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Journal of Pharmaceutical Analysis Oct 2019Jervine, a novel steroidal alkaloid from L., exhibits both antitumor effect and potential toxicity. The aim of study was to characterize the pharmacokinetic behaviors...
Jervine, a novel steroidal alkaloid from L., exhibits both antitumor effect and potential toxicity. The aim of study was to characterize the pharmacokinetic behaviors and enterohepatic circulation of jervine in rats. A rapid and simple ultra-high performance liquid chromatography-tandem mass spectrometric method was developed and validated for quantification of jervine and alpinetin (internal standard) in rat plasma. After extraction from rat plasma by a simple protein-precipitation method, the analyte was separated on a C column (2.1 mm × 50 mm, 1.7 μm) using water with 0.1% formic acid and acetonitrile as the mobile phase delivered at a flow rate of 0.4 mL/min. Jervine and alpinetin were determined in the positive mode with multiple reaction monitoring (MRM) of the ion transitions at 426.3 → 108.8 and 271.0 → 166.9, respectively. Molecular docking method was used to investigate the binding of jervine to p-glycoprotein and dehydroepiandrosterone sulfotransferase. The method was well validated within acceptance limits including specificity, matrix effect, recovery, precision, accuracy, and stability, and was successfully applied to the pharmacokinetic study of jervine after oral and intravenous administration to rats. Jervine presented a small volume of distribution, fast absorption, high oral bioavailability, and enterohepatic circulation. The enterohepatic circulation was first observed in veratrum alkaloids, and was further investigated by molecular docking studies, which was related to the binding of jervine to p-glycoprotein and dehydroepiandrosterone sulfotransferase. The pharmacokinetic properties and enterohepatic circulation of jervine in rats provided a significant basis for the drug-drug interaction and toxicity study in the future.
PubMed: 31929946
DOI: 10.1016/j.jpha.2019.04.004 -
Phytochemistry Mar 2021Fritillaria cirrhosa D. Don (Liliaceae, syn. Fritillaria roylei Hook.) is a critically endangered medicinal herb of immense importance due to its pharmaceutical...
Comparative transcriptome analysis infers bulb derived in vitro cultures as a promising source for sipeimine biosynthesis in Fritillaria cirrhosa D. Don (Liliaceae, syn. Fritillaria roylei Hook.) - High value Himalayan medicinal herb.
Fritillaria cirrhosa D. Don (Liliaceae, syn. Fritillaria roylei Hook.) is a critically endangered medicinal herb of immense importance due to its pharmaceutical bioactive compound, especially sipeimine, used for the treatment of chronic respiratory disorders. However, the industrial demand for sipeimine solely depends on its endangered natural habitat. Therefore; there is an utmost need for its biodiversity conservation as well as for the sustainable utilization of phytochemicals. Plant cell culture and transcriptomics-based molecular bioprospection of key regulatory genes involved in sipeimine biosynthesis as such will play a crucial role in exploring the unexplored traits, that are in supply crisis or nearly in extinction stage. De novo comparative transcriptome sequencing of the bulb (in vivo), callus, and regenerated plantlets (in vitro) resulted in more than 150 million high-quality paired-end clean reads that assembled into final 31,428 transcripts. Functional annotation and unigenes classification with multiple public databases such as KEGG, Refseq, Uniprot, TAIR, GO, and COG, etc. along with chemical structures and functional biocatalytic activity analysis of different steroidal alkaloids facilitated the identification of 30 unigenes specific to sipeimine biosynthesis. Additionally, ABC transporters and TFs like bHLH, MYC, MYB, and WRKY suggests their possible role in metabolite translocation and regulation in vivo as well as in vitro tissues. Differential gene expression and quantitative analysis revealed that the MVA pathway probably the predominant route for 5C intermediate (IPP & DMAPP) biosynthesis. Further, the genes involved in the downstream biosynthesis pathway viz. SQLE, CAS1, SMT1, SMO1, SMO2, SC5DL, DHCR7, DHCR24, CYP710A, 3β-HSD, CYP90D2, and CYP374A6 shown similar expression pattern with RNA-Seq and qRT-PCR findings. The positive correlation between higher expression of proposed biosynthetic pathway genes and relatively higher accumulation of sipeimine in differentiated naturally grown bulb tissues (in vivo), undifferentiated cells (callus), and de-differentiated tissues i.e. regenerated plantlets (in vitro) has been evident from the present study. Comprehensive genomic resources created in F. cirrhosa will provide strong evidence of bulb derived in vitro culture as an alternative promising source for steroidal alkaloids biosynthesis and metabolite upscaling through genetic and metabolic engineering.
