Discovery of a Potent Antiosteoporotic Drug Molecular Scaffold Derived from Angelica sinensis and Its Bioinspired Total Synthesis.

Angelica sinensis, commonly known as Dong Quai in Europe and America and as Dang-gui in China, is a medicinal plant widely utilized for the prevention and treatment of osteoporosis. In this study, we report the discovery of a new category of phthalide from Angelica sinensis, namely falcarinphthalides A and B (1 and 2), which contains two fragments, (3R,8S)-falcarindiol (3) and (Z)-ligustilide (4). Falcarinphthalides A and B (1 and 2) represent two unprecedented carbon skeletons of phthalide in natural products, and their antiosteoporotic activities were evaluated. The structures of 1 and 2, including their absolute configurations, were established using extensive analysis of NMR spectra, chemical derivatization, and ECD/VCD calculations. Based on LC-HR-ESI-MS analysis and DFT calculations, a production mechanism for 1 and 2 involving enzyme-catalyzed Diels-Alder/retro-Diels-Alder reactions was proposed. Falcarinphthalide A (1), the most promising lead compound, exhibits potent in vitro antiosteoporotic activity by inhibiting NF-κB and c-Fos signaling-mediated osteoclastogenesis. Moreover, the bioinspired gram-scale total synthesis of 1, guided by intensive DFT study, has paved the way for further biological investigation. The discovery and gram-scale total synthesis of falcarinphthalide A (1) provide a compelling lead compound and a novel molecular scaffold for treating osteoporosis and other metabolic bone diseases.

Eight new phenolic acids from the leaves of Illicium dunnianum and their osteoprotective activities.

Eight previously unreported phenolic acids (1-8), including three new phenylpropenoid glycosides (1-3), and five undescribed shikimic acid derivatives (4-8), together with six known analogues (9-14), were obtained from the dried leaves of Illicium dunnianum. The structures of these new compounds were elucidated by extensive spectroscopic analyses (1D, 2D-NMR, HRESIMS, IR, UV) and chemical methods. Compounds 1, 2, 4, and 5 were tested for their promotion effect on osteoblastogenesis of pre-osteoblastic MC3T3-E1 cells and inhibitory effect on osteoclastogenesis of RANKL-induced RAW264.7 cells. As a result, 1 and 4 exerted a promotion effect on osteoblastogenesis, but without activity on osteoclastogenesis. Our studies not only enrich the structural diversity of phenolic acids in nature, but also discover new lead compounds from folk plants with activities on osteoblastogenesis or osteoclastogenesis.

A new strategy for discovering effective substances and mechanisms of traditional Chinese medicine based on standardized drug containing plasma and the absorbed ingredients composition, a case study of Xian-Ling-Gu-Bao capsules.

ETHNOPHARMACOLOGICAL RELEVANCE: The overall therapeutic effect of traditional Chinese medicine formulae (TCMF) was achieved by the interactions of multiple components with multiple targets. However, current pharmacology research strategies have struggled to identify effective substance groups and encountered challenges in elucidating the underlying mechanisms of TCMF. AIM: In this study, a comprehensive strategy was proposed and applied to elucidate the interactions of the multiple components that underlie the functions of the famous TCMF: Xian-Ling-Gu-Bao (XLGB) capsule on bone metabolism in vivo and to elucidate the molecular mechanisms underlying the effects of XLGB on bone cells, especially on osteoblasts. METHODS: The efficacy of XLGB in the protection against bones loss in ovariectomized (OVX) rats was confirmed by Micro-CT analysis. The anti-osteoporosis mechanism involved in the systemic regulatory actions of XLGB was elucidated by transcriptome sequencing analysis on bone marrow mesenchymal stem cells isolated from OVX rats. Moreover, the components absorbed in XLGB-treated plasma were characterized by mass spectrometry analysis, and subsequently, a standardized preparation process of drug-containing plasma was established. The synergistic osteogenic effect of the multiple components in plasma was investigated by a combination and then knockout of components using pre-osteoblast MC3T3-E1 cells. In order to decipher the underlying mechanism of XLGB, the targets of the absorbed components on bone were predicted by target prediction and network pharmacology analysis, then several interactions were validated by biochemical and cell-based assay. RESULTS: A total of 18 genes, including HDC, CXCL1/2, TNF, IL6 and Il1b, were newly found to be the major target genes regulated by XLGB. Interestingly, we found that a combination of the three absorbed components, i.e. MSP, rather than their single form at the same concentration, stimulated the formation of calcified nodules in MC3T3-E1 cells, suggesting a synergistic effect of these components. Besides, target prediction and experimental validation confirmed the binding affinity of corylin and icaritin for estrogen receptor α and ß, the inhibitory activity of isobavachin and isobavachalcone on glycogen synthase kinase-3ß, and the inhibitory activity of isobavachalcone on cathepsin K. The cell-based assay further confirmed the result of the biochemical assay. A network that integrated absorbed components of XLGB-targets-perturbation genes-pathways against osteoporosis was established. CONCLUSION: Our current study provides a new systemic strategy for discovering active ingredient groups of TCM formulae and understanding their underlying mechanisms.

