Comparison of different HPLC systems for analysis of galantamine and lycorine in various species of <i>Amaryllidaceae</i> family

Yeo

et al. 2019

Biology

Lycoris radiata belongs to the Amaryllidaceae family and is a bulbous plant native to South Korea, China, and Japan. Galantamine, a representative alkaloid of Amaryllidaceae plants, including L. radiata, exhibits selective and dominant acetylcholinesterase inhibition. In spite of the economic and officinal importance of L. radiata, the molecular biological and biochemical information on L. radiata is relatively deficient. Therefore, this study provides functional information of L. radiata, describe galantamine biosynthesis in the various organs, and provide transcriptomic and metabolic datasets to support elucidation of galantamine biosynthesis pathway in future studies. The results of studies conducted in duplicate revealed the presence of a total of 325,609 and 404,019 unigenes, acquired from 9,913,869,968 and 10,162,653,038 raw reads, respectively, after trimming the raw reads using CutAdapt, assembly using Trinity package, and clustering using CD-Hit-EST. All of the assembled unigenes were aligned to the public databases, including National Center for Biotechnology Information (NCBI) non-redundant protein (NR) and nucleotide (Nt) database, SWISS-PROT (UniProt) protein sequence data bank, The Arabidopsis Information Resource (TAIR), the Swiss-Prot protein database, Gene Ontology (GO), and Clusters of Orthologous Groups (COG) database to predict potential genes and provide their functional information. Based on our transcriptome data and published literatures, eight full-length cDNA clones encoding LrPAL2, LrPAL3, LrC4H2, LrC3H, LrTYDC2, LrNNR, LrN4OMT, and LrCYP96T genes, involved in galantamine biosynthesis, were identified in L. radiata. In order to investigate galantamine biosynthesis in different plant parts of L. radiata grown in a growth chamber, gene expression levels were measured through quantitative real-time polymerase chain reaction (qRT-PCR) analysis using these identified genes and galantamine levels were quantified by high-performance liquid chromatography (HPLC) analysis. The qRT-PCR data revealed high expression levels of LrNNR, LrN4OMT, and LrCYP96T in the bulbs, and, as expected, we observed higher amounts of galantamine in the bulbs than in the root and leaves. Additionally, a total of 40 hydrophilic metabolites were detected in the different organs using gas-chromatography coupled with time-of-flight mass spectrometry. In particular, a strong positive correlation between galantamine and sucrose, which provides energy for the secondary metabolite biosynthesis, was observed.

Section: Discussionmentioning

confidence: 99%

Transcriptome Analysis and Metabolic Profiling of Lycoris Radiata

Yeo

et al. 2019

Biology

“…Lycorine, a major alkaloid found in many Amaryllidaceae species, has been shown to have a wide variety of biological activities such as antitumoral , antimalarial (Cedron et al, 2010), hepatoprotective (Ilavenil, Kaleeswaran, Ravikumar, 2012), antiviral (He et al, 2013), antifungal (Shen et al, 2014) and antiparasitic (Giordani et al, 2012) activities. Due to its diverse biological properties, it has been to the interest of phytochemists to determine the content of this alkaloid using various analytical techniques in Amaryllidaceous plants (Abou-Donia et al, 2007;Petruczynik et al, 2016).…”

Section: Introductionmentioning

confidence: 99%

Application of HPLC-DAD for the quantification of Lycorine in Galanthus elwesii Hook

Emir

Bozkurt

et al. 2017

Braz. J. Pharm. Sci.

In the present study, a reversed-phase high-performance liquid chromatographic method has been used for the quantitative determination of lycorine in the aerial parts and bulbs of G. elwesii Hook. A simple method for the extraction of lycorine in low mass plant samples was employed utilizing pre-packed columns with diatomaceous earth (Extrelut®). The chromatographic separation was performed using an isocratic system with a mobile phase of trifluoroacetic acid-water-acetonitrile (0.01:92.5:7.5, v/v/v) applied at a flow rate 1 mL min -1 using diode array detector. The content of lycorine in the bulbs and aerial parts of G. elwesii collected from Demirci (Manisa) was found as 0.130 and 0.162 %, respectively. Additionally, in the bulbs of the specimens collected from Sogucak (Balikesir), lycorine was quantified as 0.055 %, whereas in the aerial parts, it was determined as 0.006 %. The method was validated partially with respect to system specificity, linearity, accuracy, precision, limits of detection (LOD) and quantitation (LOQ). Validation procedures displayed that the method was specific, accurate and precise.Uniterms: Alkaloids. Amaryllidaceae. Plant extracts. High Performance Liquid Chromatography/method validation. High Perfomance Liquid Chromatography/quantitative analysis.

“…Our results also found that Gal was mainly accumulated in root which was different from the results in L. radiata , L. aurea , and N. pseudonarcissus , among which high amounts of Gal were accumulated in bulb, ovary and bulb, respectively ( Kilgore et al., 2014 ; Sun et al., 2018 ; Park et al., 2019 ). Such variations may due to species specificity, growth stages as well as environmental factors ( Szakiel et al., 2011 ; Quan et al., 2012 ; Petruczynik et al., 2016 ). Our current study is likely to narrow down the genes involved in this pathway by homology, expression and metabolite analysis.…”

Section: Discussionmentioning

confidence: 99%

Transcriptome Analysis of Different Tissues Reveals Key Genes Associated With Galanthamine Biosynthesis in Lycoris longituba

Yang

et al. 2020

Front. Plant Sci.

Lycoris longituba is a traditional medicinal plant containing the bioactive compound galanthamine (Gal), a type of Amaryllidaceae alkaloid and can be used to treat Alzheimer's disease. However, research on its genome or transcriptome and associated genes in the biosynthetic pathway is incomplete. In this study, we estimated the nuclear genome size of this species to be 29.33 Gb by flow cytometry. Then, RNA sequencing of the leaves, roots, and bulbs of L. longituba was carried out. After de novo assembly, 474,589 all-transcripts and 333,440 all-unigenes were finally generated. In addition, the differentially expressed genes (DEGs) were identified, and genes involved in the galanthamine metabolic pathway encoding tyrosine decarboxylase (TYDC), phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), p-coumarate 3hydroxylase (C3H), norbelladine synthase (NBS), norbelladine 4'-O-methyltransferase (OMT), and noroxomaritidine synthase (CYP96T1) were detected and validated by realtime quantitative PCR analysis. One candidate gene, Lycoris longituba O-Methyltransferase (LlOMT), was identified in the proposed galanthamine biosynthetic pathway. Sequence analysis showed that LlOMT is a class I OMT. LlOMT is localized in the cytoplasm, and biochemical analysis indicated that the recombinant LlOMT catalyzes norbelladine to generate 4′-O-methylnorbelladine. The protoplast transformation result showed that the overexpression of LlOMT could increase the Gal content. Our results indicate that LlOMT may play a role in galanthamine biosynthesis in L. longituba. This work provides a useful resource for the metabolic engineering of Amaryllidaceae alkaloids.