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Mass spectrometry imaging of small molecule in situ in Lepidium meyenii (Maca) using gold nanoparticles matrix
Microchemical Journal 150 (2019) 104190
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Mass spectrometry imaging of small molecule in situ in Lepidium meyenii (Maca) using gold nanoparticles matrix
T
Sihou Yanga,b, Lingpeng Zhanb,c, Chaozi Liub,c, Ling Fua, Rui Chena, , Zongxiu Nieb,c,d ⁎
a
College of Chemistry and Chemical Engineering, Yunnan Normal University, Kunming 650500, China Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry Chinese Academy of Sciences, Beijing 100190, China c Beijing National Laboratory for Molecular Sciences, Beijing 100190, China d Beijing Center for Mass Spectrometry, Beijing 100190, China b
ARTICLE INFO
ABSTRACT
Keywords: Lepidium meyenii (Maca) Mass spectrometry imaging Small molecule In situ Gold nanoparticles
Lepidium meyenii Walp. (Maca), a functional food, has been used as a drug and food in Peru for more than two thousand years because Maca is rich in nutrition and rational in nutritional structure. Due to their uniform size and little interference in the low molecular weight region, Au nanoparticles (AuNPs) have been applied as a matrix to analyze the components of Maca extract and to visualize them of fresh root tissue in situ by matrixassisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The result shows that imidazole alkaloids including Lepidiline A (1,3-dibenzyl-4,5-dimethylimidazolium chloride), Lepidiline B (1,3dibenzyl-2,4,5-trimethylimidazolium chloride), Lepidiline C (3-benzyl-1-(3-methoxybenzyl)-4,5-dimethylimidazolium chloride), Lepidiline D (3-benzyl-1-(3-methoxybenzyl)-2,4,5-trimethylimidazolium chloride), 1, 3-dibenzyl-2-pentyl-4,5-dimethylimidazilium and 1, 3-dibenzyl-2-phenyl-4,5-dimethylimidazilium are the products of Maca after drying proess. Additionally, the distribution of amino acids, amide alkaloids, imidazolium alkaloids and saccharide in the upper, middle, and lower parts of Maca root tissue can be clearly visualized in situ with the synthesized AuNPs matrix by MALDI mass spectrometry imaging (MSI). The report provides a meaningful reference to study active components from natural product in situ.
1. Introduction Lepidium meyenii Walp (Maca), a functional food with medicinal and economic value, has been used as food and medicine in Peru for over two thousand years. The hypocotyl of Maca contains some secondary metabolites such as alkaloid, glucosinolate, steroid, flavonoid, and natural phenols. Alkaloid, a class of naturally occurring organic nitrogen-containing bases, has diverse and important physiological effects on humans and other animals. Alkaloid in Maca contains macamides [1], macaridine (the benzylated derivative of 1,2-dihydro-Nhydroxypyridine together with the benzylated alkamides) [2,3], βcarboline [4], imidazole alkaloids [5], macahydantoin (the thiohydantoin derivatives) [6] and macapyrrolin [7]. Macamides, found only in Maca, are secondary amides formed by benzylamine and a fatty acid moiety with different hydrocarbon chain lengths and degree of unsaturation [8,9]. Some of the aphrodisiac activities of Maca have been related to the lipidic fraction of Maca which contains mainly fatty acids and macamides [1,8]. The pentane extract of Maca contains a number of macamides that may act on the endocannabinoid system by
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inhibitory activity on fatty acid amide hydrolase [10]. The methanol extract of Maca contains (1R,3S)-1-methyltetrahydro-β-carboline-3carboxylic acid, which exerts many activities on the central nervous system [4]. Imidazole alkaloids have been isolated from a root extract of Maca and identified as Lepidiline A (1,3-dibenzyl-4,5-dimethylimidazolium chloride), Lepidiline B (1,3-dibenzyl-2,4,5-trimethylimidazolium chloride), Lepidiline C (3-benzyl-1-(3-methoxybenzyl)-4,5-dimethylimidazolium chloride), Lepidiline D (3-benzyl-1-(3methoxybenzyl)-2,4,5-trimethylimidazolium chloride), and a class of sulfur-containing hexahydroimidazo[1,5-c]thiazole derivatives [5,11,12]. Macapyrrolin is the pyrrole alkaloids found in Maca with a benzyl substituent attached to the pyrrole nucleus and evaluated for its cytotoxicity against five human cancer cell lines [7]. In conclusion, due to its rich nutrient and secondary metabolites, Maca has good efficacy in anti-oxidation, anti-fatigue, neuroprotection, and in treatment of diabetes, benign prostatic hyperplasia, female menopausal syndrome, and osteoporosis in the elderly. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a “soft” ionization technique that uses
Corresponding author. E-mail address: [email protected] (R. Chen).
