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Two squalene synthase genes AlSQS1 and AlSQS2 were isolated from Atractylodes lancea and functionally characterized using in vitro enzymatic reactions. Atractylodes lancea is a traditional herb used for the treatment of rheumatic diseases, gastric disorders, and influenza. Its major active ingredients include sesquiterpenoids and triterpenes. Squalene synthase (SQS; EC 2.5.1.21) catalyzes the first enzymatic step in the central isoprenoid pathway towards sterol and triterpenoid biosynthesis. In this study, we aimed to investigate two SQSs from A. lancea using cloning and in vitro enzymatic characterization. Bioinformatics and phylogenetic analyses revealed that the AlSQSs exhibited high homology with other plant SQSs. Furthermore, AlSQS1 was observed to be localized in both the nucleus and cytoplasm, whereas AlSQS2 was localized in the cytoplasm and endoplasmic reticulum. To obtain soluble recombinant enzymes, AlSQS1 and AlSQS2 were successfully expressed as glutathione S-transferase (GST)-tagged fusion proteins in Escherichia coli Transetta (DE3). Approximately 68 kDa recombinant proteins were obtained using GST-tag affinity chromatography and Western blot analysis. Results of the in vitro enzymatic reactions established that both AlSQS1 and AlSQS2 were functional, which verifies their catalytic ability in converting two farnesyl pyrophosphates to squalene. The expression patterns of AlSQS and selected terpenoid genes were also investigated in two A. lancea chemotypes using available RNA sequencing data. AlSQS1 and AlSQS2, which showed relatively similar expression in the three tissues, were more highly expressed in the stems than in the leaves and rhizomes. Methyl jasmonate (MeJA) was used as an elicitor to analyze the expression profiles of AlSQSs. The results of qRT-PCR analysis revealed that the gene expression of AlSQS1 and AlSQS2 plummeted at lowest value at 12 h and reached its peak at 24 h. This study is the first report on the cloning, characterization, and expression of SQSs in A. lancea. Therefore, our findings contribute novel insights that may be useful for future studies regarding terpenoid biosynthesis in A. lancea.

Poria cocos (Schw.) Wolf is a saprophytic fungus that grows around the roots of old, dead pine trees. Fushen, derived from the sclerotium of P. cocos but also containing a young host pine root, has been widely used as a medicine and food in China, Japan, Korea, Southeast Asian countries, and some European countries. However, the compound variations at the different growth periods and in the different parts of Fushen have not previously been investigated. In this study, an untargeted metabolomics approach based on ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS) and targeted quantitative analysis was utilized to characterize the temporal and spatial variations in the accumulation of specialized metabolites in Fushen. There were 119 specialized metabolites tentatively identified using the UPLC-Q/TOF-MS. The nine growth periods of Fushen were divided into four groups using partial least squares discrimination analysis (PLS-DA). Four different parts of the Fushen [fulingpi (FP), the outside of baifuling (BO), the inside of baifuling (BI), and fushenmu (FM)] were clearly discriminated using a PLS-DA and orthogonal partial least squares discrimination analysis (OPLS-DA). Markers for the different growth periods and parts of Fushen were also screened. In addition, the quantitative method was successfully applied to simultaneously determine 13 major triterpenoid acids in the nine growth periods and four parts. The quantitative results indicated that the samples in January, March, and April, i.e., the late growth period, had the highest content levels for the 13 triterpenoid acids. The pachymic acid, dehydropachymic acid, and dehydrotumulosic acid contents in the FM were higher than those in other three parts in March, whereas the poricoic acid B, poricoic acid A, polyporenic acid C, dehydrotratrametenolic acid, dehydroeburicoic acid, and eburicoic acid in FP were higher beginning in October. These findings reveal characteristics in temporal and spatial distribution of specialized metabolites in Fushen and provide guidance for the identification of harvesting times and for further quality evaluations.

Peucedanum huangshanense, a new species discovered in Anhui, China, is illustrated and described. Detailed morphological comparisons have showed that it is similar to P. praeruptorum but differs from the latter by having larger compound umbels (5–14 cm across), rays up to 25, and long-ovoid mericarps with lateral ribs narrowly winged. To explore the phylogenetic position of this species, nuclear ribosomal DNA internal transcribed spacer (ITS) region was sequenced for P. huangshanense and P. praeruptorum collected in different places. The morphological and molecular evidences support the hypothesis that P. huangshanense is a new distinct species.

Background: Huangqi is a famous Chinese medicinal material whose Dao-di producing area is Hunyuan, Shanxi. Huangqi produced in Hunyuan, Shanxi, were divided into several different specifications and grades according to the diameters and different positions of root system. Objective: This article investigates the quantitative characteristics of chemical compositions in different specifications and grades of Astragalus membranaceus var. mongholicus roots, aiming to elucidate the correlation between specifications and/or grades and chemical compositions in Huangqi. Methods: Based on the field investigation, samples of Huangqi collected from Hunyuan, Shanxi, were divided into different specifications and grades. The content of seven flavonoids and five saponins in Astragalus membranaceus var. mongholicus roots of different specifications and grades were determined simultaneously by HPLC–diode–array detection–evaporative light-scattering detection (HPLC–DAD–ELSD). Results: Huangqi was processed by traditional methods, and its commercial specification was classified by different parts of the root system, such as ge-da-tou, hong-lan-qi, zheng-bai-qi, fu-bai-qi, mao-wei-zi, and qi-jian. The total content of seven flavonoids and five saponins in ge-da-tou, qi-jian were lower. The total content of seven flavonoids in hong-lan-qi was much higher, while that of five saponins was much lower. The total content of seven flavonoids in lateral roots or fibrous roots were higher, and that of five saponins was lower, such as zheng-bai-qi, fu-bai-qi, and mao-wei-zi. According to the root diameters, Huangqi was classified to special grade, grade I, grade II, grade III, grade IV, or grade V. Among six grades of Huangqi, the total content of seven flavonoids in grade III, grade IV, and grade V were lower, while the total content of five saponins in them were much higher. Conclusions: There is an obvious difference on the distribution pattern of contents of seven flavonoids and five saponins in Huangqi of different specifications and grades, which provide a certain scientific basis for the quality evaluation of Huangqi. Highlights: The content of seven flavonoids and five saponins in Huangqi were determined by HPLC-DAD-ELSD. The relationship between the commercial specification grades and chemical components of Dao-di herbs Astragalus membranaceus var. mongholicus (Huangqi ) from Hunyuan, Shanxi were revealed, which provided a chemical basis for the classification of commercial specification grades of dao-herbs Astragalus membranaceus var. mongholicus (Huangqi ) from Hunyuan, Shanxi.

