Search Results (5)

The genus Fritillaria is characterized by the production of alkaloids, primarily of the isosteroidal type. The aim of this preliminary study was to perform a histolocalization screening aimed at revealing the presence of alkaloids in the bulbs of F. meleagris, cultured in vitro under different growth regimes and at different developmental stages, as a powerful tool to identify preferable in vitro plant material for alkaloid isolation. Histochemical localization of alkaloids was performed using Wagner’s and Dragendorff’s reagents in fresh sections of bulbs cultured at 24 °C or 7 °C for 4 weeks, as well as those cultured at 24 °C following the 4-week chilling treatment, which were sampled at the beginning of sprouting. A positive reaction was observed with both reagents and was particularly intense in cold-treated bulbs. Alkaloids were mainly distributed in the bulbs and partly in the sprouts of the in vitro grown F. meleagris. The most intense staining, indicative of high alkaloid content, was observed in the bulb scales of pre-chilled bulbs that sprouted at 24 °C, rendering them preferable as in vitro plant material for alkaloid isolation. The results suggest that alkaloid production in the bulbs of F. meleagris can be improved by manipulating growth in the microenvironment of in vitro cultures, in order to meet the increasing industrial demand for medicinally or commercially important metabolites used as traditional medicines and herbal remedies. Full article

Fritillaria cirrhosa D. Don (known as Chuan-Bei-Mu in Chinese) can synthesize isosteroidal alkaloids (ISA) with excellent medicinal value, and its bulb has become an indispensable ingredient in many patented drugs. Members of the cytochrome P450 (CYP450) gene superfamily have been shown to play essential roles in regulating steroidal alkaloids biosynthesis. However, little information is available on the P450s in F. cirrhosa. Here, we performed full-length transcriptome analysis and discovered 48 CYP450 genes belonging to 10 clans, 25 families, and 46 subfamilies. By combining phylogenetic trees, gene expression, and key F. cirrhosa ISA content analysis, we presumably identify seven FcCYP candidate genes, which may be hydroxylases active at the C-22, C-23, or C-26 positions in the late stages of ISA biosynthesis. The transcript expression levels of seven FcCYP candidate genes were positively correlated with the accumulation of three major alkaloids in bulbs of different ages. These data suggest that the candidate genes are most likely to be associated with ISA biosynthesis. Finally, the subcellular localization prediction of FcCYPs and transient expression analysis within Nicotiana benthamiana showed that the FcCYPs were mainly localized in the chloroplast. This study presents a systematic analysis of the CYP450 gene family in F. cirrhosa and provides a foundation for further functional characterization of the CYPs involved in ISA biosynthesis. Full article

Fritillaria is a perennial herb with medicinal properties. There are 158 Fritillaria species worldwide, 33 of which have reported therapeutic efficacy. Alkaloids are the principal constituents in Fritillaria. Fritillaria species growing at 2700–4000 m are the sources of extract namely Chuan Beimu (the Pharmacopoeia of the People’s Republic of China, 2020 Edition), with low biomass, mainly containing more 5α-cevanine isosteroidal alkaloids with cis-configuration. In contrast, species growing below 1500 m are usually taller than 50 cm, and they mainly contain more trans-configuration isosteroidal alkaloids. There are two schemes of the biosynthetic pathways of steroidal alkaloids with different frameworks and catalytic reactions and combined high-throughput omics data. Based on the distributed elevations, Fritillaria species were divided into three major categories, which met classification features based on phylogenetic analysis or morphological features. Artificial or in vitro cultivations are effective strategies for balancing economical requirements and ecological protection. Fritillaria species growing at lower altitudes can be cultivated by bulb reproduction, but species growing at higher altitudes still rely mainly on gathering a large number of wild resources. Integration of asexual tissue culture and bulb reproduction with sexual artificial or imitated wild cultivation may create a very promising and effective way to maintain sustainable industrial development of Fritillaria. Full article

Investigations were carried out to study the effects of light-emitting diode (LED) lights on growth and development of isosteroidal alkaloids in embryogenic calli of Fritillaria cirrhosa D. Don, an important traditional Chinese medicine herb. Calli were cultured in glass bottles, each containing 100 mL of Murashige and Skoog’s basal medium supplemented with 2% sucrose and 0.4% gellan gum powder, a gelling agent. These bottles were incubated in a specially designed plant growth chamber equipped with eight different LED lights consisting of single or combinations of four different light spectra emitting blue (450 nm), green (525 nm), red (660 nm), and far-red (730 nm) light. After three months of incubation, morphological changes in embryogenic calli were recorded, and LC-MS/MS analysis of cultures was carried out for peimisine, sipeimine, peiminine, and peimine. The highest number of somatic embryos and the maximum fresh weight was recorded in calli incubated under red (9R), infrared (9IR), and a combination of red+blue+infrared (3R3B3IR), respectively, in decreasing order. The highest contents of peimisine, peiminine, and peimine were recorded under red (9R) and infrared (9IR) lights, respectively. Eight LED lights had significant effects on the morphogenesis of embryogenic calli of F. cirrhosa D. Don and contents of isosteroidal alkaloids. Full article

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 Fritillariae ussuriensis Maxim and Fritillariae thunbergii Miq powder. Chromatographic separation was achieved on an ACQUIT UPLC^® BEH C₁₈ 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 m/z 432.4→414.4 for peimine and m/z 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 Fritillaria ussuriensis Maxim and Fritillaria thunbergii Miq. Full article