico datamining and analysis. Only in 32 species, sequence of all the three defined regions was complete, while in the rest of 14 species was partial. Isodon rugosus revealed five genic regions, namely, 18S rRNA gene (partial sequence, > 1…..38), ITS-1 (complete sequence, > 39…..236), 5.8S rRNA gene (complete sequence, > 237…..400), ITS-2 (complete sequence, > 401…..607) and large subunit 26S rRNA gene (partial sequence, > 608…..672). The sequence regions were recorded with variable sites (VS), singleton (ST) and species-specific parsimonious-informative (PI) sites. The information was also collected in a phylogenetic tree obtained using the maximum likelihood (ML) method.

Molecular and morphological evidence for a new species of Isodon (Lamiaceae) from southern China

Isodon brevipedunculatus, a new species from southern China, is described and illustrated. The phylogenetic position of the new species within the genus was analyzed based on two nuclear ribosomal DNA regions and an ingroup sampling of about 80% of Asian species of Isodon. The results show that I. brevipedunculatus is recovered in a clade that consists of species mainly with glandular mericarps and that are distributed in the Sino-Japanese region. Combining molecular and geographical evidence, our study reveals that I. brevipedunculatus is most closely related to Isodon amethystoides and Isodon bifidocalyx, but differs from the former in lamina shape, number of flowers per cyme, and peduncle length, and from the latter in lamina indumentum, calyx morphology, and corolla length.

Molecular Autograph of Maturase-K Gene in Isodon rugosus (Lamiaceae)

AbstractMolecular autograph of trn/mat-K gene was used as one of the important candidate marker in addressing the questions of systematics and barcoding of medicinal plants. Features of trn/mat-K gene in Isodon rugosus (Lamiaceae) were assessed for the first time (NCBI GenBank Accession Number: MH939199.1). Sequence of 756 bp length was amplified by the universal matK primers (matK472F and matK1248R) in the cpDNA of the plant. It was reported to contain trnK gene (>1…..50; >746…..756), partial sequence; and matK gene (>51…..745), partial coding DNA sequence. Alignment search and analysis showed that only nine different Isodon species are currently available for matK sequences with repeated copy of submissions in GenBank. The matK sequences of I. rugosus was reported with 34.2 (G + C)% and 17 variable sites (VS), out of which seven were singleton (ST) and 10 sites were species-specific parsimoniously-informative (PI) that could be used to differentiate I. rugosus from other species as well as to authenticate the taxon. Phylogenetic analysis resulted into monophyletic clustering of I. rugosus near to the clade having I. coetsa by both maximum likelihood (ML) and maximum parsimony (MP) methods. Clades obtained in ML tree were more informative as compared to MP tree.

Isodon hsiwenii (Lamiaceae: Nepetoideae), A New Species From Yunnan, China

Abstract—Isodon hsiwenii, a new species from Yunnan in Southwest China, is here described and illustrated. In order to clarify the phylogenetic placement of the new species within Isodon, Bayesian and maximum likelihood analyses based on two nuclear ribosomal DNA regions and four plastid DNA regions for 71 Asian Isodon species were conducted. The results indicate that I. hsiwenii is a member of the largest and poorly resolved Clade III, as sister species of I. hirtellus. Isodon hsiwenii can be distinguished from I. hirtellus by various morphological differences.

Leaf shape deters plant processing by an herbivorous weevil.

The shapes of plant leaves are remarkably diverse, but their ecological functions are largely unknown. Reports on the effects of leaf shape on biotic interactions such as herbivory are especially scarce, partly because herbivorous insects rarely rely on leaf shape for host selection. Here, we show that leaf shape acts as a physical deterrent against a leaf-processing herbivore. Plants in the genus Isodon (Lamiaceae) host a specialized leaf-rolling weevil (Apoderus praecellens) whose ovipositing females process an entire leaf into a leaf roll to serve as larval food and shelter. Among the species of Isodon, I. umbrosus var. hakusanensis is exceptional in that it has deeply lobed leaves. Because leaf processing follows a consistent sequence of complex behaviours, the unusual shape of I. umbrosus leaves may disrupt this process. Under both natural and laboratory conditions, female weevils preferred I. trichocarpus, a close relative with non-lobed leaves, over I. umbrosus. Nutritional properties of the leaves do not explain this preference because weevil larvae developed equally well on both hosts. Modifying the non-lobed I. trichocarpus leaves to mimic the shape of I. umbrosus leaves also discouraged leaf processing. Leaf processing often terminated because weevils failed to complete the inspection routine on I. umbrosus leaves. Leaf shape may be an important but overlooked factor that affects the interactions between plants and leaf-processing herbivores.

Taxonomic notes on Isodon (Lamiaceae) in China, III: resurrection of I. brachythyrsus, I. chionanthus and I. kangtingensis

One of the main reasons that contribute to the taxonomic chaos of Isodon is insufficient field investigation. Based on extensive field observation and specimen examination, the present study aims to clarify the identities of five species of Isodon from southwest China. Our researches reveal that I. brachythyrsus and I. chionanthus, previously treated as synonyms of I. muliensis, and I. kangtingensis, previously treated as a synonym of I. flabelliformis, should be resurrected as distinct species. Taxonomic revision of the five species, including their detailed description, illustrations, as well as distribution information, is carried out.