The processing of traditional Chinese medicine is an integral part of Chinese medical heritage. The recent discovery of the earliest processed drugs may provide strong evidence for the origins of traditional Chinese medicine processing. The remains were found in wooden lacquer boxes, unearthed from Haihunhou tomb in Nanchang, Jiangxi Province. By analyzing the appearance of the unearthed samples, it was found that the outer layers of the samples were composed of an auxiliary material and the inside was composed of plant material. As can be seen from the three-dimensional image taken using nuclear magnetic resonance screening, the sample had a hollow irregular rod structure. The outer layers were composed of a substance with weak signal strength, and the inside was similar to the fiber structure of plants with high water contents. Microscopic analysis showed that periderm, secondary xylem, and broad wood rays could be seen in the transverse section of the plants inside the samples. It can be further speculated that the plants inside the samples were derived from the roots or stems of dicotyledonous herbs. Additionally, the putative roots or stems of the dicotyledonous herbs had secondary structures and contained one to three clustered, red-brown cells. No starch granules, calcium oxalate crystals, or stone cells were observed in the transverse section. According to the microscopic database of the National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, it is speculated that the plants inside the samples were derived from the roots of Rehmannia species. A comparative study of the microscopic characteristics of wild Rehmannia chingii roots and the unearthed samples was carried out. It was found that both displayed periderm, broad secondary phloem, wood rays, similar vessel morphologies, and red-brown cells. The unearthed samples and R. chingii roots were analyzed by electrospray ionization mass spectrometry (ESI-MS) in negative ion mode. The results showed that the mass spectrum peak of m / z 623 in the unearthed samples agreed with that in the roots of R. chingii , and was putatively identified as acteoside or forsythiaside. Moreover, the peak at m / z 179 in the unearthed samples was identified as caffeic acid based on the molecular weight using high resolution tandem mass spectrometry (MS/MS) data, and was usually identified as a hydrolysate of acteoside or forsythiaside. There were about two to three outer layers of excipient on the samples. They were distributed unevenly, thicker at the top and thinner at the bottom. The plants and excipient layers were observed by microscope, and starch grains were found in the cross section and powder of the excipient layers, while no starch grains were found in the cross section or powder of the plant material inside. However, the reaction between the plant tissues and iodine-potassium iodide test solution produced the characteristic blue color. This phenomenon was related to the gelatinization of starch granules. Under a polarized light microscope, a large number of crystals were seen in the excipient layers. An aqueous extract of the excipient layers was analyzed by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS), and an ion fragment peak at m / z 341.11 was detected, indicating a disaccharide. When compared to the reference substances glucose, sucrose, fructose, and maltose, the retention times and mass spectra were very similar to that of sucrose. According to ancient literary and historical records, the tomb owner suffered from severe rheumatism before his death, which is consistent with the efficacy of R. chingii . It is further speculated that the samples in the wooden lacquer boxes are the remains of processed ancient traditional Chinese medicine products. The origin of the excipients in the processed medicines might be related to improving the taste, which could provide a scientific basis for further understanding the processing and application of ancient traditional Chinese medicines.

Relationships among Fallopia multiflora (Thunb.) Haraldson., F. multiflora var. angulata (S. Y. Liu) H. J. Yan, Z. J. Fang & Shi Xiao Yu., and F. multiflora var. ciliinervis (Nakai) Yonekura & H. Ohashi. were determined based on macroscopic and microscopic morphology, molecular phylogeny, and chemical analysis. The macroscopic and microscopic morphologies of root tubers or rhizomes, stems, and leaves were compared among the three taxa. The content of 11 chemical components (catechin, polydatin, stilbene glucoside, emodin, emodin-8-O-β-D-glucopyranoside, rhein, chrysophanol, aloe-emodin, quercetin, physcion, and resveratrol) in the three taxa was determined by HPLC, and the chemical diversity was further evaluated by principal component and hierarchical cluster analyses. Molecular phylogenies were mapped using two chloroplast markers (matK and the psbA-trnH intergenic region) and a nuclear ribosomal marker [internal transcribed spacer 2 (ITS2) region]. Analyses of macroscopic and microscopic morphological characteristics revealed that the subterranean organs of F. multiflora and F. multiflora var. angulata are root tubers, whereas those of F. multiflora var. ciliinervis are rhizomes. In the phylogenetic trees, F. multiflora and F. multiflora var. angulata were clustered into a clade based on the combine matK + psbA-trnH sequence, with neighbour-joining, maximum likelihood, and Bayesian inference bootstrap support values of 99, 85, and 0.99, respectively. In addition, there were obvious differences in the chemical compositions of F. multiflora, F. multiflora var. angulata and F. multiflora var. ciliinervis. The root tubers of F. multiflora contain higher levels of stilbene glucoside and catechin, but lower levels of polydatin and anthraquinone compounds. In contrast to F. multiflora, the rhizomes of F. multiflora var. ciliinervis contain higher levels of polydatin and anthraquinone compounds, but lack stilbene glucoside. The content of all 11 assessed components was lower in F. multiflora var. angulata than in F. multiflora and F. multiflora var. cillinervis. Principal component and hierarchical cluster analyses revealed that F. multiflora and F. multiflora var. angulata individuals were clustered into a single clade, whereas F. multiflora var. ciliinervis individuals were clustered into a single clade separate from that containing F. multiflora and F. multiflora var. angulata individuals. On the basis of the results of our morphological, molecular phylogeny, and chemical analyses, we tentatively conclude that F. multiflora var. ciliinervis is an independent species, whereas F. multiflora var. angulata should be considered as a variety of F. multiflora.

