Highlights On Dried Poppy Pods
- •A TOF-SIMS based dried poppy pods imaging strategy for mapping opium alkaloids in Papaver somniferum was proposed.
- •This strategy exhibited high spatial resolution imaging, muti-components analysis, high scanning efficiency and simple sample pretreatment.
- •The spatial differentiation of opium alkaloids in Papaver somniferum was directly visualized from the macro to the micro.
- •High-resolution chemical imaging of opium alkaloids in different cell types of receptacle vascular bundles was achieved for the first time.
Abstract
Accurate mapping of opium alkaloids in Papaver somniferum (P. somniferum) is significant for comprehensively elucidating their biosynthetic pathways and metabolic regulations. Here we describe a panoramic chemical imaging strategy based on time-of-flight secondary ion mass spectrometry (TOF-SIMS) that enables a thorough and precise visualization of the spatial differentiation of five opium alkaloids (morphine, codeine, thebaine, papaverine and noscapine) in P. somniferum. Notably, the opium alkaloids exhibited enrichment in the capsule, pistil, receptacle and peduncle. Moreover, a series of cross sections of a capsule were imaged to reveal a three-dimensional horizon of internal distribution of opium alkaloids. Opium alkaloids were observed enriched within the pseudoseptum and the “tube-like” structure of the inner pod wall, witnessing a downward trend toward the stigmatic disk. Finally, in high spatial resolution mode of TOF-SIMS, direct single-cell level chemical mapping of opium alkaloids in the phloem of the vascular bundles of P. somniferum receptacle was achieved for the first time. The introduced imaging strategy can provide multi-dimensional insights into the spatial distribution of opium alkaloids in P. somniferum, and the findings are expected to facilitate shedding light on the biological activities of these pharmacologically valued alkaloids.
Graphical abstract
TOF-SIMS based panoramic chemical imaging strategy for mapping five opium alkaloids in Papaver somniferum from “plant” to “organ” to “microstructure”.
Introduction
The opium poppy, Papaver somniferum (P. somniferum) is one of the most ancient medicinal plants, and continues to attract considerable attention due to its pharmacologically important alkaloids. Diverse benzylisoquinoline alkaloids derived from L-tyrosine in P. somniferum have been reported. Among them, the opium alkaloids morphine, codeine, noscapine, thebaine and papaverine account for the highest proportions [1]. Extensive researches have demonstrated the analgesic, antitussive, and anticancer properties of these alkaloids. For instance, morphine and codeine are well-known narcotic analgesics producing analgesia effects through binding to mu-opioid, and noscapine has elucidated to be a potential anticancer drug by inducing apoptosis in tumor cells [2], [3]. To further improve the production and to sustainably exploit the medicinal value of these important alkaloids in P. somniferum, various researches in plant morphology [4], [5], genetics [6], [7] or enzymology [1], [8], [9] have been conducted for detailed information about their biosynthetic pathways, metabolic regulations and ecophysiological roles. In almost all relevant researches, the species composition, content variation and spatial differentiation of alkaloids are direct experimental evidence critical for drawing conclusions or confirming results [5], [6], [9]. Therefore, it’s vital to develop highly sensitive and spatial-resolved detection strategies for alkaloids mapping in P. somniferum.dried poppy pods
Chromatography techniques (including high-performance liquid chromatography [10], [11], [12], thin-layer chromatography [13], paper chromatography [14] and capillary electrophoresis [15]) and mass spectrometry (MS) [16], [17], [18] are the common analytical methods for the detection of alkaloids in P. somniferum. Especially, high-performance liquid chromatography (HPLC) plays a key role in the separation, qualitative and quantitative determination of opium alkaloids [10], [12]. Continuous improvement strategies involving modifying column filling materials [19], [20] and optimizing experimental conditions (such as pH value of the mobile phase, concentration of the buffer components, mobile phase organic modifier, and column temperature) [21] have been carried out to optimize the separation efficiency and detection sensitivity in HPLC analysis of alkaloids. Additionally, owing to its prominent advantage in the aspect of molecules identification and high detection sensitivity, recent years, chromatography coupled with MS technology has grown to an effective approach to detect alkaloids in P. somniferum [16], [17], [18]. For instance, Frick’s group [18] used LC-MS to obtain a different pattern of alkaloids in narcotic and condiment P. somniferum cultivars, suggesting a different gene regulation in alkaloid biosynthesis. All these efforts mentioned above enhanced the understanding of the composition and content differences of alkaloids in P. somniferum. However, systematical and direct visualization of spatial distribution characteristics of alkaloids in P. somniferum has not yet been reported. It is also poorly understood whether there are differences in the spatial distribution patterns of different alkaloids.
