Search Results (52)

Lotus (Nelumbo nucifera Gaertn.) is a quintessential medicinal and edible plant, exhibiting marked differences in therapeutic effects among its various parts. The lotus seed constitutes a key component of this plant. Notably, the entire seed and the plumule display distinct medicinal properties. To investigate the “homologous plants with different effects” phenomenon in traditional Chinese medicine, this study established a Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging (MALDI-MSI) method. This study employed immature lotus seeds as the experimental material, diverging from the mature seeds conventionally used. Conductive double-sided tape was employed for sample preparation, and complete longitudinal sections of the seeds were obtained, followed by MALDI-MSI analysis to identify and visualize the spatial distribution of characteristic secondary metabolites within the entire seeds. The results unveiled the diversity of metabolites in lotus seeds and their differential distribution across tissues, with pronounced distinctions in the plumule. A total of 152 metabolites spanning 13 categories were identified in lotus seeds, with 134, 89, 51, and 98 metabolites discerned in the pericarp, seed coat, cotyledon, and plumule, respectively. Strikingly, young lotus seeds were devoid of liensinine/isoliensinine and neferine, the dominant alkaloids of mature lotus seed plumule, revealing an early-stage alkaloid profile that sharply contrasts with the well-documented abundance found in mature seeds and has rarely been reported. We further propose a biosynthetic pathway to explain the presence of the detected benzylisoquinoline and the absence of the undetected bisbenzylisoquinoline alkaloids in this study. These findings present the first comprehensive metabolic atlas of immature lotus seeds, systematically exposing the pronounced chemical divergence from their mature counterparts, and thus lays a metabolomic foundation for dissecting the spatiotemporal mechanisms underlying the nutritional and medicinal value of lotus seeds. Full article

Annexins (ANNs) are a family of calcium (Ca²⁺)-dependent and phospholipid-binding proteins, which are implicated in the regulation of plant growth and development as well as protection from biotic and abiotic stresses. Corydalis saxicola Bunting, an endangered benzylisoquinoline alkaloid (BIA)-rich herbaceous plant, widely used in traditional Chinese medicine, is endemic to the calciphilic karst region of China. However, whether and how ANNs are involved in the biosynthesis pathway of BIAs and/or help C. saxicola plants cope with abiotic properties, such as calcareous soils, are largely unknown. Here, nine CsANN genes were identified from C. saxicola, and they were divided into three subfamilies, namely subfamilies I, II, and IV, based on the phylogenetic tree. The CsANNs clustered into the same clade, sharing similar gene structures and conserved motifs. The nine CsANN genes were located on five chromosomes, and their expansions were mainly attributed to tandem and whole-genome duplications. The CsANN transcripts displayed organ-specific and Ca²⁺-responsive expression patterns across various tissues. In addition, transient overexpression assays showed that CsANN1 could positively regulate the accumulation of BIA compounds in C. saxicola leaves, probably by directly interacting with key BIA-biosynthetic-pathway enzymes or by interacting with BIA-biosynthetic regulatory factors, such as MYBs. This study sheds light on the profiles and functions of the CsANN gene family and paves the way for unraveling the molecular mechanism of BIA accumulation, which is regulated by Ca²⁺ through CsANNs. Full article

Wounding triggers complex secondary metabolic pathways in plants, including benzylisoquinoline alkaloid (BIA) biosynthesis in opium poppy (Papaver somniferum L.). This study explores transcriptional and metabolic responses to wounding and methyl jasmonate (MeJA) treatment, focusing on BIA biosynthesis and regulatory mechanisms. Real-time expression analysis revealed significant up-regulation of transcripts in the (S)-reticuline and papaverine biosynthetic pathway, while the noscapine pathway was suppressed. The morphinan pathway also showed transcriptional activation, except in the case of codeinone reductase (COR), which remained unresponsive to both wounding and MeJA, suggesting a partially uncoupled mechanism. Metabolite profiling using HPLC-MS demonstrated a rapid accumulation of morphine post wounding, further supporting the hypothesis of independent regulatory control over COR. The role of phospholipase C (PLC) in modulating wound-induced BIA accumulation was investigated, revealing that PLC inhibition reduced morphine production and suppressed COR expression. These findings highlight the importance of phospholipid-dependent signalling in activating morphine biosynthesis, potentially at the expense of other BIAs. This study provides insights into plant stress responses and suggests strategies for enhancing BIA production through targeted interventions, offering potential applications in improving alkaloid yield. Full article