Comprehensive transcriptome analysis of different Platycodon grandiflorus tissues discovered genes related to triterpenoid saponin biosynthesis. Platycodon grandiflorus (Jacq.) A. DC. (P. grandiflorus), a traditional Chinese medicine, contains considerable triterpenoid saponins with broad pharmacological activities. Triterpenoid saponins are the major components of P. grandiflorus. Here, single-molecule real-time and next-generation sequencing technologies were combined to comprehensively analyse the transcriptome and identify genes involved in triterpenoid saponin biosynthesis in P. grandiflorus. We quantified four saponins in P. grandiflorus and found that their total content was highest in the roots and lowest in the stems and leaves. A total of 173,354 non-redundant transcripts were generated from the PacBio platform, and three full-length transcripts of β-amyrin synthase, the key synthase of β-amyrin, were identified. A total of 132,610 clean reads obtained from the DNBSEQ platform were utilised to explore key genes related to the triterpenoid saponin biosynthetic pathway in P. grandiflorus, and 96 differentially expressed genes were selected as candidates. The expression levels of these genes were verified by quantitative real-time PCR. Our reliable transcriptome data provide valuable information on the related biosynthesis pathway and may provide insights into the molecular mechanisms of triterpenoid saponin biosynthesis in P. grandiflorus.

Background Astragalus mongholicus Bunge is an important medicinal plant used in traditional Chinese medicine. It is rich in isoflavonoids and triterpenoid saponins. Although these active constituents of A. mongholicus have been discovered for a long time, the genetic basis of isoflavonoid and triterpenoid saponin biosynthesis in this plant is virtually unknown because of the lack of a reference genome. Here, we used a combination of next-generation sequencing (NGS) and single-molecule real-time (SMRT) sequencing to identify genes involved in the biosynthetic pathway of secondary metabolites in A. mongholicus. Results In this study, NGS, SMRT sequencing, and targeted compound analysis were combined to investigate the association between isoflavonoid and triterpenoid saponin content, and specific gene expression in the root, stem, and leaves of A. mongholicus. Overall, 643,812 CCS reads were generated, yielding 121,107 non-redundant transcript isoforms with an N50 value of 2124 bp. Based on these highly accurate transcripts, 104,756 (86.50%) transcripts were successfully annotated by any of the seven databases (NR, NT, Swissprot, KEGG, KOG, Pfam and GO). Levels of four isoflavonoids and four astragalosides (triterpenoid saponins) were determined. Forty-four differentially expressed genes (DEGs) involved in isoflavonoid biosynthesis and 44 DEGs from 16 gene families that encode enzymes involved in triterpenoid saponin biosynthesis were identified. Transcription factors (TFs) associated with isoflavonoid and triterpenoid saponin biosynthesis, including 72 MYBs, 53 bHLHs, 64 AP2-EREBPs, and 11 bZIPs, were also identified. The above transcripts showed different expression trends in different plant organs. Conclusions This study provides important genetic information on the A. mongholicus genes that are essential for isoflavonoid and triterpenoid saponin biosynthesis, and provides a basis for developing the medicinal value of this plant.

Orchidaceae, well known for its fascinating flowers, is one of the largest and most diverse families of flowering plants. There are many kinds of plants in this family; these are distributed practically globally and have high ornamental and medicinal values. Gastrodia elata Blume, a traditional Chinese medicinal herb, is a rootless and leafless achlorophyllous orchid. Phenolic compounds are considered to be the major bioactive constituents in G. elata, with antioxidant, antiangiogenic, neuroprotective, antidepressant, anxiolytic, and sedative activities. In this study, we determined the contents of six main phenolic components in tubers, stems and flowers from G. elata. Meanwhile, the transcriptomes of the tuber, stem and flower tissues of G. elata were obtained using the BGISEQ-500 platform. A total of 58.29 Gb of data and 113,067 unigenes were obtained, of which 74,820 unigenes were functionally annotated against seven public databases. Differentially expressed genes between tuber, stem and flower tissues were identified. A total of 76 DEGs encoding eight key enzymes were identified as candidate genes involved in the biosynthesis of phenolics in G. elata. For further validation, the expression levels of unigenes were measured using quantitative real-time PCR. Our results greatly enrich the transcriptomic data of G. elata and provide valuable information for the identification of candidate genes involved in the biosynthesis of secondary metabolites.

Programmed cell death (PCD) plays a critical role throughout the lives of plants, it is regarded as a highly regulated and active process of plant cell death during the times of biotic or abiotic stress. This study aims to provide developmental anatomical characteristics of the interxylary cork formation in the roots of Astragalus. membranaceus var. mongholicus, and to subsequently show cytomorphological evidence that PCD is involved in the development of rhytidome and interxylary cork. The developmental anatomy of rhytidome and interxylary cork of the perennial fresh main root of A. membranaceus var. mongholicus was studied using light microscopy, whereas the PCD in the development of rhytidome and interxylary cork was studied using fluorescence microscopy and transmission electron microscopy. Histologically, it was observed that the parenchyma cells of secondary phloem and xylem in roots recovered their meristematic ability, and later developed into rhytidome and interxylary cork. Cytologically, ultrastructural characteristics such as nucleus malformation, vacuole disappearance, mitochondrial degeneration, and vesicle filling were observed. In roots, the nucleus of the phloem parenchyma cells were terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive from the pre-rhytidome stage to the formation of rhytidome stage and 4',6-diamidino-2-phenylindole dihydrochloride (DAPI)-negative during the mature rhytidome stage. The TUNEL assay of the xylem parenchyma cells showed positive characteristics from the early stage of interxylary cork formation to the interxylary cork formation stage, whereas DAPI-negative characteristics were observed in the mature interxylary cork. Gel electrophoresis showed that DNA cleavage was random. Our results indicated that the formation of the rhytidome and interxylary cork involved the PCD process.

