[Comparative analysis of plastomes of Rubus chingii and R. chingii var. suavissimus].

Rubus chingii and R. chingii var. suavissimus are unique dual-purpose plant resources, with significant nutraceutical, pharmaceutical, and economic value, as well as promising prospects for further development. To investigate the genetic structure and evolutionary characteristics of these two varieties, this study conducted plastome sequencing using the Illumina HiSeq XTen sequencing platform. Subsequently, the study performed assembly, annotation, and characterization of the genomes, followed by a comparative plastome and phylogenetic analysis using bioinformatics techniques. The results revealed that the plastomes of R. chingii and R. chingii var. suavissimus exhibited a tetrad structure, comprising a large single-copy region(LSC), a small single-copy region(SSC), and two inverted repeat regions(IRs). The study identified a total of 56 simple sequence repeats(SSRs) after comparative analysis, predominantly consisting of A and T. Furthermore, the structure of the IR boundary genes in both varieties was found to be highly conserved, with only minor nucleotide variations. Additionally, the study identified three highly variable regions: rps16-trnQ-psbK, trnR-atpA, and trnT-trnL, which held promise as potential identification marks for further development and utilization. Phylogenetic analysis results obtained by the maximum likelihood and Bayesian inference methods demonstrated a close clustering of R. chingii and R. chingii var. suavissimus(100% support), with their closest relatives being R. trianthus. This study, focusing on plastome-level genetic distinctions between these two varieties, lays a foundation for future species protection, development, and utilization.

Can the heptapeptide ASSIVSF of the ß2-adrenoceptor recognize ephedrine and pseudoephedrine epimers in a complex system?

Epimer separation is crucial in the field of analytical chemistry, separation science, and the pharmaceutical industry. No reported methods could separate simultaneously epimers or even isomers and remove other unwanted, co-existing, interfering substances from complex systems like herbal extracts. Herein, we prepared a heptapeptide-modified stationary phase for the separation of 1R,2S-(-)-ephedrine [(-)-Ephe] and 1S,2S-(+)-pseudoephedrine [(+)-Pse] epimers from Ephedra sinica Stapf extract and blood samples. The heptapeptide stationary phase was comprehensively characterized by scanning electron microscopy, X-ray photoelectron spectroscopy, and Fourier transform infrared spectroscopy. The separation efficiency of the heptapeptide column was compared with an affinity column packed with full-length ß2-AR functionalized silica gel (ß2-AR column). The binding affinity of the heptapeptide with (+)-Pse was 3-fold greater than that with (-)-Ephe. Their binding mechanisms were extensively characterized by chromatographic analysis, ultraviolet spectra, circular dichroism analysis, isothermal titration calorimetry, and molecule docking. An enhanced hydrogen bonding was clearly observed in the heptapeptide-(+)-Pse complex. Such results demonstrated that the heptapeptide can recognize (+)-Pse and (-)-Ephe epimers in a complex system. This work, we believe, was the first report to simultaneously separate epimers and remove non-specific interfering substances from complex samples. The method was potentially applicable to more challenging sample separation, such as chiral separation from complex systems.

Genetic diversities in wild and cultivated populations of the two closely-related medical plants species, Tripterygium Wilfordii and T. Hypoglaucum (Celastraceae).