Imaging mass spectrometry (IMS) [22], [23] is a promising technique for biological specimen characterization, because it allows both chemical specificity and microscopic imaging without complex sample preparations. Common ionization techniques compatible with IMS include matrix-assisted laser desorption/ionization (MALDI), desorption electrospray ionization (DESI) and secondary ion mass spectrometry (SIMS). Among all IMS technologies, time-of-flight secondary ion mass spectrometry (TOF-SIMS) possesses routinely submicron-level spatial resolution [24], [25] superior to other IMS counterparts, and thus could be used to identify various biomolecules in tissues, cells and even subcellular compartments. TOF-SIMS has gained significant prominence in chemical imaging of inorganic elements and biomolecules of plants. The analysis of metallic elements distribution at the cellular and subcellular level in plants by TOF-SIMS provides insight into the nutrient transport routes [26], [27]. TOF-SIMS also suitable for identifying and locating structural components, lignin and polysaccharide in wood [28], [29], [30], [31]. Furthermore, TOF-SIMS have gradually expanded to chemical imaging of a diverse array of small molecule metabolites in plants [32], [33], [34]. Thus, we believe TOF-SIMS can make up for the shortcomings in analyzing the spatial distribution characteristics of alkaloids in P. somniferum.dried poppy pods
Herein, a chemical imaging strategy at three levels, namely “plant-organ-microstructure”, was proposed to systematically visualize the spatial distribution of five opium alkaloids (morphine, codeine, thebaine, papaverine and noscapine) in P. somniferum from macro to micro dimensions. The introduced strategy fully exploited the advantages of TOF-SIMS, such as high spatial resolution, label-free, multi-component analysis and simple sample preparations. At the “plant” level, the cross sections of different organs were obtained to characterize the total content and spatial distribution variations of the five opium alkaloids throughout the whole plant. At the “organ” level, a downward trend in the relative abundance of the five opium alkaloids in capsules from bottom toward stigmatic disk was described. Finally, at the “microstructure” level, high-resolved mode of TOF-SIMS was applied to comprehensively investigated the precise spatial distribution of the five opium alkaloids in vascular bundles. The findings could profoundly advance our comprehension of the biosynthetic pathways and metabolic regulations of opium alkaloids in P. somniferum, and promote the development of opium alkaloid extraction techniques for medical applications.
Section snippets
Materials, reagents and apparatus
UHPLC-MS grade methanol was purchased from Merck (Muskegon, MI, USA). The stock standard solutions (100 μg/mL in methanol) of five opium alkaloids, morphine, noscapine, thebaine, codeine and papaverine were obtained from Shanghai Academy of Criminal Science and Technology (Shanghai, China). Ultrapure water (over 18.2 MΩ.cm) were obtained from a Milli-Q integral system (Millipore, USA). P. somniferum and Papaver orientale (P. orientale) samples were obtained from National Anti-drug Laboratory
Detection of opium alkaloids in P. somniferum by TOF-SIMS
The five opium alkaloids morphine, codeine, thebaine, papaverine and noscapine in P. somniferum were analyzed by TOF-SIMS. As shown in Fig. 1, in the TOF-SIMS spectra of the cross sections of P. somniferum capsule, the hypothetical quasi-molecular ion ([M + H]+) peaks of morphine (C17H20NO3+, m/z 286.14), codeine (C18H22NO3+, m/z 300.16), thebaine (C19H22NO3+, m/z 312.16), papaverine (C20H22NO4+, m/z 340.15) and noscapine (C22H24NO7+, m/z 414.15) were obtained and imaged, respectively. The dried poppy pods
Conclusions
In summary, we proposed a novel TOF-SIMS based strategy to realize panoramic chemical imaging of the five opium alkaloids (morphine, codeine, thebaine, papaverine and noscapine) in P. somniferum. The results showed that, macroscopically, opium alkaloids tend to accumulate in the capsule, the pistil, the receptacle and the peduncle of P. somniferum, exhibiting a rising trend from bottom to top. At the “organ” level, a three-dimensional horizon of the internal distribution of opium alkaloids in
CRediT authorship contribution statement
Meng-Chan Xia: Conceptualization, Data curation, Methodology, Writing – original draft, Writing – review & editing. Ping He: Data curation, Writing – review & editing. Jun Ma: Investigation, Methodology. Xin Yan: Data curation. Dongmei Li: Funding acquisition, Resources. Chong Guo: Data curation. Qingli Zeng: Investigation, Resources. Lesi Cai: Conceptualization, Data curation, Supervision, Visualization. Siyuan Tan: Conceptualization, Funding acquisition. Zhanping Li: Conceptualization,
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
This work was supported by the National Key R&D Program of China (2018YFA0702600) and the National Natural Science Foundation of China (22104136, 22104149 and 21927812).
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