https://doi.org/10.1016/j.microc.2019.104190 Received 8 July 2019; Received in revised form 15 August 2019; Accepted 16 August 2019 Available online 19 August 2019 0026-265X/ © 2019 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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the matrix absorbed a laser energy to create ions from analyte with minimal fragmentation. Such technique is widely used to analyze organic compound and biomolecule in metabolomics, pharmaceutical sciences, environmental sciences and biomedicine. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is an emerging technology for study of the distribution of biomolecules in animal or plant tissues because it can visualize the localization and distribution of biomolecule in situ without marker. For example, it has been used to analyze the localization and distribution of small molecular metabolites in plant tissues at different growth stages and visualize the synthetic position of active components [13]. However, it is difficult to analyze some small molecular compounds (MW < 500) because traditional matrices have interference background peaks in the low mass region. Carbon-based material, such as graphene, was applied as MALDI matrix without background interference to analyze small molecules in Chinese herbal medicine. Because nanomaterial has the advantages of background-free, high sensitivity, good reproducibility and salt tolerance in MS analysis, nanomaterial-assisted laser desorption/ionization mass spectrometry without background interference is a powerful tool for the analysis of complex compound. For example, due to its excellent desorption/ionization efficiency, hexagonal boron nitride nanosheet was used as a multifunctional background-free matrix to detect small molecular metabolite by MALDI MS and analyze complex sample by mass spectrometry imaging (MSI) [14]. Novel metals, including Au, were found to be more efficient than organic matrices for most small metabolites tested. Water-soluble Au nanoclusters were reported to detect sugars, nucleosides, fatty acids, amino acids, and lipids in animal tissue for MSI [15]. Different types of sputter-coated metals were screened for small molecule analysis in MSI [16]. The proof of concept application was conducted on plant tissue, maize roots and seeds. Herein, due to its characteristics of high absorption coefficient [15], high resolution, little interference to low detection limit, and selective detection for some species, gold nanoparticles (AuNPs) were used as MALDI matrix to analyze and locate the components in Maca root by MALDI MS.
Fig. 1. TEM image of AuNPs.
2. Materials and methods
Fig. 2. (a) Negative ion MALDI-TOF mass spectrum of AuNPs. (b) Positive ion MALDI-TOF mass spectrum of AuNPs.
2.1. Chemicals and reagents
solution A. Then, heat the mixture until wine red colloidal gold is gained. Finally, centrifuge colloidal gold at 12000 rpm for 15 min to obtain the synthetic AuNPs solution. Fresh Maca root (10 g) and dry Maca powder (2 g) were extracted with 70% acetonitrile (10 mL) by ultrasonic wave for 60 min, respectively. The mixture was centrifuged into liquid and solid phases at 3000 rpm for 5 min. The liquid phase was directly analyzed by MALDI MS. Different concentrations of AuNPs solution were prepared for MALDI MS analysis. CHCA and DHB solutions were prepared at concentration of 10 mg/mL and 20 mg/mL in 0.2% TFA of 70% acetonitrile. Stock solutions of amino acids, fatty acids, N-benzyl-octadecanamide, 1,3-bis(2,4,6-trimethylphenyl) imidazolinium chloride and rutin were prepared at a concentration of 10 mmol/L in methanol and diluted to the desired concentration as required. Standard solutions of saccharides were prepared at a concentration of 10 mmol/L in deionized water and diluted to the desired concentration as required.
α-Cyano-4-hydroxycinnamic acid (CHCA), 2, 5-dihydroxybenzoic acid (DHB), trifluoroacetic acid (TFA), acetonitrile, methanol, HAuCl4, sodium citrate, potassium carbonate and tannic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). Inbred Maca was obtained from Lijiang at altitude of 2800 m (Lijiang, Yunnan, China). 1, 3-bis (2, 4, 6-trimethylphenyl) imidazolinium chloride, rutin were from J&K Scientific Ltd. (Beijing, China). N-benzyl-octadecanamide was purchased from Wuhan Huashite Industrial Biotechnology Development Co., Ltd. (Wuhan, Hubei, China). Amino acids (Histidine, asparagine, isoleucine, lysine, methionine, tyrosine, tryptophan, threonine, serine), fatty acids (palmitate sodium, linolenic acid, docosahexaenoic acid, eicosanoic acid), and saccharides (glucose, sucrose) were from Millipore Sigma (St. Louis, MO, USA). All chemicals were used in the highest purity and without further purification. The deionized water was prepared on a Milli-Q water system from Millipore Corp. (Billerica, MA, USA). 2.2. Preparation of matrix and sample solutions Glass apparatus for experimental was washed with aqua regia prior to use. Solution A: 1 mL of 1% HAuCl4 solution was mixed with 79 mL deionized water. Solution B: 4 mL of 1% sodium citrate buffer, 0.1 mL of 1% tannic acid solution, 15.8 mL of distilled water were mixed with 0.1 mL of 25 mmol/L potassium carbonate solution. After heating the two solutions in water bath to 60 °C, quickly add solution B to stirring
2.3. Tissue section and matrix deposition The fresh Maca root is divided into upper, middle and lower layers of 5 mm thickness. The collected tissues were frozen in liquid nitrogen, and then stored in refrigerator at −20 °C. All tissues were sectioned at 20 μm thickness sections using a Leica CM1950 cryostat (Leica 2
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Fig. 3. Positive ion MALDI-TOF mass spectra of N-benzyl-octadecanamide (a) without matrix and with (b) DHB, (c) CHCA, and (d) AuNPs as matrices.
Microsystems GmbH, Wetzlar, Germany) at −20 °C and thaw mounted onto indium tin oxide (ITO) coated glass slides. The glass slides were then placed into a vacuum desiccator for 30 min before matrix application. The matrix was sprayed on tissue sections using an automatic matrix sprayer (ImagePrep, Bruker Daltonics). The instrumental parameters were as follows: 40 cycles; 60% spray intensity; 1 s spray time; 20 s incubating time; and 60 s drying time. AuNPs solution was sprayed on tissue sections by self-made matrix sprayer. The average weight of the matrix on tissue sections was 5 mg.