BACKGROUND: The sustainable supply of medicinal plants is important, and cultivating and domesticating them has been suggested as an optimal strategy. However, this can lead to a loss of genetic diversity. Tripterygium wilfordii Hook. f. is a medicinal plant commonly used in traditional Chinese medicine, but its wild populations are dwindling due to excessive harvesting. To protect the species and meet the increasing demand, it is urgent to cultivate it on a large scale. However, distinguishing between T. wilfordii and T. hypoglaucum, two similar species with different medicinal properties, is challenging. Therefore, it is crucial to understand the genetic diversity and population structure of these species for their sustainable utilization. RESULTS: In this study, we investigated the genetic diversity and population structure of the two traditional medicinal semiwoody vines plant species, Tripterygium wilfordii and T. hypoglaucum, including wild and cultivated populations using chloroplast DNA (cpDNA) sequences and microsatellite loci. Our results indicated that the two species maintain a high level of genetic divergence, indicating possible genetic bases for the different contents of bioactive compounds of the two species. T. wilfordii showed lower genetic diversity and less subdivided population structures of both markers than T. hypoglaucum. The potential factors in shaping these interesting differences might be differentiated pollen-to-seed migration rates, interbreeding, and history of population divergence. Analyses of cpDNA and microsatellite loci supported that the two species are genetically distinct entities. In addition, a significant reduction of genetic diversity was observed for cultivated populations of the two species, which mainly resulted from the small initial population size and propagated vegetative practice during their cultivation. CONCLUSION: Our findings indicate significant genetic divergence between T. wilfordii and T. hypoglaucum. The genetic diversity and population structure analyses provide important insights into the sustainable cultivation and utilization of these medicinal plants. Accurate identification and conservation efforts are necessary for both species to ensure the safety and effectiveness of crude drug use. Our study also highlighted the importance of combined analyses of different DNA markers in addressing population genetics of medicinal plants because of the contrasts of inheritance and rates of gene flow. Large-scale cultivation programs should consider preserving genetic diversity to enhance the long-term sustainability of T. wilfordii and T. hypoglaucum. Our study proposed that some populations showed higher genetic diversity and distinctness, which can be considered with priority for conservation and as the sources for future breeding and genetic improvement.

[Research progress in microevolutionary process of excellent traits and quality of Dao-di herbs].

Dao-di herbs are the treasure of Chinese materia medica and one of the characteristic research objects of traditional Chinese medicine(TCM). Probing into the microevolution of Dao-di herbs can help to reveal their biological essence and quality formation mechanisms. The progress in molecular biology and omics provides the possibility to elucidate the phylogenetic and quality forming characteristics of Dao-di herbs at the molecular level. In particular, genomics serves as a powerful tool to decipher the genetic origins of Dao-di herbs, and molecular markers have been widely used in the research on the genetic diversity and population structure of Dao-di herbs. Focusing on the excellent traits and quality of Dao-di herbs, this paper reviews the studies about the microevolution process of quality formation mechanisms of Dao-di herbs with the application of molecular markers and omics, aiming to underpin the protection and utilization of TCM resources.

Authenticating the geographic origins of Atractylodes lancea rhizome chemotypes in China through metabolite marker identification.

Introduction: Atractylodes lancea is widely distributed in East Asia, ranging from Amur to south-central China. The rhizome of A. lancea is commonly used in traditional Chinese medicine, however, the quality of products varies across different regions with different geochemical characteristics. Method: This study aimed to identify the chemotypes of A. lancea from different areas and screen for chemical markers by quantifying volatile organic compounds (VOCs) using a targeted metabolomics approach based on GC-MS/MS. Results: The A. lancea distributed in Hubei, Anhui, Shaanxi, and a region west of Henan province was classified as the Hubei Chemotype (HBA). HBA is characterized by high content of ß-eudesmol and hinesol with lower levels of atractylodin and atractylon. In contrast, the Maoshan Chemotype (MA) from Jiangsu, Shandong, Shanxi, Hebei, Inner Mongolia, and other northern regions, exhibited high levels of atractylodin and atractylon. A total of 15 categories of VOCs metabolites were detected and identified, revealing significant differences in the profiles of terpenoid, heterocyclic compound, ester, and ketone among different areas. Multivariate statistics indicated that 6 compounds and 455 metabolites could serve as candidate markers for differentiating A. lancea obtained from the southern, northern, and Maoshan areas. Discussion: This comprehensive analysis provides a chemical fingerprint of selected A. lancea. Our results highlight the potential of metabolite profiling combined with chemometrics for authenticating the geographical origin of A. lancea.

[Species identification of Ligustrum lucidum].