Atractylodes lancea (Thunb.) DC. is a traditional Chinese medicine rich in sesquiterpenes that has been widely used in China and Japan for the treatment of viral infections. Despite its important pharmacological value, genomic information regarding A. lancea is currently unavailable. In the present study, the whole genome sequence of A. lancea was obtained using an Illumina sequencing platform. The results revealed an estimated genome size for A. lancea of 4,159.24 Mb, with 2.28% heterozygosity, and a repeat rate of 89.2%, all of which indicate a highly heterozygous genome. Based on the genomic data of A. lancea, 27,582 simple sequence repeat (SSR) markers were identified. The differences in representation among nucleotide repeat types were large, e.g., the mononucleotide repeat type was the most abundant (54.74%) while the pentanucleotide repeats were the least abundant (0.10%), and sequence motifs GA/TC (31.17%) and TTC/GAA (7.23%) were the most abundant among the dinucleotide and trinucleotide repeat motifs, respectively. A total of 93,434 genes matched known genes in common databases including 48,493 genes in the Gene Ontology (GO) database and 34,929 genes in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. This is the first report to sequence and characterize the whole genome of A. lancea and will provide a theoretical basis and reference for further genome-wide deep sequencing and SSR molecular marker development of A. lancea.

Shihu is a famous Chinese Materia Medica derived from species of the Dendrobium. In order to differentiate 24 Dendrobium species, microscopic features of the 24 Dendrobium species were systemically observed, including the trend of vascular bundle number with different internodes and the transverse section characteristics of stem. The results indicate that the thickness of cuticle, the level of thickening of epidermal cells and cell types, the number of vascular bundles, the shape of vascular bundle sheath, the type and distribution of calcium oxalate crystals, the presence or absence of silica masses can be used to authenticate the 24 Dendrobium species. In addition, the number of vascular bundles in stems of 24 Dendrobium species also have changed at different internodes, and showed a trend of first increasing and then decreasing. For some specific species, the vascular bundle numbers had typical differences, which can provide some support for the identification of Dendrobium plants. Thus, using the multiple microscopic characteristics of stems can identify the Dendrobium plants effectively and quickly.

“Qidian”, “Qiai”, “Qishe”, and “Qigui” were regarded as the “Four Treasures of Kichow” in Li Shizhen’s hometown. From the Tang Dynasty onwards, “Qidian” had been a tribute for the central authority. “Qidian” has been highly praised in the poetry and prose of the Tang and Song dynasties. The Qi Chun county annals records that the craftsman Ye Caihua’s bamboo weaving skill was exceptional in Qichun, and his classic “Shuangfeng Chaoyang” Zhudian won the Golden award in the Panama world fair in 1915. However, “Qidian” had disappeared and the manufacturing process had also been lost. In 1974, a valuable “Qidian” sample was unearthed in Liu Jiaju’s tomb, Wangyao Village, Qizhou town, Qichun County, Hubei Province. The tomb dated back to 1516–1558 (the Ming dynasty). The front of the “Qidian” sample is yellow brown, and its back is yellowish white; it is about 183 cm long and 65 cm wide. Features of “Qidian” as light as cotton and soft like cloth, it can be folded at will, which indicates that its toughness is very good. Based on the existing research of the flexibility and uses of bamboos in Flora Hubeiensisi , we speculate that “Qidian” originated from Phyllostachys , Neosinocalamus or Bambusa . The “Qidian” sample and many bamboo species from Qichun were used to manufacture bamboo charcoal and were examined by means of scanning the ultrastructure with a scanning electron microscope (SEM). A total of 12 genera were recorded in Flora Hubeiensisi , these include Bambusa , Brachystachyum , Chimonobambusa , Fargesia , Indocalamus , Lingnania , Phyllostachys , Pleioblastus , Pseudosasa , Qiongzhuea , Sinarundinaria , and Sinocalamus . Based on its description of bamboos in Bamboo culm anatomy of China , pits of different genera showed significant differences. Pits were arranged alternately in Bambusa, Lingnania , and Sinocalamus , while pits were arranged stepwise in Brachystachyum and Qiongzhuea . However, the pits were arranged alternately, took on a ladder shape, or faced in the opposite direction in Chimonobambusa , Indocalamus , Phyllostachys , Pleioblastus , Pseudosasa , and Sinarundinaria . “Qidian” has been hypothesised to originate from Phyllostachys or Pleioblastus according to arrangement of pits. The experimental results showed that some pits were longitudinally extended in “Qidian” and Phyllostachys heteroclada Oliver, pits of other bamboo samples were ladder-shaped or alternately arranged in patterns. Therefore, we speculate that “Qidian” originates from Phyllostachys heteroclada Oliver. and its related species. The investigation of ancient poetry indicated the characteristics of the unearthed “Qidian” relics were in accordance with this description. The processing technology of “Qidian” has been discussed on the basis of section analysis. Based on the comparison of the ultrastructural features of “Qidian” and Phyllostachys heteroclada Oliver, which were observed through the scanning electron microscope, it can be concluded that “Qidian” can be extracted from the ground tissues in the outer side of Phyllostachys heteroclada Oliver. As one of the four treasures of Qizhou, “Qidian” is a typical part of China’s material and cultural heritage. It has obvious cultural characteristics and profound academic value. The archaeological research on the origin and production technology of “Qidian” in the Ming dynasty provides a basis for the cultural restoration of the cultural relic of “Qidian” in Qichun.