MALDI target plate, following by drying under a stream of nitrogen gas at room temperature. For MALDI-MSI analysis, imaging spatial resolution was set to 100 μm for root tissues from Maca, and each spectrum consists of 200 laser shots. Regions of interest (ROIs) were manually defined in the imaging software using both the optical image and MSI data image. The MS data was analyzed using FlexAnalysis 3.4, and MSI data FlexImaging 3.0, whose normalization method is total ion count (TIC). ROIs were manually defined in the imaging software using both the optical image and MSI data image. The data of FT-ICR MS and MS/MS were processed using DataAnalysis at the scan range of m/z 100–1000 Da.
2.4. MALDI-TOF MS or MSI analysis Profiling and imaging of tissue sections were performed on a MALDI TOF/TOF MS (Bruker Daltonics, Billerica, MA) equipped with a Smartbeam II Nd:YAG 355 nm laser. The laser is fired at a repetition rate of 2000 kHz, and the mass analyzer was operated in positive ion mode. The mass spectra in the reflector mode were collected with a pulsed ion extraction time of 80 ns, an accelerating voltage of 20.00 kV, an extraction voltage of 17.90 kV, a lens voltage of 5.85 kV, and a reflector voltage of 21.15 kV. The laser power was optimized at the beginning of each operation, then fixed in the whole experiment. For MALDI analysis, the dried-droplet sample preparation method was used as follows: 1 μL of sample solution was mixed with 1 μL of matrix solution, and 1 μL of the resulting mixture was then pipetted on the
2.5. Structural confirmation MS/MS fragmentations using the LIFT technique on the Ultra-flextreme MALDI-TOF/TOF MS together with the Solarix Fourier transform ion cyclotron resonance (FTICR) MS (Bruker Daltonics, Bremen, Germany) as well as Orbitrap MS (Thermo Scientific, Massachusetts, USA) were used for further confirmation of the identified components. The parameters for FTICR MS were as follows: time of flight: 80 ns, voltage of capillary exit: 200.0 V, voltage of deflector plate: 180.0 V, voltage of funnel 1120.0 V, voltage of skimmer 1 25.0 V, voltage of collision: −2.0 V, voltage of DC extract bias: 0.7 V, RF frequency: 1.4 MHz, and full resolution: 4 M. 3
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interference of matrix increase at the same experimental conditions. As the best compromise between signal intensity and background interference, 5 mg/mL was considered as the optimal concentration of AuNPs matrix for MALDI analysis. Fig. A. 2 shows positive ion MALDI-TOF mass spectra of amino acids, including histidine (His), isoleucine (Ile), lysine (Lys), asparagine (Asp), methionine (Met), tyrosine (Tyr), tryptophan (Trp), threonine (Thr) and serine (Ser) with AuNPs as matrix. The peaks at m/z 178, 154, 169, 155, 172, 204, 227, 142 and 128 are assigned to the sodium adducts of above amino acids, respectively. The signals of m/z 200, 176, 191, 177, 194, 226, 249, 166 and 150 are assigned to (M + 2Na-H)+ of the amino acids in MALDI mass spectra. Sucrose is a signal molecule in a signal-transduction pathway that regulates the symporter [17]. Using AuNPs as a matrix, glucose and sucrose were further analyzed in positive ion MALDI and the peaks of (M + Na)+ appear in the mass spectra (Fig. A. 3 in Supporting Information). The signals of (M + Na)+ can also be detected using AuNPs as a matrix to analyze some free fatty acids, such as palmitate sodium, linolenic acid, docosahexaenoic acid and eicosanoic acid (Fig. A. 4 in Supporting Information). Prior to analysis of the chemical components in Maca, we also evaluate the feasibility of this matrix to analyze natural products. Peak at m/z 633 associated with (M + Na)+ of rutin, a natural product, can be determined using AuNPs matrix in mass spectrum (Fig. A. 5a in Supporting Information). In the mass spectrum of 1,3-bis(2,4,6-trimethylphenyl) imidazolinium chloride, the peak of (M-Cl)+ clearly appears at m/z 305 with AuNPs as matrix (Fig. A. 5b in Supporting Information). It was reported that peaks of (M-Cl)+ clearly appear in the mass spectra of Lepidiline A and Lepidiline B in Maca [5]. In conclusion, imidazolium chloride alkaloids can be detected by MALDI MS as imidazolium ion. The limit of detection (LOD) and relative standard deviation for these analytes were shown in Table A. 1. For example, the LOD of 1,3-Bis(2,4,6-trimethylphenyl) imidazolium chloride was 0.5 pmol according to the peak detection criteria above a signal-tonoise (S/N) to 3 [18]. All these results are suggested that AuNPs can be used as a MALDI matrix to analyze small molecules. Using CHCA, DHB, AuNPs as matrices and without matrix, the mass spectra of N-benzyl-octadecanamide, one of the main macamides [8], are presented at the same experimental condition in Fig. 3. No peak of N-benzyl-octadecanamide is detected in mass spectrum with matrixfree. Although the peaks of (M + Na)+ and (M + K)+ at m/z 368 and 384 could be detected with DHB and CHCA as matrix, the intensity of the peaks is low. With the use of AuNPs as a matrix, the analyte is readily detected with high peak intensity, and, more importantly, the interference from matrix ion is eliminated completely. No fragment ion of the analyte is observed, suggesting that AuNPs is an effective positive ion-mode matrix for MALDI-TOF MS analysis of small molecules. The positive ion MALDI TOF mass spectra of extracts of fresh Maca and dry Maca are shown in Fig. 4. The compounds were identified on the basis of published literature, precise m/z and fragment ion
Fig. 4. Positive ion MALDI-TOF MS spectra of extract from (a) fresh Maca and (b) dry Maca using AuNPs as a matrix. The compounds detected in dry Maca but not in fresh Maca were labelled with red. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