Ligustrum lucidum is a woody perennial plant of genus Ligustrum in family Oleaceae. Its dried fruit has high medicinal value. In this study, the authors evaluated the variability and species identification efficiency of three specific DAN barcodes(rbcL-accD, ycf1a, ycf1b) and four general DAN barcodes(matK, rbcL, trnH-psbA, ITS2) for a rapid and accurate molecular identification of Ligustrum species. The results revealed that matK, rbcL, trnH-psbA, ITS2 and ycf1a were inefficient for identifying the Ligustrum species, and a large number of insertions and deletions were observed in rbcL-accD sequence, which was thus unsuitable for development as specific barcode. The ycf1b-2 barcode had DNA barcoding gap and high success rate of PCR amplification and DNA sequencing, which was the most suitable DNA barcode for L. lucidum identification and achieved an accurate result. In addition, to optimize the DNA extraction experiment, the authors extracted and analyzed the DNA of the exocarp, mesocarp, endocarp and seed of L. lucidum fruit. It was found that seed was the most effective part for DNA extraction, where DNAs of high concentration and quality were obtained, meeting the needs of species identification. In this study, the experimental method for DNA extraction of L. lucidum was optimized, and the seed was determined as the optimal part for DNA extraction and ycf1b-2 was the specific DNA barcode for L. lucidum identification. This study laid a foundation for the market regulation of L. lucidum.

Genetic diversity and population divergence of Leonurus japonicus and its distribution dynamic changes from the last interglacial to the present in China.

BACKGROUND: Leonurus japonicus, a significant medicinal plant known for its therapeutic effects on gynecological and cardiovascular diseases, has genetic diversity that forms the basis for germplasm preservation and utilization in medicine. Despite its economic value, limited research has focused on its genetic diversity and divergence. RESULTS: The avg. nucleotide diversity of 59 accessions from China were 0.00029 and hotspot regions in petN-psbM and rpl32-trnL(UAG) spacers, which can be used for genotype discrimination. These accessions divided into four clades with significant divergence. The four subclades, which split at approximately 7.36 Ma, were likely influenced by the Hengduan Mountains uplift and global temperature drop. The initial divergence gave rise to Clade D, with a crown age estimated at 4.27 Ma, followed by Clade C, with a crown age estimated at 3.39 Ma. The four clades were not showed a clear spatial distribution. Suitable climatic conditions for the species were identified, including warmest quarter precipitation 433.20 mm ~ 1,524.07 mm, driest month precipitation > 12.06 mm, and coldest month min temp > -4.34 °C. The high suitability distribution showed contraction in LIG to LGM, followed by expansion from LGM to present. The Hengduan Mountains acted as a glacial refuge for the species during climate changes. CONCLUSIONS: Our findings reflected a clear phylogenetic relationships and divergence within species L. japonicus and the identified hotspot regions could facilitate the genotype discrimination. The divergence time estimation and suitable area simulation revealed evolution dynamics of this species and may propose conservation suggestions and exploitation approaches in the future.

Chang qing formula ameliorates colitis-associated colorectal cancer via suppressing IL-17/NF-κB/STAT3 pathway in mice as revealed by network pharmacology study.

Colitis-associated colorectal cancer (CAC) is a specific type of colorectal cancer (CRC) with high mortality and morbidity, the chronic inflammation in the intestinal mucosal is the characteristic of CAC. Chang Qing formula (CQF) is a Chinese herbal formula used clinically for the treatment of CAC with remarkable clinical efficacy, but its mechanism remains unclear. In the present work, Combined network pharmacology and transcriptomics were used to analyze the potential active ingredients and elucidate molecular mechanism of CQF in treating CAC. Firstly, the constituents migrating to blood of CQF were analyzed and identified by UPLC-Q-TOF-MS/MS, and core genes and pathways were screened by network pharmacology analysis. Encyclopedia of Genes and Genomes (KEGG) analysis showed that the IL-17 signaling pathway involved in CAC may be closely associated with the potential mechanismof action of CQF. Subsequently, the results from animal studies indicated that CQF profoundly reduced tumor numbers and tumor size in AOM/DSS mice. The RNA-seq data was analysed utilizing Ingenuity Pathway Analysis (IPA), and the results supported the idea that CQF exerts a tumour-suppressive effect via the IL-17 signalling pathway. Further studies demonstrated that CQF significantly reduced IL-17A levels, which in turn inhibited NF-κB/IL-6/STAT3 signaling cascade, suppressed MMP9 expression and promoted tumor cell apoptosis. In conclusion, the current study demonstrated that CQF remarkably improved inflammatory tumor microenvironment, and hindered the transformation of inflammation into cancer. These findings may help to design future strategies for the treatment of CAC.