Aristolochia contorta Bunge has been widely used as traditional Chinese medicine materials. However, its utility faces a great challenge due to the presence of aristolochic acids (AAs), a class of benzylisoquinoline alkaloid (BIA) derivatives. The first step in BIA skeleton formation is catalysis by norcoclaurine synthase (NCS). To gain knowledge of BIA and AA biosynthesis in A. contorta, genome-wide characterizations of NCS genes were carried out. This resulted in the identification of 15 A. contorta NCSs, namely, AcNCS1–AcNCS15. The AcNCS1–AcNCS8 proteins contained one catalytic domain, whereas the AcNCS9–AcNCS15 proteins had two. Phylogenetic analysis shows that AcNCS proteins can be classified into two clades. Gene expression analysis shows that five AcNCSs, including AcNCS2, AcNCS4, AcNCS5, AcNCS14, and AcNCS15, exhibited relatively high expression in roots and flowers, where norcoclaurine accumulated. An enzyme catalytic activity assay shows that all five of the AcNCSs can catalyze norcoclaurine formation with AcNCS14 and AcNCS15, exhibiting higher catalytic efficiency. Precolumn derivatization analysis shows that the formed norcoclaurine included (S)- and (R)-norcoclaurine, with more (S)-configuration. The results provide useful information for further understanding BIA and AA biosynthesis in A. contorta and for AA elimination and bioactive compound improvement in AA-containing medicinal materials. Full article

Background/Objectives: Natural substances have been a widely studied source of both pharmaceutical excipients and drugs. Berberine (BRB) is a benzylisoquinoline alkaloid isolated from different plant sources. It possesses various pharmacological properties including antibacterial, antitumor, antidiabetic, neuroprotective, hepatoprotective, anti-inflammatory, antioxidant, etc. However, the limited aqueous solubility hinders its application. Nanosized drug delivery systems are an innovative approach for addressing various challenges regarding drug delivery via different routes of administration. Their utilization could improve the solubility of active constituents. Methods: A melt-emulsification and ultrasonication technique was applied for the preparation of nanostructured lipid carriers (NLCs). They were thoroughly physicochemically characterized by the means of Dynamic Light Scattering, TEM, FTIR, DSC, TGA, and In Vitro release. The In Vitro efficacy and safety were evaluated on cholangiocarcinoma, colorectal adenocarcinoma, hepatocellular carcinoma, lymphoma, fibroblast, and cardioblast cells, as well as rat liver microsomes by means of cytotoxicity assays and the comet assay. Results: The obtained nanoparticles had a spherical shape and size around 158.2 ± 1.8 nm with negative zeta potential. They revealed successful drug loading and improved dissolution of berberine in physiological conditions. The In Vitro safety studies showed that loading BRB in NLCs resulted in improved or retained cytotoxicity to tumor cell lines and reduced cytotoxicity to normal cell lines and liver microsomes. The NLC itself increased microsomal malondialdehyde (MDA) and comet formation. Conclusions: A successful preparation of NLCs with berberine is presented. The nanocarriers show favorable physicochemical and biopharmaceutical properties. The cellular experiments show that the NLC loading of berberine could improve its anticancer efficacy and safety. These findings highlight the potential applicability of berberine in gastrointestinal neoplasms and build the foundation for future practical translation. Full article

Background/Objectives: Stephania epigaea is a plant from the Menispermaceae family. Its root is an important traditional folk medicine, which is called Diburong in China. Diburong is rich in benzylisoquinoline alkaloids (BIAs), including cepharanthine, which has been demonstrated to exhibit significant anti-inflammatory, antiviral, antineoplastic, and anti-SARS-CoV-2 activities, as well as raising leukocytes. Cepharanthine is composed of (R)- and (S)-1-benzylisoquinoline alkaloid (1-BIA). (S)-norcoclaurine-6-O-methyltransferase (6OMT) is a rate-limiting enzyme in BIA biosynthesis. However, its role in the cepharanthine biosynthetic pathway, particularly with the (R) stereoisomer substrate, remains largely unexplored. This study aimed to identify Se6OMTs involved in the cepharanthine biosynthetic pathway and elucidate the O-methyltransferases (OMTs) responsible for the production of (R)- and (S)-stereoisomer BIAs. Methods: In this study, three OMTs were cloned from S. epigaea and functionally characterized using nine 1-BIAs of (R)- and (S)-configurations as substrates. Results: Two O-methyltransferases, Se6OMT1 and Se6OMT3, showed efficient catalytic activity at the C6 position of both (R)- and (S)-norcoclaurine. Furthermore, Se6OMT3 demonstrated high catalytic activity at the C7 and C4′ positions of other (R)- and (S)-configuration 1-BIAs, which resulted in the generation of multiple products. Conclusions: This study focused on 6OMT enzymes in S. epigaea, identifying Se6OMTs involved in the cepharanthine biosynthetic pathway, determining the OMTs involved in the production of (R)- and (S)-stereoisomer BIAs. This research provides valuable insights into the substrate promiscuity of Se6OMTs on (R)- and (S)-configured 1-BIAs in S. epigaea and highlights the genetic components necessary for the metabolic engineering and synthetic biology approaches to cepharanthine production. Full article