We present the 1.06 Gb sequenced genome of Gastrodia elata, an obligate mycoheterotrophic plant, which contains 18,969 protein-coding genes. Many genes conserved in other plant species have been deleted from the G. elata genome, including most of those for photosynthesis. Additional evidence of the influence of genome plasticity in the adaptation of this mycoheterotrophic lifestyle is evident in the large number of gene families that are expanded in G. elata, including glycoside hydrolases and urease that likely facilitate the digestion of hyphae are expanded, as are genes associated with strigolactone signaling, and ATPases that may contribute to the atypical energy metabolism. We also find that the plastid genome of G. elata is markedly smaller than that of green plant species while its mitochondrial genome is one of the largest observed to date. Our report establishes a foundation for studying adaptation to a mycoheterotrophic lifestyle., Gastrodia elata is an obligate mycoheterotrophic plant with highly reduced leaves and bracts in scape. Here, Yuan et al sequence and analyze its 1.06 Gb genome which provides insights in adaptation to a lifestyle of heterotrophy.

This study aimed to distinguish the rhizomes of Paris polyphylla var. yunnanensis (Franch) Hand Mazz (PPY) and Paris veitnamensis (Takht.) H. Li (PV) using metabolomics-based ultra high-performance liquid chromatography coupled with quadrupole time-of-fligh mass spectrometry (UHPLC/Q-TOF MS). First, the UHPLC/Q-TOF MS approach was optimized for metabolite profiling. Then, the MS data were processed using UNIFI™ combined with an in-house library to automatically characterize the metabolites. Based on the exact mass information, the fragmentation characteristics, and the retention time of compounds, and the fragmentation mechanism and retention behavior of steroidal glycosides in the references, the structures identified by UNIFI were further verified. Overall, 146 metabolites, including 42 potential new compounds, were identified or tentatively identified. Pattern recognition analysis of the PPY and PV MS data revealed that they were clearly separated, and 15 potential biomarkers for differentiating between them were selected. These biomarkers were subsequently used to successfully predict the genus of PPY and PV samples. These results indicated that metabolite profiling by UHPLC/Q-TOF MS is an effective, robust approach for determining the characteristic biomarkers that differentiate between TCM species with multiple botanical origins.

Modern clinical studies have found that Pulsatilla radix contains a variety of active ingredients; however, its medicinal parts and microstructure have been controversial. Based on morphological observation and microscopic identification of different structures, we chose Anhui, Henan, Jilin, and Shanxi Province to study Pulsatilla chinensis (Bunge) Regel populations. We specifically addressed different diameters and anatomical structures of different parts of underground organs. We also found that P. chinensis (Bunge) Regel had a "joint point" structure in its underground organ. Above the "joint point" is the rhizome, below the "joint point" is the root. The main medicinal organ is the rhizome. The protective tissues of the endothelial layer of P. chinensis (Bunge) Regel changed in the process of its development. The protective tissues were replaced by epidermis, cortex, endodermis, and phloem. With the secondary growth, the endothelial cells have been radial division. This study showed that morphology and microscopic identification was an important mean of medicinal material identifications, and it had the characteristics of a convenient, fast, and intuitive method to identify the composition and structural characteristics of P. chinensis (Bunge) Regel medicinal parts.

The accumulation of chemical constituents of some medicinal plants, such as Paeonia ostii T. Hong et J. X. Zhang, Houpo&euml, a officinalis (Rehder and E. H. Wilson) N. H. Xia and C. Y. Wu. and Atractylodes lancea (Thunb.) DC, can precipitate on the surface and form frosts after natural or artificial intervention. The characteristics of these three medicinal plants and their frosts were analyzed by light microscope, polarizing microscope, stereomicroscope, and metalloscope. The results of ordinary Raman of P. ostii and H. officinalis showed that the frosts of P. ostii matched paeonol, while that of H. officinalis matched magnolol and honokiol. In P. ostii and its frost, 19 peaks were identified by UPLC-Q/TOF-MS, and the main component was paeonol. Eleven components were identified in H. officinalis and its frosts, and the main components were magnolol and honokiol. A. lancea and its frosts were analyzed by gas chromatography-mass spectrometry (GC-MS), 21 were identified, and its main components were hinesol and &beta, eudesmol. These three medicinal plants accumulate compounds and precipitate frosts on the surface. The results show that the components of the frosts provide a basis for quality evaluation and research on similar medicinal plants, and reveals the scientific connotation of &ldquo, taking the medicinal materials&rsquo, precipitated frosts as the best&rdquo, of P. ostii, H. officinalis, and A. lancea, to some extent.

Background and AimsCompensatory base changes (CBCs) that occur in stems of ribosomal internal transcribed spacer 2 (ITS2) can have important phylogenetic implications because they are not expected to occur within a single species and also affect selection of appropriate DNA substitution models. These effects have been demonstrated when studying ancient lineages. Here we examine these effects to quantify their importance within a more recent lineage by using both DNA- and RNA-specific models.MethodsWe examined the phylogenetic implications of the CBC process by using a comprehensive sampling of ITS2 from ten closely related species of Corydalis. We predicted ITS2 secondary structures by using homology modelling, which was then used for a structure-based alignment. Paired and unpaired regions were analysed separately and in combination by using both RNA-specific substitution models and conventional DNA models. We mapped all base-pair states of CBCs on the phylogenetic tree to infer their evolution and relative timing.Key ResultsOur results indicate that selection acted to increase the thermodynamic stability of the secondary structure. Thus, the unpaired and paired regions did not evolve under a common substitution model. Only two CBCs occurred within the lineage sampled and no striking differences in topology or support for the shared clades were found between trees constructed using DNA- or RNA-specific substitution models.ConclusionsAlthough application of RNA-specific substitution models remains preferred over more conventional DNA models, we infer that application of conventional DNA models is unlikely to be problematic when conducting phylogenetic analyses of ITS2 within closely related lineages wherein few CBCs are observed. Each of the two CBCs was found within the same lineages but was not observed within a given species, which supports application of the CBC species concept.