Investigating the Mechanisms of Jieduquyuziyin Prescription Improves Lupus Nephritis and Fibrosis via FXR in MRL/lpr Mice.

Lupus nephritis (LN) is one of the most serious complications of systemic lupus erythematosus (SLE) and one of the leading causes of death. An alternative effective treatment to ameliorate and relieve LN and delay the process of renal tissue fibrosis is urgently needed in the clinical setting. Jieduquyuziyin prescription (JP) has been successfully used to treat SLE, but its potential mechanisms are not sufficiently understood. In this study, we treated MRL/lpr mice with JP for 8 weeks and treated human renal tubular epithelial cells (human kidney 2 (HK-2)) with drug-containing serum to observe the antagonistic effects of JP on inflammation and fibrosis, as well as to investigate the possible mechanisms. Results demonstrated that JP significantly reduced urinary protein and significantly improved pathological abnormalities. Metabolomics combined with ingenuity pathway analysis illustrated that the process of kidney injury in lupus mice may be closely related to farnesoid X receptor (FXR) pathway abnormalities. Microarray biomimetic analysis and LN patients indicated that FXR may play a protective role as an effective therapeutic target for LN and renal fibrosis. JP significantly increased the expression of FXR and inhibited the expression of its downstream targets, namely, nuclear transcription factor κB (NF-κB) and α-smooth muscle actin (α-SMA), in the kidney of MRL/lpr mice and HK-2 cells, as confirmed by in vitro and in vivo experiments. In conclusion, JP may mediate the activation of renal FXR expression and inhibit NF-κB and α-SMA expression to exert anti-inflammatory and antifibrotic effects for LN prevention and treatment.

Phylogenomics and Genetic Diversity of Arnebiae Radix and Its Allies (Arnebia, Boraginaceae) in China.

Arnebiae Radix is a traditional medicine with pleiotropic properties that has been used for several 100 years. There are five species of Arnebia in China, and the two species Arnebia euchroma and Arnebia guttata are the source plants of Arnebiae Radix according to the Chinese Pharmacopoeia. Molecular markers that permit species identification and facilitate studies of the genetic diversity and divergence of the wild populations of these two source plants have not yet been developed. Here, we sequenced the chloroplast genomes of 56 samples of five Arnebia species using genome skimming methods. The Arnebia chloroplast genomes exhibited quadripartite structures with lengths from 149,539 and 152,040 bp. Three variable markers (rps16-trnQ, ndhF-rpl32, and ycf1b) were identified, and these markers exhibited more variable sites than universal chloroplast markers. The phylogenetic relationships among the five Arnebia species were completely resolved using the whole chloroplast genome sequences. Arnebia arose during the Oligocene and diversified in the middle Miocene; this coincided with two geological events during the late Oligocene and early Miocene: warming and the progressive uplift of Tianshan and the Himalayas. Our analyses revealed that A. euchroma and A. guttata have high levels of genetic diversity and comprise two and three subclades, respectively. The two clades of A. euchroma exhibited significant genetic differences and diverged at 10.18 Ma in the middle Miocene. Three clades of A. guttata diverged in the Pleistocene. The results provided new insight into evolutionary history of Arnebia species and promoted the conservation and exploitation of A. euchroma and A. guttata.

[Complete chloroplast genome of Ligustrum lucidum and highly variable marker identification for Ligustrum].

Ligustri Lucidi Fructus, the sun-dried mature fruit of Ligustrum lucidum, is cool, plain, sweet, and bitter, which can be used as both food and medicine, with the effects of improving vision, blacking hair, and tonifying liver and kidney. It takes effect slowly. However, little is known about the genetic information of the medicinal plant and it is still a challenge to distinguish Ligustrum species. In this study, the complete chloroplast genome of L. lucidum was obtained by genome skimming and then compared with that of five other Ligustrum species, which had been reported. This study aims to evaluate the interspecific variation of chloroplast genome within the genus and develop molecular markers for species identification of the genus. The result showed that the chloroplast genome of L. lucidum was 162 162 bp with a circular quadripartite structure of two single-copy regions separated by a pair of inverted repeats. The Ligustrum chloroplast genomes were conserved with small interspecific difference. Comparative analysis of six Ligustrum chloroplast genomes revealed three variable regions(rbcL-accD, ycf1a, and ycf1b), and ycf1a and ycf1b can be used as the species-specific DNA barcode for Ligustrum. Phylogeny analysis provided the best resolution of Ligustrum and supported that L. lucidum was sister to L. gracile. This study clarified the genetic diversity of L. lucidum from provenance, which can serve as a reference for further analysis of pharmacological differences and breeding of excellent varieties with stable drug effects.

