Full-length transcriptome profiling of Acanthopanax gracilistylus provides new insight into the kaurenoic acid biosynthesis pathway.

Acanthopanax gracilistylus is a deciduous plant in the family Araliaceae, which is commonly used in Chinese herbal medicine, as the root bark has functions of nourishing the liver and kidneys, removing dampness and expelling wind, and strengthening the bones and tendons. Kaurenoic acid (KA) is the main effective substance in the root bark of A. gracilistylus with strong anti-inflammatory effects. To elucidate the KA biosynthesis pathway, second-generation (DNA nanoball) and third-generation (Pacific Biosciences) sequencing were performed to analyze the transcriptomes of the A. gracilistylus leaves, roots, and stems. Among the total 505,880 isoforms, 408,954 were annotated by seven major databases. Sixty isoforms with complete open reading frames encoding 11 key enzymes involved in the KA biosynthesis pathway were identified. Correlation analysis between isoform expression and KA content identified a total of eight key genes. Six key enzyme genes involved in KA biosynthesis were validated by real-time quantitative polymerase chain reaction. Based on the sequence analysis, the spatial structure of ent-kaurene oxidase was modeled, which plays roles in the three continuous oxidations steps of KA biosynthesis. This study greatly enriches the transcriptome data of A. gracilistylus and facilitates further analysis of the function and regulation mechanism of key enzymes in the KA biosynthesis pathway. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-024-01436-7.

Comparative transcriptome analysis of different tissues of Solanum khasianum reveals candidate genes involved in steroidal glycoalkaloid biosynthesis.

Fruits and leaves of Solanum khasianum C. B. Clarke have long been used as a common Chinese herbal medicine. Steroidal glycoalkaloids (SGAs), the main active ingredient in S. khasianum, exhibit various pharmacological effects. However, genes involved in the SGA biosynthetic pathway in S. khasianum have not yet been identified. Genes encoding potential key SGA biosynthesis enzymes were identified through comprehensive RNA sequencing analysis (RNA-seq) of S. khasianum leaves, stems, and fruits. A total of 123,704 unigenes were obtained, of which 109,775 (88.74%) were annotated in seven public databases. Among these, 54 unigenes potentially involved in SGA biosynthesis were identified. Additionally, 23,636 differentially expressed genes were identified by comparing gene expression levels among the fruits, stems, and leaves of S. khasianum. The structural characteristics and phylogenetic relationship of cycloartenol synthase involved in SGA biosynthesis were further analyzed. Solasodine constituent was detected by high-performance liquid chromatography. This is the first study to report the comparative transcriptome analysis of different tissues of S. khasianum that identifies valuable genes potentially involved in SGA biosynthesis in this species.

[Identification of key enzyme genes involved in biosynthesis pathways of lignan and lignin in Eucommia ulmoides based on transcriptome assembly].

Lignan is the main medicinal component of Eucommia ulmoides, and lignin is involved in the defense of plants against diseases and insect pests.They are synthesized from coniferyl alcohol with the help of dirigent(DIR) and peroxidase(POD), respectively.In this study, transcriptome assembly of stems and leaves of E.ulmoides was performed, yielding 112 578 unigenes.Among them, 70 459 were annotated in seven databases.A total of 59 unigenes encodes 11 key enzymes in the biosynthesis pathways of lignin and lignin, of which 11 encode POD and 8 encode DIR.A total of 13 unigenes encoding transcription factors are involved in phenylpropanoid metabolism. Compared with leaves of E.ulmoides, 7 575 unigenes were more highly expressed in stems, of which 462 were involved in phenylpropanoid biosynthesis.Our results extend the public transcriptome dataset of E.ulmoides, which provide valuable information for the analysis of biosynthesis pathways of lignan and lignin in E.ulmoides and lay a foundation for further study on the functions and regulation mechanism of key enzymes in lignan and lignin biosynthesis pathways.

Probing the transcriptome of Boehmeria nivea reveals candidate genes associated with the biosynthesis of chlorogenic acid.

Boehmeria nivea (L.) Gaudich is used in traditional Chinese medicine. Chlorogenic acids are major medically active components of Boehmeria nivea, which can be used clinically to treat hyperglycemia, pneumonia, and cancer. To identify the genes involved in chlorogenic acid biosynthesis, we analyzed transcriptome data from leaf, root, and stem tissues of Boehmeria nivea using the Illumina Hi-Seq 4000 platform. A total of 146,790 unigenes were obtained from Boehmeria nivea, of which 106,786 were annotated in public databases. In analyses of the KEGG (Kyoto Encyclopedia of Genes and Genome) database, 484 unigenes that encode the five key enzymes involved in chlorogenic acid biosynthesis were identified, and shikimate O-hydroxycinnamoyl transferase was spatially simulated. Some of these key enzyme unigenes expression levels were verified by RT-qPCR (real-time quantitative Polymerase Chain Reaction). Furthermore, multiple genes encoding plant resistance proteins or transcription factors were identified and analyzed. Differentially expressed genes were identified by performing pairwise comparison of genes between tissues. This study increases the number of public transcript datasets of this species and identifies candidate genes related to the biosynthesis of chlorogenic acid, laying a foundation for the further exploration of this pathway in Boehmeria nivea.