Frequent chloroplast capture among Isodon (Lamiaceae) species in Japan revealed by phylogenies based on variation in chloroplast and nuclear DNA

AbstractA recent phylogenetic study based only on chloroplast DNA (cpDNA) variation revealed that populations of an Isodon species are frequently embedded paraphyletically among other Isodon species. This phylogenetic discrepancy between species taxonomy and molecular phylogeny was considered to have resulted from chloroplast DNA captures and/or incomplete lineage sorting. To elucidate which of these factors was mainly responsible for the observed phylogenetic pattern, we performed phylogenetic analyses of multiple populations of Isodon species in Japan using cpDNA variation, three single‐copy nuclear genes, and double‐digest restriction‐site‐associated DNA sequencing (ddRAD‐seq). Although a species often shared chlorotypes with other species, our phylogenetical analyses based on variation in the three single‐copy nuclear genes and the ddRAD‐seq data showed that most populations belonging to the same species were monophyletic at the species level, suggesting that chloroplast capture may have frequently occurred between Isodon species. Some populations of an intraspecific taxon were embedded paraphyletically within the species, regardless of the large amount of phylogenetic information in nuclear DNA; this incongruity may have resulted from incomplete lineage sorting.

Comparative transcriptome analysis and simple sequence repeat marker development for two closely related Isodon species used as ‘Xihuangcao’ herbs

Purpose : To facilitate the molecular identification of original plants, resolve taxonomic problems and identify standards for ‘Xihuangcao’-based products on the market. Methods : A transcriptomic analysis of two closely related species, i.e., Isodon serra (Maxim.) (IS) and I. lophanthoides (Buch.-Ham. ex D. Don) Hara, was conducted by using the Illumina HiSeq 2500 platform, and expressed sequence tag-derived simple sequence repeat (EST-SSR) markers were developed based on these transcriptomes. Results : In total, 149,650 and 103,221 contigs were obtained, with N50 values of 1,400 and 1,516, from the IS and I. lophanthoides RNA-Seq datasets, respectively. These contigs were clustered into 107,777 and 68,220 unigenes, which were functionally annotated to identify the genes involved in therapeutic components. In total, 14,138 and 11,756 EST-SSR motifs were identified, and of these motifs, 7,453 and 6,428 were used to design primers for IS and I. lophanthoides, respectively. After PCR verification and fluorescence-based genotyping, 24 SSR markers with bright bands, high polymorphism, and single amplification were obtained and used to identify closely related Isodon species/varieties. Conclusion : These data could help herbal scientists identify high-quality herbal plants and provide a reference for genetic improvement and population genetic and phylogenetic studies investigating ‘Xihuangcao’ herbs. Keywords : Xihuangcao, Transcriptome, EST-SSR, Molecular markers

Acetyl-macrocalin B, an ent-kaurane diterpenoid, initiates apoptosis through the ROS-p38-caspase 9-dependent pathway and induces G2/M phase arrest via the Chk1/2-Cdc25C-Cdc2/cyclin B axis in non-small cell lung cancer

ABSTRACTNon-small cell lung cancer (NSCLC) is the leading cause of cancer-related death worldwide, and novel effective drugs against NSCLC are urgently needed. Isodon species are rich in ent-kaurane diterpenoids that have been reported to have antitumor bioactivity. Acetyl-macrocalin B (A-macB) is a novel ent-kaurane diterpenoid isolated from Isodon silvatica, and its antitumor efficacy against NSCLC and the underlying mechanisms were scrutinized in depth. The viability of cells treated with A-macB was detected by CCK-8 and colony formation assays. Apoptosis and cell cycle distribution were analyzed by flow cytometry. The mechanisms were investigated by detecting ROS and performing western blotting and verification experiments with specific inhibitors. The in vivo effect of A-macB was explored in a nude mouse xenograft model. A-macB effectively inhibited H1299 and A549 cell viability, triggered apoptosis and delayed cells in the G2/M phase. A-macB induced cellular ROS production and then activated the p38 MAPK-mediated, caspase 9-dependent apoptotic pathway. Both the ROS scavenger NAC and the specific p38 inhibitor SB203580 inactivated the function of p38 induced by A-macB, thus preventing cells from apoptosis. A-macB activated the Chk1/2-Cdc25C-Cdc2/cyclin B1 axis to induce G2/M phase arrest. AZD7762 abrogated the function of Chk1/2, abolished the G2/M delay and enhanced the cytotoxicity of A-macB. Moreover, A-macB efficiently suppressed tumor growth in a mouse xenograft model without noticeable toxicity to normal tissues. Having both efficacy and relative safety, A-macB is a potential lead compound that is worthy of further exploration for development as an anticancer agent.