Topics: Cevanes; Fritillaria; Gene Expression Profiling; Gene Expression Regulation, Plant; Liliaceae; Plants, Medicinal; Transcriptome
PubMed: 33370713
DOI: 10.1016/j.phytochem.2020.112631 -
International Journal of Molecular... Nov 2015Protein transduction using cell-penetrating peptides (CPPs) is useful for the delivery of large protein molecules, including some transcription factors. This method is... (Review)
Review
Protein transduction using cell-penetrating peptides (CPPs) is useful for the delivery of large protein molecules, including some transcription factors. This method is safer than gene transfection methods with a viral vector because there is no risk of genomic integration of the exogenous DNA. Recently, this method was reported as a means for the induction of induced pluripotent stem (iPS) cells, directing the differentiation into specific cell types and supporting gene editing/correction. Furthermore, we developed a direct differentiation method to obtain a pancreatic lineage from mouse and human pluripotent stem cells via the protein transduction of three transcription factors, Pdx1, NeuroD, and MafA. Here, we discuss the possibility of using CPPs as a means of directing the differentiation of iPS cells and other stem cell technologies.
Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Cell Differentiation; Cell-Penetrating Peptides; Cellular Reprogramming; Cinnamates; Gene Expression; Glucagon-Like Peptide 1; Homeodomain Proteins; Humans; Induced Pluripotent Stem Cells; Insulin-Secreting Cells; Intercellular Signaling Peptides and Proteins; Maf Transcription Factors, Large; Mice; Nerve Tissue Proteins; Niacinamide; Trans-Activators; Tretinoin; Veratrum Alkaloids
PubMed: 26561805
DOI: 10.3390/ijms161125986 -
Molecules (Basel, Switzerland) Sep 2018is a rich source of steroidal alkaloids such as cyclopamine, a known inhibitor of the Hedgehog (Hh) signaling pathway. Here we provide a detailed analysis of the...
is a rich source of steroidal alkaloids such as cyclopamine, a known inhibitor of the Hedgehog (Hh) signaling pathway. Here we provide a detailed analysis of the alkaloid composition of by plant part through quantitative analysis of cyclopamine, veratramine, muldamine and isorubijervine in the leaf, stem and root/rhizome of the plant. To determine whether additional alkaloids in the extracts contribute to Hh signaling inhibition, the concentrations of these four alkaloids present in extracts were replicated using commercially available standards, followed by comparison of extracts to alkaloid standard mixtures for inhibition of Hh signaling using Shh-Light II cells. Alkaloid combinations enhanced Hh signaling pathway antagonism compared to cyclopamine alone, and significant differences were observed in the Hh pathway inhibition between the stem and root/rhizome extracts and their corresponding alkaloid standard mixtures, indicating that additional alkaloids present in these extracts are capable of inhibiting Hh signaling.
Topics: Alkaloids; Animals; Biomass; Hedgehog Proteins; Mice; NIH 3T3 Cells; Plant Extracts; Plant Leaves; Plant Roots; Plant Stems; Reference Standards; Signal Transduction; Veratrum
PubMed: 30200443
DOI: 10.3390/molecules23092222 -
International Journal of Molecular... May 2022Clear cell renal cell carcinoma (ccRCC) is the most common and aggressive subtype of kidney cancer, with high mortality rates worldwide. The sonic hedgehog (SHH)...
Clear cell renal cell carcinoma (ccRCC) is the most common and aggressive subtype of kidney cancer, with high mortality rates worldwide. The sonic hedgehog (SHH) molecular cascade is altered in various malignancies in tumorigenesis, and several SHH pathway inhibitors have been considered as potential anticancer drugs. The aim of the present study was to determine the expression profile of SHH signaling components and their target genes in ccRCC. Additionally, the present study examined the effects of SHH pathway inhibitory drugs (RU‑SKI43, cyclopamine and GLI‑antagonist 61) on cell viability, cell cycle progression, expression levels of SHH target genes and migration ability in 786‑O, ACHN and HK2 cells. The study also included paired tumor and normal samples from 62 patients with ccRCC. The mRNA levels in clinical samples and cell lines were measured via reverse transcription‑quantitative PCR. Cell viability was examined using a sulforhodamine B assay. Flow cytometry was used to investigate cell cycle progression and the migratory rate of cells was assessed using a wound healing assay. High mRNA levels of , smoothened (), glioma‑associated zinc finger protein (), BCL2 apoptosis regulator (), MYC proto‑oncogene (), vascular endothelial growth factor A () and cyclin D1 () were observed in the tumor tissues, especially in early ccRCC, according to the TNM stage or World Health Organization/International Society of Urological Pathology (ISUP) grade. High expression levels of , as well as low mRNA expression, were associated with short overall survival, and increased expression was an independent prognostic factor of a poor outcome in patients with advanced ISUP grade (Cox hazard ratio test). Cyclopamine treatment was found to arrest 786‑O cells in the G/M phase and decreased the expression levels of , , and . RU‑SKI43 inhibited cell migration and decreased the expression levels of , and in ACHN cells. Overall, the results of the present study suggested that SHH signaling may be involved in the early development of ccRCC, and the expression levels of and may serve as prognostic factors of this disease. Cyclopamine and RU‑SKI43 appear to be potential anti‑renal cell carcinoma drugs; however, this hypothesis requires verification by further studies.