A novel strategy for exploring food originated anti-adipogenesis substances and mechanism by structural similarity evaluation, ADME prediction, network pharmacology and experimental validation.

Screening potential functional substances based on active compounds is still a challenge faced by researchers since hundreds and thousands of possible compounds exist in natural products (food, herb, etc.). In this study, an integrated strategy by a combination of structural similarity evaluation, ADME (absorption, distribution, metabolism, excretion) prediction, network pharmacology and experimental validation (SANE strategy) was proposed and applied to explore anti-adipogenesis substances. This strategy was divided into four parts: first, potential compounds were screened based on representative active compounds by similarity evaluation and ADME prediction. Second, the activity of targeted compounds was evaluated in vitro based on the molecular biology method. Third, network pharmacology was used to explore potential targets and pathways. Last, the core pharmacological mechanism was confirmed by modern pharmacology methods. As a result, 8-prenylgenistein (8PG) was screened with chemical structure similarity with genistein and improved ADME propriety. Meanwhile, 8PG was found to present significant anti-adipogenesis effects in pre-adipocyte 3T3-L1 cells and primary human bone marrow stromal cells (hBMSC). Through using methods including: chemical staining, functional assays, and Real time PCR, 8PG was found to present more potency than genistein in suppressing the adipocyte differentiation. Further, the potential pharmacological mechanism was predicted, and significant effects of 8PG on activating the Wnt/ß-catenin pathway in 3T3-L1 cells and hBMSC were confirmed by  immunoblotting in the absence/presence of signaling pathway blocker and immunofluorescence staining. A new insight for exploring more potent compounds based on accurate effect compounds is provided in our work. Moreover, a potential compound (8PG), suppressing adipogenesis, was also supplied.

Discovery of a New Class of Cathepsin K Inhibitors in Rhizoma Drynariae as Potential Candidates for the Treatment of Osteoporosis.

Rhizoma Drynariae (RD), as one of the most common clinically used folk medicines, has been reported to exert potent anti-osteoporotic activity. The bioactive ingredients and mechanisms that account for its bone protective effects are under active investigation. Here we adopt a novel in silico target fishing method to reveal the target profile of RD. Cathepsin K (Ctsk) is one of the cysteine proteases that is over-expressed in osteoclasts and accounts for the increase in bone resorption in metabolic bone disorders such as postmenopausal osteoporosis. It has been the focus of target based drug discovery in recent years. We have identified two components in RD, Kushennol F and Sophoraflavanone G, that can potentially interact with Ctsk. Biological studies were performed to verify the effects of these compounds on Ctsk and its related bone resorption process, which include the use of in vitro fluorescence-based Ctsk enzyme assay, bone resorption pit formation assay, as well as Receptor Activator of Nuclear factor κB (NF-κB) ligand (RANKL)-induced osteoclastogenesis using murine RAW264.7 cells. Finally, the binding mode and stability of these two compounds that interact with Ctsk were determined by molecular docking and dynamics methods. The results showed that the in silico target fishing method could successfully identify two components from RD that show inhibitory effects on the bone resorption process related to protease Ctsk.

Isolation and identification of metabolites of bakuchiol in rats.

Bakuchiol, the main active component of Psoralea corylifolia, showed a range of significant pharmacological activities, including antimicrobial, antiinflammatory, reduction of bone loss and estrogenic activities. In this research, 12 metabolites, including 11 new compounds, were isolated from the urine and feces of rats after oral administration of bakuchiol, and their structures were elucidated by extensive spectroscopic analysis. The possible metabolic pathways of bakuchiol in rats were proposed, and a rare bile acid conjugation reaction was found. In addition, bakuchiol and its metabolites M1-M3 were studied for their alkaline phosphatase (ALP) activities on MC3T3-E1 cells and cytotoxicity on HKC-8 cells. The data showed that bakuchiol exerted significant effects on ALP activity of MC3T3-E1 cells and cytotoxicity on HKC-8 cells, while M1-M3 only showed ALP activities at 10(-5)M on MC3T3-E1 cells and no cytotoxicity on HKC-8 cells.