3. Results and discussion The morphology and dimension of synthesized AuNPs were characterized using a JEM 2011 transmission electron microscope (TEM) (JEOL Ltd., Japan) (Fig. 1). The size of AuNPs is uniform and the diameter of them is about 7–10 nm. Due to their high absorption coefficient, AuNPs were used as a matrix for MALDI MS analysis of small molecules [15]. Fig. 2 presents the MALDI mass spectra of AuNPs acquired in both positive and negative ion modes. The negative ion spectrum exhibits five prominent peaks at m/z 197, 394, 591, 788 and 985 arising from Au−, Au2−, Au3−, Au4− and Au5−, respectively. In the positive ion spectrum, however, no peak is detected in the range of m/z 100–1000 Da. In addition, no distinctive ions associated with AuNPs and their fragments were found. This remarkable feature renders AuNPs well-suited for use as a MALDI matrix for MS analysis of small molecules. To optimize the concentration of AuNPs matrix, serial AuNPs solutions of 0.2 mg/mL, 1 mg/mL, 2 mg/mL, 5 mg/mL, and 10 mg/mL were used as a matrix to analyze N-benzyl-octadecanamide by MALDI MS (Fig. A. 1 in Supporting Information). The ion signal, (N-benzyloctadecanamide+Na)+, can be readily identified in the mass spectra of this sample. With the concentration of AuNPS increasing from 0.2 mg/ mL to 10 mg/mL, the signal intensity of sample and the background
Fig. 5. Ion images of the localization and spatial distribution of compounds in Maca root tissue with AuNPs and CHCA as matrices, respectively. 4
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Fig. 6. MALDI-MSI localization and spatial distribution of components in the upper (a), middle (b), and lower (c) parts of Maca root tissue using AuNPs as a matrix.
information (Table A. 2 in Supporting Information). Amino acids were detected in Maca by FTICR MS using AuNPs as a matrix. Peaks at m/z 156, 175 and 192 are assigned to the adducts of aspartic acid, arginine and glutamate, respectively. Peak at m/z 212 and 381, assigned to (Nbenzyl benzamide+H)+ and (sucrose+K)+, can also be detected in both Maca extracts by MALDI MS [19]. In Fig. 4(a), the peaks of m/z 252 and 274 were identified as (1-benzyl-5-(methoxymethyl)-1H-pyrrole-2-carbaldehyde+Na)+ and (1-benzyl-5-(methoxymethyl)-1H-pyrrole-2-carbaldehyde+2Na-H)+, which is in agreement with previous findings [7]. In Fig. 4(b), peak of m/z 422 is assigned to the sodium adduct of N-(3-methoxybenzyl)-(9Z,12Z)-octadecadienamide, which is in agreement to the reported literature that N-(3-methoxybenzyl)(9Z,12Z)-octadecadienamide is only detected in dry Maca [9]. Some signals of imidazole alkaloids including Lepidiline A (m/z 277), Lepidiline B (m/z 291), Lepidiline C (m/z 307), Lepidiline D (m/z 321), 1, 3dibenzyl-2-pentyl-4,5-dimethylimidazilium (m/z 347) and 1, 3-dibenzyl-2-phenyl-4,5-dimethylimidazilium (m/z 353) are detected in the MALDI mass spectra of dry Maca. Compared the result of Fig. 4, it is reasonably speculated that these imidazole alkaloids may also be the products of Maca after drying process. For comparison, AuNPs and CHCA were used as matrices to analyze sucrose and alkaloids in dry Maca root in situ by MALDI-MSI in Fig. 5. The result suggests that imaging obtained from AuNPs matrix not only has stronger pixels and clearer spatial distribution in analysis of sucrose (m/z 381), but obtains a lower detection limit in analysis of alkaloids in situ. No signal of Lepidiline A (m/z 277), DPDI (1, 3-dibenzyl-2-propenyl-4, 5-dimethyl imidazilium, m/z 317), DIDI(1, 3-dibenzyl-2-isopropyl-4, 5-dimethyl imidazilium, m/z 319) and DBDI (1, 3-dibenzyl-2butyl-4, 5-dimethyl imidazilium, m/z 333) is detected in fresh Maca root using both CHCA and AuNPs as matrices. The reason that the imidazole alkaloids can be detected in dry Maca may be that they are formed during storage and drying process [9,20]. In conclusion, there were two advantages of AuNPs used as a matrix to analyze tissue
sample by MALDI-MSI: (1) devoid of matrix interfering ions in the low m/z region; (2) the clear distribution of imidazole alkaloids in the Maca root tissue. There are different chemical components in the three parts of the cell layer in fresh Maca root (Fig. A. 6 in Supporting Information). The localization of these compounds in tissues in situ can be visualized by MALDI-MSI using AuNPs as a matrix in Fig. 6. The upper part of Maca, closer to Maca stem, contains amino acids, amides alkaloids, imidazolium alkaloids, saccharide, and so forth. Compared to the component in the upper part of Maca, no signal of DBDI and DPDI was detected in the middle part of Maca. In addition, proline (m/z 116), 1-benzyl-5(methoxymethyl)-1H-pyrrole-2-carbaldehyde (m/z 252) and benzylnitrile (m/z 104) were not detected in the lower part of fresh Maca root. Arginine is mainly distributed in the xylem of the three parts of fresh Maca root. Leucine is distributed in the xylem and cortex in the upper part, but rarely in the medulla. Sucrose is distributed in the xylem in the upper part, in the cortex and xylem in the middle part, and uniformly in the whole tissue in the lower part. Lepidiline A is in the cortex of the upper part, a little in the cortex of the middle part, but uniformly in the lower part. The distribution of some component in the tissue of Maca fresh roots is shown in Fig. 6. In conclusion, the localization of components in Maca root tissue can be clearly observed in situ by MALDIMSI with AuNPs as matrix. The conclusion provides a meaningful reference to improve the extraction efficiency of active components and to further study the effect of active components in Maca root. AuNPs was successfully synthesized via a double solution method. Due to its characteristic of uniform size and interference-free background, AuNPs was used as a matrix to analyze small molecule and to visualize the localization of amino acids, amides alkaloids, imidazolium alkaloids and saccharide in situ in the upper, middle and lower parts of Maca root tissue. The result shows that the distribution of the components in the Maca root can be clearly visualized by MALDI-MSI using AuNPs as a matrix. The synthetic sites of active component and the 5
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localization of active component in Maca at different growth stages are currently in progress in our laboratories.