Topics: Carcinoma, Renal Cell; Hedgehog Proteins; Humans; Kidney Neoplasms; Vascular Endothelial Growth Factor A; Veratrum Alkaloids; Zinc Finger Protein GLI1
PubMed: 35266008
DOI: 10.3892/ijmm.2022.5114 -
Plants (Basel, Switzerland) Feb 2020L. (Liliaceae), a monocotyledonous species distributed throughout the Changbai mountains of Northeast China, is pharmaceutically important, due to the capacity to...
L. (Liliaceae), a monocotyledonous species distributed throughout the Changbai mountains of Northeast China, is pharmaceutically important, due to the capacity to produce the anticancer drug cyclopamine. An efficient transformation system of mediated with is presented. Murashige and Skoog (MS) medium containing 8 mg/L picloram was used to induce embryogenic calli from immature embryos with 56% efficiency. LBA4404 carrying the bar gene driven by the cauliflower mosaic virus 35S promoter was employed for embryogenic callus inoculation. cell density OD660 = 0.8 for inoculation, half an hour infection period, and three days of co-culture duration were found to be optimal for callus transformation. Phosphinothricin (PPT, 16 mg/L) was used as the selectable agent, and a transformation efficiency of 15% (transgenic plants/100 infected calli) was obtained. The transgenic nature of the regenerated plants was confirmed by PCR and Southern blot analysis, and expression of the bar gene was detected by RT-PCR and Quick PAT/bar strips. The steroid alkaloids cyclopamine, jervine, and veratramine were detected in transgenic plants, in non-transformed and control plants collected from natural sites. The transformation system constitutes a prerequisite for the production of the pharmaceutically important anticancer drug cyclopamine by metabolic engineering of .
PubMed: 32033134
DOI: 10.3390/plants9020191 -
Natural Product Reports May 2016Covering: 1950s to 2015During the 1950s, sheep ranchers in the western United States experienced episodic outbreaks of cyclopic lambs. In this highlight I describe how... (Review)
Review
Covering: 1950s to 2015During the 1950s, sheep ranchers in the western United States experienced episodic outbreaks of cyclopic lambs. In this highlight I describe how these mysterious incidents were traced to the grazing of Veratrum californicum wildflowers by pregnant ewes, leading to the discovery of cyclopamine () as a plant-derived teratogen. The precise mechanism of cyclopamine action remained enigmatic for 30 years, until this steroid alkaloid was found to be the first specific inhibitor of Hedgehog (Hh) signalling and a direct antagonist of the transmembrane receptor Smoothened (SMO). In addition to being a valuable probe of Hh pathway function, cyclopamine has been used to demonstrate the therapeutic potential of Hh pathway inhibitors. I discuss the development of SMO antagonists as anticancer therapies and emerging challenges.
Topics: Animals; Drug Delivery Systems; Female; Hedgehog Proteins; Holoprosencephaly; Humans; Molecular Structure; Pregnancy; Sheep; Signal Transduction; Smoothened Receptor; Veratrum; Veratrum Alkaloids
PubMed: 26787175
DOI: 10.1039/c5np00153f -
Molecules (Basel, Switzerland) Jun 2018A simple and high sensitive ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the simultaneous...
A simple and high sensitive ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was developed and validated for the simultaneous determination of peimine and peiminine in beagle dog plasma after the oral administration of Maxim and Miq powder. Chromatographic separation was achieved on an ACQUIT UPLC BEH C column (1.7 μm, 2.1 × 100 mm) in a gradient elution way with a mobile phase consisting of acetonitrile and water containing 0.1% formic acid at a flow rate of 0.4 mL/min. The plasma samples were prepared by a liquid⁻liquid extraction (LLE) method with ethyl acetate. The analytes were detected with a triple quadrupole tandem mass spectrometry (MS) in multiple reaction monitoring (MRM) mode and a positive ion electrospray ionization (ESI) of the transitions at / 432.4→414.4 for peimine and / 430.3→412.3 for peiminine. The method was linear for two analytes over the investigated range with all determined correlation coefficients exceeding 0.9900. The lower limit of quantification (LLOQ) was 0.988 ng/mL for peimine and 0.980 ng/mL for peiminine. The mean extraction recoveries of peimine and peiminine at three quality control samples (QC) levels were ranged from 82.56 to 88.71%, and matrix effects ranged from 92.06 to 101.2%. The intra-day and inter-day precision and accuracy were within the acceptable limits at LLOQ and QC levels. The method was effectively and successfully applied to the pharmacokinetics of peimine and peiminine after oral administration of powder to beagle dogs. The obtained results may be help to guide the clinical application of Maxim and Miq.