The medicinal plant Stephania yunnanensis is rich in aporphine alkaloids, a type of benzylisoquinoline alkaloid (BIA), with aporphine being the representative and most abundant compound, but our understanding of the biosynthesis of BIAs in this plant has been relatively limited. Previous research reported the genome of S. yunnanensis and preliminarily identified the norcoclaurine synthase (NCS), which is involved in the early stages of the BIA biosynthetic pathways. However, the key genes promoting the formation of the aporphine skeleton have not yet been reported. In this study, based on the differences in the content of crebanine and several other BIAs in different tissues, we conducted transcriptome sequencing of roots, stems, and leaves. We then identified candidate genes through functional annotation and sequence alignment and further analyzed them in combination with the genome. Based on this analysis, we identified three CYP80 enzymes (SyCYP80Q5-1, SyCYP80Q5-3, and SyCYP80G6), which exhibited different activities toward (S)- and (R)-configured substrates in S. yunnanensis and demonstrated strict stereoselectivity enroute to aporphine. This study provides metabolomic and transcriptomic information on the biosynthesis of BIAs in S. yunnanensis, offers valuable insights into the elucidation of BIA biosynthesis, and lays the foundation for the complete analysis of pathways for more aporphine alkaloids. Full article

Zanthoxylum simulans Hance, commonly known as Sichuan pepper, is a well-known medicinal plant recognized for its potential as a source of bioactive specialized metabolites. As part of our interest in natural antifungal compounds, the present study describes the discovery of an unreported N-alcoxycarbonylbenzo[c]phenanthridinium salt, N-methoxycarbonyl-9,12-dimethoxy-norchelerythrine 1 (a type-III benzo[c]phenanthridine), isolated from Z. simulans seedlings, which were propagated under controlled greenhouse conditions. Six-month seedlings were harvested and subjected to cold acid–base extraction. Chromatographic techniques achieved the isolation of 1 from raw alkaloid extract. The structural elucidation of 1 was accomplished through comprehensive spectroscopic analysis, including nuclear magnetic resonance and high-resolution mass spectrometry. Fusarium oxysporum, a fungal pathogen responsible for substantial agricultural losses, was exposed to different concentrations of the novel compound, exhibiting potent antifungal efficacy (IC₅₀ < 3 µM) and fungicide effects. These findings highlight the potential of benzophenanthridines as antifungal leads and underscore the importance of exploring natural products for agricultural applications. Full article

Aristolochia contorta Bunge is an academically and medicinally important plant species. It belongs to the magnoliids, with an uncertain phylogenetic position, and is one of the few plant species lacking a whole-genome duplication (WGD) event after the angiosperm-wide WGD. A. contorta has been an important traditional Chinese medicine material. Since it contains aristolochic acids (AAs), chemical compounds with nephrotoxity and carcinogenicity, the utilization of this plant has attracted widespread attention. Great efforts are being made to increase its bioactive compounds and reduce or completely remove toxic compounds. MicroRNAs (miRNAs) and natural antisense transcripts (NATs) are two classes of regulators potentially involved in metabolism regulation. Here, we report the identification and characterization of 223 miRNAs and 363 miRNA targets. The identified miRNAs include 51 known miRNAs belonging to 20 families and 172 novel miRNAs belonging to 107 families. A negative correlation between the expression of miRNAs and their targets was observed. In addition, we identified 441 A. contorta NATs and 560 NAT-sense transcript (ST) pairs, of which 12 NATs were targets of 13 miRNAs, forming 18 miRNA-NAT-ST modules. Various miRNAs and NATs potentially regulated secondary metabolism through the modes of miRNA-target gene–enzyme genes, NAT-STs, and NAT-miRNA-target gene–enzyme genes, suggesting the complexity of gene regulatory networks in A. contorta. The results lay a solid foundation for further manipulating the production of its bioactive and toxic compounds. Full article