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Declaration of competing interest We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted. Acknowledgements This project is supported by grants from the National Natural Sciences Foundation of China (Grant No 21565033). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.microc.2019.104190. References [1] B.L. Zheng, K. He, C.H. Kim, L. Rogers, Y. Shao, Z.Y. Huang, Y. Lu, S.J. Yan, L.C. Qien, Q.Y. Zheng, Effect of a lipidic extract from Lepidium meyenii on sexual behavior in mice and rats, Urology 55 (2000) 598–602. [2] I. Muhammad, J. Zhao, D.C. Dunbar, I.A. Khan, Constituents of Lepidium meyenii ‘Maca’, Phytochemistry 59 (2002) 105–110. [3] J.P. Zhao, I. Muhammad, D.C. Dunbar, J. Mustafa, I.A. Khan, New alkamides from Maca (Lepidium meyenii), J. Agric. Food Chem. 53 (2005) 690–693. [4] S. Piacente, V. Carbone, A. Plaza, A. Zampelli, C. Pizza, Investigation of the tuber constituents of Maca (Lepidium meyenii Walp.), J. Agric. Food Chem. 50 (2002) 5621–5625. [5] B. Cui, B.L. Zheng, K. He, Q.Y. Zheng, Imidazole alkaloids from Lepidium meyenii, J. Nat. Prod. 66 (2003) 1101–1103. [6] M. Yu, X.-J. Qin, L.-D. Shao, X.-R. Peng, L. Li, H. Yang, M.-H. Qiu, Macahydantoins a and B, two new thiohydantoin derivatives from Maca (Lepidium meyenii): structural elucidation and concise synthesis of macahydantoin a, Tetrahedron Lett. 58 (2017) 1684–1686.
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Mass spectrometry imaging of small molecule in situ in Lepidium meyenii (Maca) using gold nanoparticles matrix
T
Sihou Yanga,b, Lingpeng Zhanb,c, Chaozi Liub,c, Ling Fua, Rui Chena, , Zongxiu Nieb,c,d ⁎
a
College of Chemistry and Chemical Engineering, Yunnan Normal University, Kunming 650500, China Key Laboratory of Analytical Chemistry for Living Biosystems, Institute of Chemistry Chinese Academy of Sciences, Beijing 100190, China c Beijing National Laboratory for Molecular Sciences, Beijing 100190, China d Beijing Center for Mass Spectrometry, Beijing 100190, China b
ARTICLE INFO
ABSTRACT
Keywords: Lepidium meyenii (Maca) Mass spectrometry imaging Small molecule In situ Gold nanoparticles
Lepidium meyenii Walp. (Maca), a functional food, has been used as a drug and food in Peru for more than two thousand years because Maca is rich in nutrition and rational in nutritional structure. Due to their uniform size and little interference in the low molecular weight region, Au nanoparticles (AuNPs) have been applied as a matrix to analyze the components of Maca extract and to visualize them of fresh root tissue in situ by matrixassisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The result shows that imidazole alkaloids including Lepidiline A (1,3-dibenzyl-4,5-dimethylimidazolium chloride), Lepidiline B (1,3dibenzyl-2,4,5-trimethylimidazolium chloride), Lepidiline C (3-benzyl-1-(3-methoxybenzyl)-4,5-dimethylimidazolium chloride), Lepidiline D (3-benzyl-1-(3-methoxybenzyl)-2,4,5-trimethylimidazolium chloride), 1, 3-dibenzyl-2-pentyl-4,5-dimethylimidazilium and 1, 3-dibenzyl-2-phenyl-4,5-dimethylimidazilium are the products of Maca after drying proess. Additionally, the distribution of amino acids, amide alkaloids, imidazolium alkaloids and saccharide in the upper, middle, and lower parts of Maca root tissue can be clearly visualized in situ with the synthesized AuNPs matrix by MALDI mass spectrometry imaging (MSI). The report provides a meaningful reference to study active components from natural product in situ.
