Chromosomal level genome of Ilex asprella and insight into antiviral triterpenoid pathway.

Ilex asprella is a widely used herbs in Traditional Chinese Medicine for treating viral infection and relieving inflammation. Due to the earlier fruiting period of I. asprella, it is the major food source for frugivores in summer. Despite its pharmacological and ecological importance, a reference genome for I. asprella is lacking. By using Illumina, stLFR and Omni-C sequencing data, we present the first chromosomal-level assembly for I. asprella. The genome assembly size is 804 Mbp, with Benchmarking Universal Single-Copy Orthologs (BUSCO) score 94.4% for eudicotyledon single copy genes. Transcriptomes of leaves, stems, flowers, premature fruits and roots were analyzed, providing 39,215 gene models. The complete set of genes involved in the triterpenoids production is disclosed for the first time. We have also found the oxidosqualene cyclases (OSCs), CYP716s and UDP-glycosyltransferases (UGTs), which are responsible for the modification of triterpenoid backbones, resulting in the high variety of triterpenoid saponins.

Comparative analysis and phylogenetic investigation of Hong Kong Ilex chloroplast genomes.

Ilex is a monogeneric plant group (containing approximately 600 species) in the Aquifoliaceae family and one of the most commonly used medicinal herbs. However, its taxonomy and phylogenetic relationships at the species level are debatable. Herein, we obtained the complete chloroplast genomes of all 19 Ilex types that are native to Hong Kong. The genomes are conserved in structure, gene content and arrangement. The chloroplast genomes range in size from 157,119 bp in Ilex graciliflora to 158,020 bp in Ilex kwangtungensis. All these genomes contain 125 genes, of which 88 are protein-coding and 37 are tRNA genes. Four highly varied sequences (rps16-trnQ, rpl32-trnL, ndhD-psaC and ycf1) were found. The number of repeats in the Ilex genomes is mostly conserved, but the number of repeating motifs varies. The phylogenetic relationship among the 19 Ilex genomes, together with eight other available genomes in other studies, was investigated. Most of the species could be correctly assigned to the section or even series level, consistent with previous taxonomy, except Ilex rotunda var. microcarpa, Ilex asprella var. tapuensis and Ilex chapaensis. These species were reclassified; I. rotunda was placed in the section Micrococca, while the other two were grouped with the section Pseudoaquifolium. These studies provide a better understanding of Ilex phylogeny and refine its classification.

Bioassay-guided isolation of norviburtinal from the root of Rehmannia glutinosa, exhibited angiogenesis effect in zebrafish embryo model.

ETHNOPHARMACOLOGICAL RELEVANCE: The root of Rehmannia glutinosa (RR) is commonly used as a wound-healing agent in various traditional Chinese herbal formulae; while angiogenesis is one of the crucial aspects in wound-healing. AIM OF THE STUDY: The objective of the present study was to investigate the angiogenesis effects of RR aqueous crude extract and its active component(s) using zebrafish model. MATERIALS AND METHODS: The in vivo angiogenesis effect was studied using transgenic TG(fli1:EGFP)(y1)/+(AB) zebrafish embryos by observing the capillary sprouts formation in sub-intestinal vessel (SIV) of zebrafish embryos after 72 h post-fertilization under fluorescence microscopy. RESULTS: Our results indicated that RR aqueous crude extract (250 µg/ml) exhibited significant angiogenesis effect, with an increase in capillary sprouts formation in SIV. Following sequential solvent partition of the RR aqueous crude extract with dichloromethane, ethyl acetate and n-butanol successively, the dichloromethane fraction (DCM) was found to have the most sprouts formation in the SIV region. Subjected to column chromatography, DCM fraction was further fractionated into six sub-fractions and among these tested, the sub-fraction C2 exhibited the most potent angiogenesis effect. The major component, C2A, was isolated and identified as norviburtinal using nuclear magnetic resonance (NMR) and mass spectrometry (MS). The compound norviburtinal (at 50 µg/ml) was shown to possess significant angiogenesis effect in zebrafish model (p < 0.001). CONCLUSIONS: Norviburtinal was, for the first time, found in the extract of RR and possessed novel angiogenesis effect. Bioassay-guided fractionation suggested that norviburtinal was not the only active component responsible for the angiogenesis effect of RR.

In vitro & in vivo assessment of a herbal formula used topically for bone fracture treatment.

AIM OF THE STUDY: A novel topical paste used for fracture healing (FH), consisting of the extracts of six herbs, Radix Dipsaci, Ramulus Sambucus Williamsii, Rhizoma Notoginseng, Flos Carthami, Rhizoma Rhei and Fructus Gardeniae, was developed according to the classical theory of traditional Chinese medicine. This study aimed to determine the effectiveness of this formula, and some of its important chemical components in the promotion of fracture healing. The transdermal transport of FH was also examined. MATERIALS AND METHODS: The osteogenic, angiogenic and nitric oxide suppressing effects of FH and its important chemical marker components were assessed by using osteoblastosacroma UMR-106 cells, human umbilical vein endothelial cells (HUVEC) and murine macrophage RAW264.7 cells, respectively. The bone healing effects of the FH paste and its transdermal absorption were determined using a rabbit fracture model. The callus sizes, bone specific alkaline phosphatase levels and biomechanical properties of the healed bone were assessed. RESULTS: FH significantly increased the cell proliferation in UMR-106 and HUVEC cells and inhibited the nitric oxide production in murine macrophage in dose-dependent manner. Its important chemical components asperosaponin VI, ginsenoside Rg1 and emodin were shown to be acting positively in the respective in vitro studies. FH paste significantly improved the bone healing in the rabbit fracture model, as was indicated by the increases in callus size at weeks 2-5, and the elevations in bone specific alkaline phosphatase activities at weeks 5-6. The analysis using LC/MS/MS also showed the presence of important chemical marker components of the FH formula in the plasma after 8 weeks of topical treatment. CONCLUSION: This study presents the first scientific evidence of the efficacy of a herbal paste in the promotion of fracture healing. There were evidences of transdermal transport of the chemical components, control the inflammation through nitric oxide inhibition, promotion of angiogenesis, and bone healing in the in vitro tests, as well as in the experimental animal.