Heshouwu, derived from root tubers of Fallopia multiflora (Thunb.) Harald., is a well-known herb used for millennia in traditional Chinese medicine. However, different forms of root tubers of Heshouwu have occurred in current Chinese herbal market and used in clinic, although it is still unknown whether their quality is consistent. In the present study, a mass spectrometry imaging and laser microdissection combined with UPLC-Q/TOF-MS were therefore used for the metabolite profiling on the whole and different parts of root tubers of F. multiflora and F. multiflora var. angulata. Our results suggested that the character of "woody heart" root tubers of F. multiflora was similar to that of F. multiflora var. angulata, but the latter had more phloem fibers and larger diameter vessel in the normal vascular bundle. Moreover, 140 compounds including stilbenes, anthraquinones, phenolic acids, naphthalenes, and other compounds were identified or putatively characterized from F. multiflora and F. multiflora var. angulata. Both unsupervised principal component analysis (PCA) and supervised Orthogonal Partial Least Squares Discrimination Analysis (OPLS-DA) multivariate statistics allowed discriminating F. multiflora and F. multiflora var. angulata. And a total of 32 potential markers were identified. The tissue-specific study indicated that the compounds in the phelloderm of F. multiflora and F. multiflora var. angulata were the most abundant. This is the first study on metabolite profiling and comparison of root tubers between F. multiflora and F. multiflora var. angulata, which would provide reasonable basis for further quality evaluation and safe medication of F. multiflora.

Platycodi Radix (PR) is the root of Platycodon grandiflorum (Jacq.) A. DC., which has been used for a long time in China to treat pulmonary diseases. The present study aimed to evaluate the quality of PR samples collected from 23 regions of 11 provinces in China. Eight saponins were quantified using HPLC coupled with evaporative light scattering detection (HPLC-ELSD). The samples with the highest total contents of saponins were from southern China, such as Yunnan, Guangxi, Jiangxi, and Guangzhou. The fingerprint analysis of PR samples was conducted by HPLC-UV method. Nineteen common peaks were selected and the similarity values varied from 0.607 to 0.921. These findings indicated that the saponins contents of PR from different regions varied significantly, with PR samples from southern China having the highest contents of saponins. These comprehensive methods were successful in evaluating the quality of PR samples from northern and southern China, which will serve as a guide for the development of PR as a clinical medication.

Many species of Corydalis (Papaveraceae) have been used as medicinal plants in East Asia, and the most well-known species are Corydalis yanhusuo and C. decumbens in the Pharmacopoeia of China. However, authentication of these species remains problematic because of their high morphological variation. Here, we selected 14 closely related species and five genomic regions (chloroplast: matK, trnG, rbcL, psbA-trnH, nuclear: ITS) to explore the utility of DNA barcoding for authenticating these herbs. In addition, the Poisson tree process (PTP) and automatic barcode gap discovery (ABGD) were also used and compared with DNA barcoding. Our results showed that the ITS region was not suitable for molecular analysis because of its heterogeneous nature in Corydalis. In contrast, matK was an ideal region for species identification because all species could be resolved when matK was used along with the other three chloroplast regions. We found that at least five traditional identified species were lumped into one genetic species by ABGD and PTP methods, thus, highlighting the overestimation of species diversity using the morphological approach. In conclusion, our first attempt of molecular analysis of Corydalis herbs presented here successfully resolved the identification of medicinal species and encouraged their taxonomic re-assessment.

The Daodi herb, Huangqi from Shanxi, is derived from the root of Astragalus membranaceus (Fisch.) Bge. var. mongholicus (Bge.) Hsiao. Huangqi was divided into different commercial specifications and grades in producing area according to macroscopic features. Developmental anatomy of anomalous structure of the perennial fresh main root of A. membranaceus var. mongholicus was investigated using conventional paraffin section to explore the relationship between anomalous structure and classification of commercial specifications and grades of Huangqi. The results of developmental anatomy showed that parenchyma cells in secondary phloem and secondary xylem of the perennial fresh main root regained meristematic ability, and resulted in anomalous structure. Several layers of additional periderm could sometimes be found in the secondary phloem near the initial periderm, which was called rhytidome. Additional periderm could sometimes be observed in the center of secondary xylem, which was described as "rotten heart." The area of rhytidome and "rotten heart" increased gradually with increasing age. The results of different commercial specifications and grades of Huangqi revealed rhytidome and "rotten heart" mainly existed in Ge-Da-Tou, Er-Dao-Tou, Hong-Lan-Qi, Special Class, First Class, and Second Class. The additional periderm in secondary phloem of Ge-Da-Tou, Er-Dao-Tou, Hong-Lan-Qi, and Special Class could reach five layers; however, that of First Class and Second Class was three layers at most. In summary, this study demonstrated the rules of the development of rhytidome and "rotten heart" in A. membranaceus var. mongholicus. The relationship between anomalous structure and different commercial specifications and grades of Daodi herb Huangqi, was clarified. RESEARCH HIGHLIGHTS: The rhytidome and the development of "rotten heart" have not been reported in Astragalus membranaceus var. mongholicus. At present article, we demonstrated the developmental anatomy of the rhytidome and "rotten heart" existed in the secondary phloem and secondary xylem of the root of A. membranaceus var. mongholicus respectively. The rhytidome and "rotten heart" is different among six commercial specifications and grades of Huangqi.