Recent Advances in the Synthesis of Isodon Diterpenoids and Schinortriterpenoids

The plants of the genus Isodon and the schisandraceae family are two economically and medicinally important phytogroups, and the research of chemical constituents from these two phytogroups has been recognized as one of the most outstanding achievements in natrural product research recently. So far, over 1200 diterpenoids classified into 11 different groups have been reported from the Isodon species and more than 200 schinortriterpenoids (SNTs) involving more than 20 skeletons have been isolated from the schisandraceae species. Their diverse scffolds and significant bioactivities have aroused great interest among the community of organic synthetic chimistry. In this review, the advances in the synthesis of Isodon diterpenoids and schinortriterpenoids during the past decade will be reviewed.

Taxonomic notes onIsodon(Nepetoideae, Lamiaceae) in China I: revision of four species

A taxonomic revision of four species of Isodon (I. alborubrus, I. lungshengensis, I. rubescens and I. scrophularioides) from China is presented. Isodon alborubrus, previously regarded as conspecific with I. sculponeatus, is resurrected as a distinct species. Seven synonyms are newly proposed: I. gibbosus as a synonym of I. lungshengensis, I. henryi, Rabdosia rubescens var. rubescens f. lushanensis, R. rubescens var. taihangensis, R. rubescens var. lushiensis and I. rubescens var. eglandulosus of I. rubescens var. rubescens, and R. megathyrsoides of I. scrophularioides. Information about nomenclature, description, phenology, distribution and habitat, taxonomic notes as well as specimens examined is provided for each species.

Rapid differentiation of Xihuangcao from the three Isodon species by UPLC-ESI-QTOF-MS/MS and chemometrics analysis.

Background Isodon lophanthoides, I. lophanthoides var. graciliflorus and I. serra are the three botanical sources of Xihuangcao, which are often used indiscriminately in herbal products. The aim of this study was to develop a rapid and accurate analytical method to identify the three different botanical sources of Xihuangcao by combining UPLC-ESI-QTOF-MS with chemometrics analysis.MethodsFifteen batches of plants were collected as reference materials and their chemical profiles were analyzed by UPLC-ESI-QTOF-MS. These data were subsequently processed by statistical methods, including principal component analysis (PCA), hierarchical cluster analysis (HCA) and orthogonal partial least squared discriminant analysis (OPLS-DA). An automated sample class prediction model was also built using Naive Bayes as a class prediction algorithm to rapidly determine the source species of twenty-seven batches of commercial Xihuangcao samples.ResultsThe base peak chromatograms of the three authenticated species showed different patterns and twenty-seven peaks were chosen, including six diterpenoids, one phenolic acid and two glycosides to distinguish among these three species. The results showed good differentiation among the three species by PCA, HCA and OPLS-DA. Isodon lophanthoides var. graciliflorus was found to be the major botanical source of the commercial samples.ConclusionUPLC-ESI-QTOF-MS and subsequent chemometrics analysis were demonstrated effective to differentiate among the three different species of plants used as Xihuangcao.Electronic supplementary materialThe online version of this article (doi:10.1186/s13020-016-0120-y) contains supplementary material, which is available to authorized users.

Oridonin, a Promising ent-Kaurane Diterpenoid Lead Compound.

Oridonin belongs to ent-kaurane tetracyclic diterpenoid and was first isolated from Isodon species. It exhibits inhibitory activities against a variety of tumor cells, and pharmacological study shows that oridonin could inhibit cell proliferation, DNA, RNA and protein synthesis of cancer cells, induce apoptosis and exhibit an antimutagenic effect. In addition, the large amount of the commercially-available supply is also very important for the natural lead oridonin. Moreover, the good stability, suitable molecular weight and drug-like property guarantee its further generation of a natural-like compound library. Oridonin has become the hot molecule in recent years, and from the year 2010, more than 200 publications can be found. In this review, we summarize the synthetic medicinal chemistry work of oridonin from the first publication 40 years ago and share our research experience of oridonin for about 10 years, which may provide useful information to those who are interested in this research field.

Chromosome Complements of Isodon (Family: Lamiaceae–Subfamily: Nepetoideae): Trends in Karyotype Evolution

The use of Isodon in folk medicines has a long tradition. The tetracyclic compound ent-kaurane diterpenoids present in the genus have varied biological activities including antibacterial, anti-inflammatory and antitumor activity and makes it a good candidate in drug research. Being a cosmopolitan and important genus of the family Lamiaceae with considerable taxonomic dispute, a review on chromosome complements and its evolution is of great significance. The prevalence of confusion regarding the members of the genus makes it obligatory to include the synonymised members also. Hence, the review is made inclusive of Rabdosia, Isodon and members of Plectranthus which have now been included under the genus Isodon. The chromosome counts of all the reported species of Isodon have been examined and the haploid chromosome number was found to be n = 12. Except for the two species Isodon lophanthoides and Isodon ramosissimus for which the chromosome counts were reported as 2n = 36 and 2n = 42 respectively, all other species had 2n = 24 as its diploid count. The secondary basic chromosome number of x2 = 12 prevalent in majority of the taxa might have evolved from the primary base number x1 = 6 via protoautoploidy.