1. Introduction Lepidium meyenii Walp (Maca), a functional food with medicinal and economic value, has been used as food and medicine in Peru for over two thousand years. The hypocotyl of Maca contains some secondary metabolites such as alkaloid, glucosinolate, steroid, flavonoid, and natural phenols. Alkaloid, a class of naturally occurring organic nitrogen-containing bases, has diverse and important physiological effects on humans and other animals. Alkaloid in Maca contains macamides [1], macaridine (the benzylated derivative of 1,2-dihydro-Nhydroxypyridine together with the benzylated alkamides) [2,3], βcarboline [4], imidazole alkaloids [5], macahydantoin (the thiohydantoin derivatives) [6] and macapyrrolin [7]. Macamides, found only in Maca, are secondary amides formed by benzylamine and a fatty acid moiety with different hydrocarbon chain lengths and degree of unsaturation [8,9]. Some of the aphrodisiac activities of Maca have been related to the lipidic fraction of Maca which contains mainly fatty acids and macamides [1,8]. The pentane extract of Maca contains a number of macamides that may act on the endocannabinoid system by
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inhibitory activity on fatty acid amide hydrolase [10]. The methanol extract of Maca contains (1R,3S)-1-methyltetrahydro-β-carboline-3carboxylic acid, which exerts many activities on the central nervous system [4]. Imidazole alkaloids have been isolated from a root extract of Maca and identified as Lepidiline A (1,3-dibenzyl-4,5-dimethylimidazolium chloride), Lepidiline B (1,3-dibenzyl-2,4,5-trimethylimidazolium chloride), Lepidiline C (3-benzyl-1-(3-methoxybenzyl)-4,5-dimethylimidazolium chloride), Lepidiline D (3-benzyl-1-(3methoxybenzyl)-2,4,5-trimethylimidazolium chloride), and a class of sulfur-containing hexahydroimidazo[1,5-c]thiazole derivatives [5,11,12]. Macapyrrolin is the pyrrole alkaloids found in Maca with a benzyl substituent attached to the pyrrole nucleus and evaluated for its cytotoxicity against five human cancer cell lines [7]. In conclusion, due to its rich nutrient and secondary metabolites, Maca has good efficacy in anti-oxidation, anti-fatigue, neuroprotection, and in treatment of diabetes, benign prostatic hyperplasia, female menopausal syndrome, and osteoporosis in the elderly. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a “soft” ionization technique that uses
Corresponding author. E-mail address: [email protected] (R. Chen).
https://doi.org/10.1016/j.microc.2019.104190 Received 8 July 2019; Received in revised form 15 August 2019; Accepted 16 August 2019 Available online 19 August 2019 0026-265X/ © 2019 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).
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the matrix absorbed a laser energy to create ions from analyte with minimal fragmentation. Such technique is widely used to analyze organic compound and biomolecule in metabolomics, pharmaceutical sciences, environmental sciences and biomedicine. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is an emerging technology for study of the distribution of biomolecules in animal or plant tissues because it can visualize the localization and distribution of biomolecule in situ without marker. For example, it has been used to analyze the localization and distribution of small molecular metabolites in plant tissues at different growth stages and visualize the synthetic position of active components [13]. However, it is difficult to analyze some small molecular compounds (MW < 500) because traditional matrices have interference background peaks in the low mass region. Carbon-based material, such as graphene, was applied as MALDI matrix without background interference to analyze small molecules in Chinese herbal medicine. Because nanomaterial has the advantages of background-free, high sensitivity, good reproducibility and salt tolerance in MS analysis, nanomaterial-assisted laser desorption/ionization mass spectrometry without background interference is a powerful tool for the analysis of complex compound. For example, due to its excellent desorption/ionization efficiency, hexagonal boron nitride nanosheet was used as a multifunctional background-free matrix to detect small molecular metabolite by MALDI MS and analyze complex sample by mass spectrometry imaging (MSI) [14]. Novel metals, including Au, were found to be more efficient than organic matrices for most small metabolites tested. Water-soluble Au nanoclusters were reported to detect sugars, nucleosides, fatty acids, amino acids, and lipids in animal tissue for MSI [15]. Different types of sputter-coated metals were screened for small molecule analysis in MSI [16]. The proof of concept application was conducted on plant tissue, maize roots and seeds. Herein, due to its characteristics of high absorption coefficient [15], high resolution, little interference to low detection limit, and selective detection for some species, gold nanoparticles (AuNPs) were used as MALDI matrix to analyze and locate the components in Maca root by MALDI MS.
Fig. 1. TEM image of AuNPs.
2. Materials and methods
Fig. 2. (a) Negative ion MALDI-TOF mass spectrum of AuNPs. (b) Positive ion MALDI-TOF mass spectrum of AuNPs.
2.1. Chemicals and reagents
solution A. Then, heat the mixture until wine red colloidal gold is gained. Finally, centrifuge colloidal gold at 12000 rpm for 15 min to obtain the synthetic AuNPs solution. Fresh Maca root (10 g) and dry Maca powder (2 g) were extracted with 70% acetonitrile (10 mL) by ultrasonic wave for 60 min, respectively. The mixture was centrifuged into liquid and solid phases at 3000 rpm for 5 min. The liquid phase was directly analyzed by MALDI MS. Different concentrations of AuNPs solution were prepared for MALDI MS analysis. CHCA and DHB solutions were prepared at concentration of 10 mg/mL and 20 mg/mL in 0.2% TFA of 70% acetonitrile. Stock solutions of amino acids, fatty acids, N-benzyl-octadecanamide, 1,3-bis(2,4,6-trimethylphenyl) imidazolinium chloride and rutin were prepared at a concentration of 10 mmol/L in methanol and diluted to the desired concentration as required. Standard solutions of saccharides were prepared at a concentration of 10 mmol/L in deionized water and diluted to the desired concentration as required.