Authentication is the first priority when evaluating the quality of Chinese herbal medicines, particularly highly toxic medicines. The most commonly used authentication methods are morphological identification and microscopic identification. Unfortunately, these methods could not effectively evaluate some herbs with complex interior structures, such as root of Aconitum species with a circular conical shape and an interior structure with successive changes. Defining the part that should be selected as the standard plays an essential role in accurate microscopic identification. In this study, we first present a visual 3D model of Aconitum carmichaeli Debx. constructed obtained from microscopic analysis of serial sections. Based on this model, we concluded that the point of largest root diameter should be used as the standard for comparison and identification. The interior structure at this point is reproducible and its shape and appearance can easily be used to distinguish among species. We also report details of the interior structures of parts not shown in the 3D model, such as stone cells and cortical thickness. To demonstrate the usefulness of the results from the 3D model, we have distinguished the microscopic structures, at their largest segments, of the other three Aconitum species used for local habitat species of Caowu. This work provides the basis for resolution of some debate regarding the microstructural differences among these species. Thus, we conclude that the 3D model composed of serial sections has enabled the selection of a standard cross-section that will enable the accurate identification of Aconitum species in Chinese medicine. Microsc. Res. Tech. 78:425–432, 2015. © 2015 Wiley Periodicals, Inc.

Growth years are closely related to the quality of Chinese medicinal materials, and they can provide a reliable basis for quality evaluation on crude drugs in accordance with the suitable growth years. Herbchronology, the numerical and pattern analysis of annual growth rings in herbaceous perennials, has emerged to be a recognized science that is applied in age determination of perennial forbs. Fifty medicinal species growing in temperate and subtropical zones of China were examined by microstructure observation to determine the presence of growth rings in the secondary root xylem of medicinal perennial dicotyledonous herbs. Nearly half of the surveyed species showed clearly or relatively clearly demarcated growth rings in their roots, and additional anatomical patterns have been observed in different species. Clear variations in growth rings of different genus or species were characterized in this study, and these findings proved the feasibility of applying herbaceous plant growth rings in age identification of traditional Chinese medicine.

The impact of various doses of erythropoietin (EPO) on liver regeneration after partial hepatectomy (PH) in different animal models is still under debate. We investigated the impact of low doses of EPO on liver regeneration in a rat model of subtotal hepatectomy.We established a 90 % PH rat model with perioperative injections of low-dose EPO (1,000 IU/kg). We analyzed survival and hepatocyte proliferation in animals treated with or without EPO and assessed liver function by blood ammonia measurement and the indocyanine green 15-min retention test.Low doses of EPO treatment improved the survival of rats after 90 % PH. Unexpectedly, during the first 24 h after the operation, liver regeneration in the EPO-treated rats was inhibited. DNA synthesis, cell proliferation, and the expression of cyclins and p-STAT3 peaked 48 h after PH, which was delayed by about 24 h vs. the control rats. Furthermore, EPO treatment increased the serum level of IL-6 and protected the hepatocytes from apoptosis.Low doses of EPO do not stimulate early hepatocyte proliferation in the regenerating liver, but contribute to liver protection by inducing IL-6 and inhibiting apoptosis.

“Huoshan shihu,” derived from Dendrobium huoshanense, is a well-known, valuable, and rare Daodi herb. Because of its higher price and scarce resources, its related species D. officinale and D. moniliforme are usually presented as “Huoshan shihu” for sale. Fengdou, the processed form of D. huoshanense, its identification is particularly important. To effectively identify Fengdou of D. huoshanense and protect the Daodi herb, the morphological and microscopic characteristics of the stems of three Dendrobium species were examined. The results showed that macroscopic and microscopic features helpful for identification of the three species were the total lengths and internode numbers of stems, diameter and length of each internode, number of vascular bundles, and presence or absence of silica masses. The key features useful for distinguishing between D. huoshanense and its related species, and also between their Fengdou, were the stem lengths and the trends of change in diameter in different internodes. The results of the study indicated that a combination of macroscopic and microscopic identification techniques might conveniently and effectively be applied for identification of Dendrobium species. “Longtou Fengwei” is the main feature of the Daodi herb “Huoshan shihu,” (D. huoshanense Fengdou) and reflects the wisdom behind the protection of Daodi herbs in ancient times.

Basically, Dao-di hers are produced in specific area which has a long history, good quality, good medicine, curative effect. However genuine medicinal material area in history is not static, this makes the establishment of genuine medicinal material origin and the in-depth research be very difficult. This paper has profoundly analyzed the origin of different historical periods taking Astragalus membranaceus and Salvia miltiorrhiza as examples, and then summarized the reasons of herbal origin changes from the humanities, social and natural three aspects. This paper provides a basis for establishment and the further research of high-quality genuine producing area.

To determine the evolutionary relationships among all members of the genus Atractylodes (Compositae, Cardueae), we conducted molecular phylogenetic analyses of one nuclear DNA (nrDNA) region (internal transcribed spacer, ITS) and one chloroplast DNA (cpDNA) region (intergenic spacer region of trnL-F). In ITS and ITS + trnL-F trees, all members of Atractylodes form a monophyletic clade. Atractylodes is a sister group of the Carlina and Atractylis branch. Atractylodes species were distributed among three clades: (1) A. carlinoides (located in the lowest base of the Atractylodes phylogenetic tree), (2) A. macrocephala, and (3) the A. lancea complex, including A. japonica, A. coreana, A. lancea, A. lancea subsp. luotianensis, and A. chinensis. The taxonomic controversy over the classification of species of Atractylodes is mainly concentrated in the A. lancea complex. In base on molecular results, the intraspecific division of Atractylodes lancea is not supported, and A. coreana should be treated as a synonym A. chinensis.