[Evaluation of genetic diversity of ginseng fruit color germplasm resources: based on SSR analysis].

Illumina Xten was employed for shallow sequencing of Panax ginseng(ginseng) samples, MISA for screening of SSR loci, and Primer 3 for primer design. Polymorphic primers were screened from 180 primers. From the successfully amplified polymorphic primers, 15 primers which featured clear peak shape, good polymorphism, and ease of statistics were selected and used to evaluate the genetic diversity and germplasm resources of 36 ginseng accessions with different fruit colors from Jilin province. The results showed that red-fruit ginseng population had high genetic diversity with the average number of alleles(N_a) of 1.031 and haploid genetic diversity(h) of 0.172. The neighbor-joining cluster analysis demonstrated that the germplasms of red-fruit and yellow-fruit ginseng populations were obviously intermixed, and pick-fruit ginseng germplasms clustered into a single clade. The results of STRUCTURE analysis showed high proportion of single genotype in pick-fruit ginseng germplasm and abundant genotypes in red-fruit and yellow-fruit ginseng germplasms with obvious germplasm mixing. AMOVA revealed that genetic variation occurred mainly within populations(62.00%, P<0.001), and rarely among populations(39%, P<0.001), but homogenization was obvious among different populations. In summary, pink-fruit ginseng population may contain rare genotypes, which is the basis for breeding of high-quality high-yield, and multi-resistance varieties, genetic improvement of varieties, and sustainable development and utilization of ginseng germplasm resources.

Heterogeneous Genetic Diversity Estimation of a Promising Domestication Medicinal Motherwort Leonurus Cardiaca Based on Chloroplast Genome Resources.

Leonurus cardiaca has a long history of use in western herbal medicine and is applied for the treatment of gynaecological conditions, anxiety, and heart diseases. Because of its botanical relationship to the primary Chinese species, L. japonicus, and extensive medical indications that go beyond the traditional indications for the Chinese species, it is a promising medicinal resource. Therefore, the features of genetic diversity and variability in the species have been prioritized. To explore these issues, we sequenced the chloroplast genomes of 22 accessions of L. cardiaca from different geographical locations worldwide using high-throughput sequencing. The results indicate that L. cardiaca has a typical quadripartite structure and range from 1,51,236 bp to 1,51,831 bp in size, forming eight haplotypes. The genomes all contain 114 distinct genes, including 80 protein-coding genes, 30 transfer RNA genes and four ribosomal RNA genes. Comparative analysis showed abundant diversity of single nucleotide polymorphisms (SNPs), indels, simple sequence repeats (SSRs) in 22 accessions. Codon usage showed highly similar results for L. cardiaca species. The phylogenetic and network analysis indicated 22 accessions forming four clades that were partly related to the geographical distribution. In summary, our study highlights the advantage of chloroplast genome with large data sets in intraspecific diversity evaluation and provides a new tool to facilitate medicinal plant conservation and domestication.

Chloroplast genome variation and phylogenetic relationships of Atractylodes species.