Topics: Administration, Oral; Animals; Cevanes; Chromatography, Liquid; Dogs; Drugs, Chinese Herbal; Fritillaria; Male; Tandem Mass Spectrometry
PubMed: 29958456
DOI: 10.3390/molecules23071573 -
Journal of Dental Research Apr 2017Whereas neurovascular interactions in spinal neuropathic pain models have been well characterized, little attention has been given to such neurovascular interactions in...
Whereas neurovascular interactions in spinal neuropathic pain models have been well characterized, little attention has been given to such neurovascular interactions in orofacial neuropathic pain models. This study investigated in male Sprague-Dawley rats the vascular changes following chronic constriction injury (CCI) of the infraorbital nerve (IoN), a broadly validated preclinical model of orofacial neuropathic pain. Following IoN-CCI, an early downregulation of tight junction proteins Claudin-1 and Claudin-5 was observed within the endoneurium and perineurium, associated with increased local accumulation of sodium fluorescein (NaFlu) within the IoN parenchyma, as compared with sham animals. These events were evidence of local blood-nerve barrier disruption and increased vascular permeability. A significant upregulation of immunocytes (CD3, CD11b) and innate immunity (TLR2, TLR4) mRNA markers was also observed, suggestive of increased local inflammation. Finally, a significant downregulation of Hedgehog pathway readouts Patched-1 and Gli-1 was observed within the IoN after CCI and local injections of cyclopamine, a Hedgehog pathway inhibitor, replicated in naïve rats the molecular, vascular, and behavioral changes observed following IoN-CCI. These results suggest a major role of Hedgehog pathway inhibition in mediating local increased endoneurial and perineurial vascular permeability following trigeminal nerve injury, thus facilitating immunocytes infiltration, neuroinflammation development, and neuropathic pain-like aversive behavior.
Topics: Animals; Capillary Permeability; Claudin-1; Claudin-5; Disease Models, Animal; Hedgehog Proteins; Immunity, Innate; Immunohistochemistry; Male; Microscopy, Fluorescence; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; Tight Junctions; Trigeminal Nerve Injuries; Trigeminal Neuralgia; Veratrum Alkaloids
PubMed: 27856965
DOI: 10.1177/0022034516679395 -
Scientific Reports Jul 2020Voltage-gated Na (Na) channels regulate homeostasis in bacteria and control membrane electrical excitability in mammals. Compared to their mammalian counterparts,...
Voltage-gated Na (Na) channels regulate homeostasis in bacteria and control membrane electrical excitability in mammals. Compared to their mammalian counterparts, bacterial Na channels possess a simpler, fourfold symmetric structure and have facilitated studies of the structural basis of channel gating. However, the pharmacology of bacterial Na remains largely unexplored. Here we systematically screened 39 Na modulators on a bacterial channel (NaChBac) and characterized a selection of compounds on NaChBac and a mammalian channel (human Na1.7). We found that while many compounds interact with both channels, they exhibit distinct functional effects. For example, the local anesthetics ambroxol and lidocaine block both Na1.7 and NaChBac but affect activation and inactivation of the two channels to different extents. The voltage-sensing domain targeting toxin BDS-I increases Na1.7 but decreases NaChBac peak currents. The pore binding toxins aconitine and veratridine block peak currents of Na1.7 and shift activation (aconitine) and inactivation (veratridine) respectively. In NaChBac, they block the peak current by binding to the pore residue F224. Nonetheless, aconitine has no effect on activation or inactivation, while veratridine only modulates activation of NaChBac. The conservation and divergence in the pharmacology of bacterial and mammalian Na channels provide insights into the molecular basis of channel gating and will facilitate organism-specific drug discovery.
Topics: Aconitine; Anesthetics, Local; Bacterial Proteins; Drug Interactions; Electrophysiological Phenomena; HEK293 Cells; Humans; Ion Channel Gating; NAV1.7 Voltage-Gated Sodium Channel; Small Molecule Libraries; Sodium Channels; Toxins, Biological; Veratridine; Voltage-Gated Sodium Channel Agonists
PubMed: 32612253
DOI: 10.1038/s41598-020-67761-5