Genetic relationships were studied among eight species of three taxa in the genus Chaenomeles by nuclear ribosomal internal transcribed spacer (ITS) analysis. A genetic distance matrix based on ITS sequences was estimated according to the formula of Kimura-2 parameter and a neighbour-joining phenogram, which were obtained with Clustalx4.1 software. The results showed that the germplasms of Mugua originate from Ch. speciosa (Sweet) Nakai, not including Ch. sinensis (Thouin) Kochne and Ch. cathayensis (Hemsl.) Schneid. The results also showed that ‘Yao Mugua’ and ‘Ornamental Mugua’ are the most distantly related species in germplasms.

Ginseng, which is the root of Panax ginseng (Araliaceae), has been used in Oriental medicine as a stimulant and dietary supplement for more than 7,000 years. Older ginseng plants are substantially more medically potent, but ginseng age can be simulated using unscrupulous cultivation practices. Telomeres progressively shorten with each cell division until they reach a critical length, at which point cells enter replicative senescence. However, in some cells, telomerase maintains telomere length. In this study, to determine whether telomere length reflects ginseng age and which tissue is best for such an analysis, we examined telomerase activity in the main roots, leaves, stems, secondary roots and seeds of ginseng plants of known age. Telomere length in the main root (approximately 1 cm below the rhizome) was found to be the best indicator of age. Telomeric terminal restriction fragment (TRF) lengths, which are indicators of telomere length, were determined for the main roots of plants of different ages through Southern hybridization analysis. Telomere length was shown to be positively correlated with plant age, and a simple mathematical model was formulated to describe the relationship between telomere length and age for P. ginseng.

A method of comprehensive chemical pattern recognition of Atractylodis Rhizoma was established by GC-MS fingerprint, principal component analysis, cluster analysis and discriminant analysis. A DB-wax column (0.25 mm x 60 m, 0.25 microm) with El ion source and 70 V electron multiplier were used for GC-MS analysis. Using principal component analysis, cluster analysis, and discriminant analysis, 15 common peaks of sample fingerprints for chemical pattern recognition research were analysed. The same results were obtained from the fingerprint, principal component analysis and cluster analysis, which could use to distinguish genuine Atractylodes lancea, ungenuine A. lancea and A. chinensis. Thus, this method could be used for the quality control and comprehensive evaluation of Atractylodis Rhizoma.

Polygonum multiflorum is one of the most commonly used Chinese medicines. In this study, an effective pressurized water extraction and HPLC method was developed for first simultaneous determination of 8 hydrophilic compounds, including gallic acid, Hypaphorine, Catechin, Proanthocyanidin B1, Epicatechin, Proanthocyanidin B2, Emodin-8-O-β- d -glucopyranoside, stilbene glycosides, in P. multiflorum . The analysis was performed on a Zorbax SB-AQ column with gradient elution of 0.05% phosphoric acid aqueous solution and acetonitrile in 45 min. All calibration curves showed good linearity ( R 2 > 0.9994) within test ranges. The LOD and LOQ were lower than 0.2 and 1.0 μg/mL on column, respectively. RSD for intra- and inter-day of 8 analytes were less than 4.1% and 4.0%, respectively, and the overall recovery was 96.0–100.7%. The validated method was successfully applied to quantification of 8 hydrophilic compounds in samples of P. multiflorum from different locations of China. Chemometrics such as principal component analysis (PCA) and hierarchical clustering analysis (HCA) were used to evaluate homogeneity of P. multiflorum in China, which suggested that their quality homogeneity was good.

Objective To elucidate the composition structure of "annual rings" and the formation process of anomalous structures in Sophora flavescens, and further discuss the medicinal parts of S. flavescens. Method Based on investigation on S. flavescens in its producing areas, the morphology of root systems was observed, and the developmental anatomy of roots was researched. Result Creeping underground rhizomes of S. flavescen existed in some parts of the north place, there were many differences in appearance characters and microscopic features between these roots and rhizomes. Parenchyma cells in secondary xylem regained meristematic ability, became into anomalous cambia, and then developed into anomalous structures. "Annual rings" in transverse section of S. flavescens were not actually growth rings, they were made up of anomalous parenchyma girdle in secondary xylem and normal secondary structure. Conclusion Roots are the medicinal parts of S. flavescens. This paper suggests that "annual rings" in the decoction pieces of S. flavescens should be called "annular structure".

Growth rings were used to determine the root age of medicinal Paeonia lactiflora from four producing areas, and their corresponding paeoniflorin content were measured based on the identification of ages. Different P. lactiflora root samples of different ages were collected from the four major growing areas in China: Bozhou, Anhui Province; Pan'an, Zhejiang Province; Zhongjiang, Sichuan Province; and Heze, Shandong Province. The relationship between the number of growth rings and age was analyzed using hand sections and paraffin sections. The paeoniflorin content in the roots of different P. lactiflora cultivars from different growing areas was measured using high-performance liquid chromatography (HPLC). The growth rings in the P. lactiflora roots were consistent with the age of the plant from Heze, Zhongjiang, Pan'an, whereas that for the P. lactiflora from Bozhou was one less than the age of the plant. The HPLC results show that the paeoniflorin content was highest in P. lactiflora 'Baihuachuanshaoyao,' followed by 'Baihuahangshaoyao,' 'Honghuachuanshaoyao,' and 'Honghuahangshaoyao,' 'Bozhoushaoyao' had the lowest levels of paeoniflorin. With increasing age, the paeoniflorin in the roots of the different P.lactiflora cultivars slowly declined or remained the same. In summary, the age of the roots of P. lactiflora from different growing areas can be determined using growth rings. The paeoniflorin content in the roots of P. lactiflora is correlated with cultivar and it was slowly declined with increasing age.