BACKGROUND: Atractylodes DC is the basic original plant of the widely used herbal medicines "Baizhu" and "Cangzhu" and an endemic genus in East Asia. Species within the genus have minor morphological differences, and the universal DNA barcodes cannot clearly distinguish the systemic relationship or identify the species of the genus. In order to solve these question, we sequenced the chloroplast genomes of all species of Atractylodes using high-throughput sequencing. RESULTS: The results indicate that the chloroplast genome of Atractylodes has a typical quadripartite structure and ranges from 152,294 bp (A. carlinoides) to 153,261 bp (A. macrocephala) in size. The genome of all species contains 113 genes, including 79 protein-coding genes, 30 transfer RNA genes and four ribosomal RNA genes. Four hotspots, rpl22-rps19-rpl2, psbM-trnD, trnR-trnT(GGU), and trnT(UGU)-trnL, and a total of 42-47 simple sequence repeats (SSR) were identified as the most promising potentially variable makers for species delimitation and population genetic studies. Phylogenetic analyses of the whole chloroplast genomes indicate that Atractylodes is a clade within the tribe Cynareae; Atractylodes species form a monophyly that clearly reflects the relationship within the genus. CONCLUSIONS: Our study included investigations of the sequences and structural genomic variations, phylogenetics and mutation dynamics of Atractylodes chloroplast genomes and will facilitate future studies in population genetics, taxonomy and species identification.

Phylogenomic and evolutionary dynamics of inverted repeats across Angelica plastomes.

BACKGROUND: Angelica L. (family Apiaceae) is an economically important genus comprising ca. One hundred ten species. Angelica species are found on all continents of the Northern Hemisphere, and East Asia hosts the highest number of species. Morphological characters such as fruit anatomy, leaf morphology and subterranean structures of Angelica species show extreme diversity. Consequently, the taxonomic classification of Angelica species is complex and remains controversial, as the classifications proposed by previous studies based on morphological data and molecular data are highly discordant. In addition, the phylogenetic relationships of major clades in the Angelica group, particularly in the Angelica s. s. clade, remain unclear. Chloroplast (cp) genome sequences have been widely used in phylogenetic studies and for evaluating genetic diversity. RESULTS: In this study, we sequenced and assembled 28 complete cp genomes from 22 species, two varieties and two cultivars of Angelica. Combined with 36 available cp genomes in GenBank from representative clades of the subfamily Apioideae, the characteristics and evolutionary patterns of Angelica cp genomes were studied, and the phylogenetic relationships of Angelica species were resolved. The Angelica cp genomes had the typical quadripartite structure including a pair of inverted repeats (IRs: 5836-34,706 bp) separated by a large single-copy region (LSC: 76,657-103,161 bp) and a small single-copy region (SSC: 17,433-21,794 bp). Extensive expansion and contraction of the IR region were observed among cp genomes of Angelica species, and the pattern of the diversification of cp genomes showed high consistency with the phylogenetic placement of Angelica species. Species of Angelica were grouped into two major clades, with most species grouped in the Angelica group and A. omeiensis and A. sinensis grouped in the Sinodielsia with Ligusticum tenuissimum. CONCLUSIONS: Our results further demonstrate the power of plastid phylogenomics in enhancing the phylogenetic reconstructions of complex genera and provide new insights into plastome evolution across Angelica L.

The complete chloroplast genome of the Euphorbia maculata L. (Euphorbiaceae): characterization and phylogeny.

Euphorbia maculata is an important medicinal plants of the family Euphorbiaceae. The complete chloroplast genome reported here is 162,685 bp in length, including two inverted repeats (IRs) of 26,822 bp, which are separated by a large single-copy (LSC) and a small single-copy (SSC) of 90,514 bp and 18,527 bp, respectively. The whole chloroplast genome of E. maculata contains 111 genes, including 77 protein-coding genes, 4 transfer RNA, and 30 ribosome RNA. Phylogenetic analysis indicated that E. maculata is closely related to E. milii and E. tirucalli.

The complete chloroplast genome of a medicinal plant, Abutilon theophrasti Medik. (Malvaceae).

Abutilon theophrasti Medik. is an annual weed, widely distributed in Asia and Europe. The complete chloroplast genome reported here is 160,446 bp in length, including two inverted repeats (IRs) of 25,064 bp, which are separated by a large single-copy (LSC) and a small single-copy (SSC) of 89,089 and 21,229 bp, respectively. The whole chloroplast genome of A. theophrasti contains 113 distinct genes, including 79 protein-coding genes, 30 transfer RNA, and four ribosome RNA. Phylogenetic analysis indicated that A. theophrasti is located in the basal position in Malveae.