Ocotea, the largest genus in the Lauraceae family, encompasses numerous species of scientific interest. However, most Ocotea species have only been described morphologically. This study used an untargeted metabolomics workflow with UHPLC-HRMS and GNPS-FBMN to provide the first chemical evaluation of the polar specialized metabolites of O. delicata leaves. Leaves from three O. delicata specimens were extracted using ultrasound-assisted extraction with 70% ethanol. Among the examined samples, 44 metabolites, including alkaloids and flavonoids, were identified. In contrast to other Ocotea species, O. delicata has a wider diversity of kaempferol derivatives than quercetin. The biomass of the specimens showed a significant correlation with the chemical profile. The similarity among specimens was mostly determined by the concentrations of quinic acid, kaempferol glycosides, and boldine. The evaluated specimens exhibited chemical features similar to those of species classified as New World Ocotea, with the coexistence of aporphine and benzylisoquinoline alkaloids. Full article

Tetranychus urticae Koch, a phytophagous mite, is one of the most significant crop pests globally. The primary method employed for controlling T. urticae involves chemical means, utilizing synthesized products, posing the risk of developing resistance. The urgency for novel strategies integrated into pest management programs to combat this mite is becoming increasingly imperative. Botanical pesticides emerge as a promising tool to forestall arthropod resistance. Among these, extracts from Rutaceae plants, abundant in bioactive specialized metabolites, have demonstrated potential as insecticides and miticides. In this study, various concentrations of alkaloidal extracts sourced from the bark of Zanthoxylum schreberi J.F.Gmel. (Rutaceae) were evaluated against T. urticae adult females. Furthermore, the extract’s combination with three distinct commercial acaricides (i.e., chlorfenapyr, cyflumetofen, and abamectin) was also assessed for this mite. Chemical characterization of the extract via LC-MS allowed for the annotation of various compounds related to ten benzylisoquinoline-derived alkaloids. The extract, both alone and in combination with commercial insecticides, yielded varying responses, inducing over 40% mortality at 2% w/w, demonstrating a 90% repellency rate at the same concentration, and exerting a moderate impact on fecundity. These treatments extended beyond phenotypic responses, delving into the biochemical effects on treated T. urticae females through an exploration of the impact on four enzymes, i.e., acetylcholinesterase (AChE), glutathione S-transferase (GST), esterases (GE), and P450-like monooxygenases (PMO). Employing consensus docking studies and in vitro enzymatic evaluations, it was discovered that the Z. schreberi-derived extract and its constituents significantly affected two key enzymes, AChE and GST (IC₅₀ < 6 µM), which were associated with the phenotypic observations of T. urticae females. The evaluation of alkaloid-rich botanicals showcases promising potential as a relevant biotechnological strategy in addressing mite-related concerns, offering a pathway toward innovative and sustainable pest management solutions. Full article

Bisbenzylisoquinoline and aporphine alkaloids are the two main pharmacological compounds in the ancient sacred lotus (Nelumbo nucifera). The biosynthesis of bisbenzylisoquinoline and aporphine alkaloids has attracted extensive attention because bisbenzylisoquinoline alkaloids have been reported as potential therapeutic agents for COVID-19. Our study showed that NnCYP80A can catalyze C-O coupling in both (R)-N-methylcoclaurine and (S)-N-methylcoclaurine to produce bisbenzylisoquinoline alkaloids with three different linkages. In addition, NnCYP80G catalyzed C-C coupling in aporphine alkaloids with extensive substrate selectivity, specifically using (R)-N-methylcoclaurine, (S)-N-methylcoclaurine, coclaurine and reticuline as substrates, but the synthesis of C-ring alkaloids without hydroxyl groups in the lotus remains to be elucidated. The key residues of NnCYP80G were also studied using the 3D structure of the protein predicted using Alphafold 2, and six key amino acids (G39, G69, A211, P288, R425 and C427) were identified. The R425A mutation significantly decreased the catalysis of (R)-N-methylcoclaurine and coclaurine inactivation, which might play important role in the biosynthesis of alkaloids with new configurations. Full article