α-Cyano-4-hydroxycinnamic acid (CHCA), 2, 5-dihydroxybenzoic acid (DHB), trifluoroacetic acid (TFA), acetonitrile, methanol, HAuCl4, sodium citrate, potassium carbonate and tannic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). Inbred Maca was obtained from Lijiang at altitude of 2800 m (Lijiang, Yunnan, China). 1, 3-bis (2, 4, 6-trimethylphenyl) imidazolinium chloride, rutin were from J&K Scientific Ltd. (Beijing, China). N-benzyl-octadecanamide was purchased from Wuhan Huashite Industrial Biotechnology Development Co., Ltd. (Wuhan, Hubei, China). Amino acids (Histidine, asparagine, isoleucine, lysine, methionine, tyrosine, tryptophan, threonine, serine), fatty acids (palmitate sodium, linolenic acid, docosahexaenoic acid, eicosanoic acid), and saccharides (glucose, sucrose) were from Millipore Sigma (St. Louis, MO, USA). All chemicals were used in the highest purity and without further purification. The deionized water was prepared on a Milli-Q water system from Millipore Corp. (Billerica, MA, USA). 2.2. Preparation of matrix and sample solutions Glass apparatus for experimental was washed with aqua regia prior to use. Solution A: 1 mL of 1% HAuCl4 solution was mixed with 79 mL deionized water. Solution B: 4 mL of 1% sodium citrate buffer, 0.1 mL of 1% tannic acid solution, 15.8 mL of distilled water were mixed with 0.1 mL of 25 mmol/L potassium carbonate solution. After heating the two solutions in water bath to 60 °C, quickly add solution B to stirring
2.3. Tissue section and matrix deposition The fresh Maca root is divided into upper, middle and lower layers of 5 mm thickness. The collected tissues were frozen in liquid nitrogen, and then stored in refrigerator at −20 °C. All tissues were sectioned at 20 μm thickness sections using a Leica CM1950 cryostat (Leica 2
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Fig. 3. Positive ion MALDI-TOF mass spectra of N-benzyl-octadecanamide (a) without matrix and with (b) DHB, (c) CHCA, and (d) AuNPs as matrices.
Microsystems GmbH, Wetzlar, Germany) at −20 °C and thaw mounted onto indium tin oxide (ITO) coated glass slides. The glass slides were then placed into a vacuum desiccator for 30 min before matrix application. The matrix was sprayed on tissue sections using an automatic matrix sprayer (ImagePrep, Bruker Daltonics). The instrumental parameters were as follows: 40 cycles; 60% spray intensity; 1 s spray time; 20 s incubating time; and 60 s drying time. AuNPs solution was sprayed on tissue sections by self-made matrix sprayer. The average weight of the matrix on tissue sections was 5 mg.
MALDI target plate, following by drying under a stream of nitrogen gas at room temperature. For MALDI-MSI analysis, imaging spatial resolution was set to 100 μm for root tissues from Maca, and each spectrum consists of 200 laser shots. Regions of interest (ROIs) were manually defined in the imaging software using both the optical image and MSI data image. The MS data was analyzed using FlexAnalysis 3.4, and MSI data FlexImaging 3.0, whose normalization method is total ion count (TIC). ROIs were manually defined in the imaging software using both the optical image and MSI data image. The data of FT-ICR MS and MS/MS were processed using DataAnalysis at the scan range of m/z 100–1000 Da.
2.4. MALDI-TOF MS or MSI analysis Profiling and imaging of tissue sections were performed on a MALDI TOF/TOF MS (Bruker Daltonics, Billerica, MA) equipped with a Smartbeam II Nd:YAG 355 nm laser. The laser is fired at a repetition rate of 2000 kHz, and the mass analyzer was operated in positive ion mode. The mass spectra in the reflector mode were collected with a pulsed ion extraction time of 80 ns, an accelerating voltage of 20.00 kV, an extraction voltage of 17.90 kV, a lens voltage of 5.85 kV, and a reflector voltage of 21.15 kV. The laser power was optimized at the beginning of each operation, then fixed in the whole experiment. For MALDI analysis, the dried-droplet sample preparation method was used as follows: 1 μL of sample solution was mixed with 1 μL of matrix solution, and 1 μL of the resulting mixture was then pipetted on the
2.5. Structural confirmation MS/MS fragmentations using the LIFT technique on the Ultra-flextreme MALDI-TOF/TOF MS together with the Solarix Fourier transform ion cyclotron resonance (FTICR) MS (Bruker Daltonics, Bremen, Germany) as well as Orbitrap MS (Thermo Scientific, Massachusetts, USA) were used for further confirmation of the identified components. The parameters for FTICR MS were as follows: time of flight: 80 ns, voltage of capillary exit: 200.0 V, voltage of deflector plate: 180.0 V, voltage of funnel 1120.0 V, voltage of skimmer 1 25.0 V, voltage of collision: −2.0 V, voltage of DC extract bias: 0.7 V, RF frequency: 1.4 MHz, and full resolution: 4 M. 3