Role of Acupuncture in Amblyopia Treatment.

CONTEXT: Amblyopia is an ophthalmological developmental disorder that occurs during early childhood due to disrupted binocular vision. It leads to reduced best-corrected visual acuity in one eye (seldom in both eyes) in which the visual acuity of one eye is 2 rows weaker than the other eye on a Snellen vision testing chart. Many studies have reported the outcomes of acupuncture in the treatment of amblyopia. Evidence suggests that acupuncture improves outcomes in children with amblyopia, but these studies are associated with several limitations which may affect the evaluation of the efficacy and reproducibility of studies on acupuncture. One important limitation is the lack of sham acupuncture as a control which has not been used in any clinical trial. The use of nonacupoint acupuncture is suggested as a placebo to improve the quality of evidence and comparability?

Bioactive minor components of the total salvianolic acids injection prepared from Salvia miltiorrhiza Bge.

The total salvianolic acids are main effective constituents of Salvia miltiorrhiza Bge., a traditional Chinese medicine used for thousands of years. The purpose of present study was to make clear the composition and bioactivities of the minor components of the total salvianolic acids injection. As a result, three new minor phenolic acids (1-3) together with six known compounds (4-9) were characterized from the total salvianolic acids injection. Their structures were elucidated by extensive analysis of the spectral data. The absolute configuration of compounds 1-3 were confirmed by their J7',8' observed in (1)H NMR spectra, absorption band at approximately 250-260nm in their CD spectra as well as chemical shifts of C-8″ and C-8‴ displayed in (13)C NMR spectra. Then DPPH free radical scavenging assay and NAD(P)H: quinine oxidoreductase 1 (NQO1) inducing activity test were employed to evaluate the antioxidant effect of new minor compounds 1 and 2. Compound 2 showed significant NQO1 inducing activity at 20µM with IR value 2.6. Meanwhile, DPPH scavenging assay revealed that the inhibition rates of compounds 1 and 2 were 84.3% and 74.9% at 2mM, respectively.

Detection and chemical profiling of Ling-Gui-Zhu-Gan decoction by ultra performance liquid chromatography-hybrid linear ion trap-Orbitrap mass spectrometry.

Ling-Gui-Zhu-Gan decoction (LGZGD), a well-known traditional Chinese medicine (TCM) formula, has been extensively used for the treatment of cardiovascular disease in clinic. However, the chemical constituents in LGZGD had not been investigated so far. In this study, an ultra performance liquid chromatography-hybrid electrospray ionization linear ion trap-Orbitrap mass spectrometry (UPLC-LTQ-Oribitrap-MS/MS) method was established for rapid separation and structural identification of the constituents in LGZGD. Separation was performed on an ACQUITY(TM) UPLC BEH C18 column (50 × 2.1 mm, 1.7 µm) by gradient elution mode, using acetonitrile-water containing 0.1% formic acid as mobile phase at the flow rate of 0.2 mL/min. Accurate mass measurement for molecular ions and characteristic fragment ions could represent identification criteria for these compounds. As a result, 95 compounds including triterpene acids, triterpene saponins, flavonoids, coumarins, coumestans, benzofurans, phenylpropanoids and sesquiterpenoid lactones were detected, and 90 of them were tentatively identified. All compounds were further assigned in the individual raw material. In conclusion, the UPLC-LTQ-Orbitrap-MS/MS is a highly efficient technique to separate and identify constituents in complex matrices of TCMs. These results obtained in this research will provide a basis for quality control and further in vivo study of LGZGD.

Pharmacokinetics, tissue distribution and excretion study of dictamnine, a major bioactive component from the root bark of Dictamnus dasycarpus Turcz. (Rutaceae).

Dictamnine is an herbal ingredient isolated from the root bark of Dictamnus dasycarpus Turcz. (Rutaceae). The present study was aimed at the development of an ultra-high performance liquid chromatography-tandem mass spectrometry method to quantify the concentration of dictamnine in rat plasma and tissues for the in vivo pharmacokinetics, tissue distribution and excretion study. Biological samples were processed with protein precipitation. Skimmianine was chosen as internal standard. The chromatographic separation was carried out on a Thermo Syncronis C18 column (2.1mm×50mm, 1.7µm) with an isocratic mobile phase consisting of methanol and 0.1% formic acid water (75:25, v/v). The detection was accomplished by using positive ion electrospray ionization in multiple reaction monitoring (MRM) mode. The MS/MS ion transitions were monitored at m/z 200.0→129.0 for dictamnine and 260.3→227.1 for IS, respectively. An excellent linearity was observed over the concentration range from 0.5 to 250ng/mL. The lower limit of quantification (LLOQ) was 0.5ng/mL for dictamnine. The developed method was rapid, accurate, and highly sensitive and selective. It was successfully applied to the in vivo pharmacokinetics, tissue distribution and excretion study of dictamnine in rats after oral or intravenous administration of dictamnine.