REPI is a pivotal point enzyme in plant benzylisoquinoline alkaloid metabolism as it promotes the evolution of the biosynthetic branch of morphinan alkaloids. Experimental studies of its activity led to the identification of two modules (DRS and DRR) that catalyze two sequential steps of the epimerization of (S)- to (R)-reticuline. Recently, special attention has been paid to its genetic characterization and evolutionary history, but no structural analyses of the REPI protein have been conducted to date. We present here a computational structural characterization of REPI with heme and NADP cofactors in the apo state and in three complexes with substrate (S)-reticuline in DRS and intermediate 1,2-dehydroreticuline in DRS and in DRR. Since no experimental structure exists for REPI, we used its AlphaFold model as a scaffold to build up these four systems, which were submitted to all-atom molecular dynamics (MD) simulations. A comparison of MD results for the four systems revealed key dynamic changes associated with cofactor and ligand binding and provided a dynamic picture of the evolution of their structures and interactions. We also explored the possible dynamic occurrence of tunnels and electrostatic highways potentially involved in alternative mechanisms for channeling the intermediate from DRS to DRR. Full article

Opioids are important analgesics, and their pharmaceutical application is increasing worldwide. Many opioids are based on benzylisoquinoline alkaloids (BIA) and are still industrially produced from Papaver somniferum (opium poppy). (S)-norlaudanosoline ((S)-NLS) is a complex BIA and an advanced intermediate for diverse pharmaceuticals. The efficient synthesis of this scaffold could pave the way for a plant-independent synthesis platform. Although a promising biocatalytic route to (S)-NLS using norcoclaurine synthase (NCS) and ω-transaminase (TAm) has already been explored, the cost-effectiveness of this process still needs much improvement. Therefore, we investigated whether the synthesis could also be performed using whole cells to avoid the use of (partially) purified enzymes. With an optimized mixing ratio of TAm- and NCS-containing cells in batch biotransformations, 50 mM substrate was converted within 3 h with more than 90% yield and a high enantiomeric excess of the product (95%). To further increase the space–time yield, the cells were immobilized to enable their retainment in fixed-bed reactors. A comparison of glass beads, Diaion HP-2MG and alginate revealed that the addition of Diaion during bacterial growth led to the most active immobilisates. To facilitate sustained production of (S)-NLS, a fixed-bed setup was constructed based on lithographically printed columns from biocompatible PRO-BLK 10 plastic. The continuous production at two scales (5 mL and 50 mL columns) revealed insufficient system stability originating from biocatalyst leaching and inactivation. Thus, while the use of whole cells in batch biotransformations represents an immediate process improvement, the transfer to flow catalysis needs further optimization. Full article

The aim of this study was to compile a detailed phytochemical profile and assess the antioxidant properties of bee-collected pollen (PBP) obtained from corn poppy (Papaver rhoeas L.) plants. To achieve this, a lipid fraction was prepared for quantifying fatty acids using GC-FID. Extractable and alkaline-hydrolysable PBP fractions (obtained from a defatted sample) were used to determine the qualitative and quantitative profiles of phenolic compounds, phenylamides and alkaloids using UHPLC/Q-ToF-MS. Additionally, various spectrophotometric assays (TAC, FRP, CUPRAC, DPPH^⦁) were conducted to evaluate the antioxidant properties. Phenolic compounds were more present in the extractable fraction than in the alkaline-hydrolysable fraction. Luteolin was the predominant compound in the extractable fraction, followed by tricetin and various derivatives of kaempferol. This study presents one of the first reports on the quantification of tricetin aglycone outside the Myrtaceae plant family. The alkaline-hydrolysable fraction exhibited a different phenolic profile, with a significantly lower amount of phenolics. Kaempferol/derivatives, specific compounds like ferulic and 5-carboxyvanillic acids, and (epi)catechin 3-O-gallate were the predominant compounds in this fraction. Regarding phenylamides, the extractable fraction demonstrated a diverse range of these bioactive compounds, with a notable abundance of different spermine derivatives. In contrast, the hydrolysable fraction contained six spermine derivatives and one spermidine derivative. The examined fractions also revealed the presence of seventeen different alkaloids, belonging to the benzylisoquinoline, berberine and isoquinoline classes. The fatty-acid profile confirmed the prevalence of unsaturated fatty acids. Furthermore, both fractions exhibited significant antioxidant activity, with the extractable fraction showing particularly high activity. Among the assays conducted, the CUPRAC assay highlighted the exceptional ability of PBP’s bioactive compounds to reduce cupric ions. Full article