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interference of matrix increase at the same experimental conditions. As the best compromise between signal intensity and background interference, 5 mg/mL was considered as the optimal concentration of AuNPs matrix for MALDI analysis. Fig. A. 2 shows positive ion MALDI-TOF mass spectra of amino acids, including histidine (His), isoleucine (Ile), lysine (Lys), asparagine (Asp), methionine (Met), tyrosine (Tyr), tryptophan (Trp), threonine (Thr) and serine (Ser) with AuNPs as matrix. The peaks at m/z 178, 154, 169, 155, 172, 204, 227, 142 and 128 are assigned to the sodium adducts of above amino acids, respectively. The signals of m/z 200, 176, 191, 177, 194, 226, 249, 166 and 150 are assigned to (M + 2Na-H)+ of the amino acids in MALDI mass spectra. Sucrose is a signal molecule in a signal-transduction pathway that regulates the symporter [17]. Using AuNPs as a matrix, glucose and sucrose were further analyzed in positive ion MALDI and the peaks of (M + Na)+ appear in the mass spectra (Fig. A. 3 in Supporting Information). The signals of (M + Na)+ can also be detected using AuNPs as a matrix to analyze some free fatty acids, such as palmitate sodium, linolenic acid, docosahexaenoic acid and eicosanoic acid (Fig. A. 4 in Supporting Information). Prior to analysis of the chemical components in Maca, we also evaluate the feasibility of this matrix to analyze natural products. Peak at m/z 633 associated with (M + Na)+ of rutin, a natural product, can be determined using AuNPs matrix in mass spectrum (Fig. A. 5a in Supporting Information). In the mass spectrum of 1,3-bis(2,4,6-trimethylphenyl) imidazolinium chloride, the peak of (M-Cl)+ clearly appears at m/z 305 with AuNPs as matrix (Fig. A. 5b in Supporting Information). It was reported that peaks of (M-Cl)+ clearly appear in the mass spectra of Lepidiline A and Lepidiline B in Maca [5]. In conclusion, imidazolium chloride alkaloids can be detected by MALDI MS as imidazolium ion. The limit of detection (LOD) and relative standard deviation for these analytes were shown in Table A. 1. For example, the LOD of 1,3-Bis(2,4,6-trimethylphenyl) imidazolium chloride was 0.5 pmol according to the peak detection criteria above a signal-tonoise (S/N) to 3 [18]. All these results are suggested that AuNPs can be used as a MALDI matrix to analyze small molecules. Using CHCA, DHB, AuNPs as matrices and without matrix, the mass spectra of N-benzyl-octadecanamide, one of the main macamides [8], are presented at the same experimental condition in Fig. 3. No peak of N-benzyl-octadecanamide is detected in mass spectrum with matrixfree. Although the peaks of (M + Na)+ and (M + K)+ at m/z 368 and 384 could be detected with DHB and CHCA as matrix, the intensity of the peaks is low. With the use of AuNPs as a matrix, the analyte is readily detected with high peak intensity, and, more importantly, the interference from matrix ion is eliminated completely. No fragment ion of the analyte is observed, suggesting that AuNPs is an effective positive ion-mode matrix for MALDI-TOF MS analysis of small molecules. The positive ion MALDI TOF mass spectra of extracts of fresh Maca and dry Maca are shown in Fig. 4. The compounds were identified on the basis of published literature, precise m/z and fragment ion
Fig. 4. Positive ion MALDI-TOF MS spectra of extract from (a) fresh Maca and (b) dry Maca using AuNPs as a matrix. The compounds detected in dry Maca but not in fresh Maca were labelled with red. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
3. Results and discussion The morphology and dimension of synthesized AuNPs were characterized using a JEM 2011 transmission electron microscope (TEM) (JEOL Ltd., Japan) (Fig. 1). The size of AuNPs is uniform and the diameter of them is about 7–10 nm. Due to their high absorption coefficient, AuNPs were used as a matrix for MALDI MS analysis of small molecules [15]. Fig. 2 presents the MALDI mass spectra of AuNPs acquired in both positive and negative ion modes. The negative ion spectrum exhibits five prominent peaks at m/z 197, 394, 591, 788 and 985 arising from Au−, Au2−, Au3−, Au4− and Au5−, respectively. In the positive ion spectrum, however, no peak is detected in the range of m/z 100–1000 Da. In addition, no distinctive ions associated with AuNPs and their fragments were found. This remarkable feature renders AuNPs well-suited for use as a MALDI matrix for MS analysis of small molecules. To optimize the concentration of AuNPs matrix, serial AuNPs solutions of 0.2 mg/mL, 1 mg/mL, 2 mg/mL, 5 mg/mL, and 10 mg/mL were used as a matrix to analyze N-benzyl-octadecanamide by MALDI MS (Fig. A. 1 in Supporting Information). The ion signal, (N-benzyloctadecanamide+Na)+, can be readily identified in the mass spectra of this sample. With the concentration of AuNPS increasing from 0.2 mg/ mL to 10 mg/mL, the signal intensity of sample and the background
Fig. 5. Ion images of the localization and spatial distribution of compounds in Maca root tissue with AuNPs and CHCA as matrices, respectively. 4
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Fig. 6. MALDI-MSI localization and spatial distribution of components in the upper (a), middle (b), and lower (c) parts of Maca root tissue using AuNPs as a matrix.
information (Table A. 2 in Supporting Information). Amino acids were detected in Maca by FTICR MS using AuNPs as a matrix. Peaks at m/z 156, 175 and 192 are assigned to the adducts of aspartic acid, arginine and glutamate, respectively. Peak at m/z 212 and 381, assigned to (Nbenzyl benzamide+H)+ and (sucrose+K)+, can also be detected in both Maca extracts by MALDI MS [19]. In Fig. 4(a), the peaks of m/z 252 and 274 were identified as (1-benzyl-5-(methoxymethyl)-1H-pyrrole-2-carbaldehyde+Na)+ and (1-benzyl-5-(methoxymethyl)-1H-pyrrole-2-carbaldehyde+2Na-H)+, which is in agreement with previous findings [7]. In Fig. 4(b), peak of m/z 422 is assigned to the sodium adduct of N-(3-methoxybenzyl)-(9Z,12Z)-octadecadienamide, which is in agreement to the reported literature that N-(3-methoxybenzyl)(9Z,12Z)-octadecadienamide is only detected in dry Maca [9]. Some signals of imidazole alkaloids including Lepidiline A (m/z 277), Lepidiline B (m/z 291), Lepidiline C (m/z 307), Lepidiline D (m/z 321), 1, 3dibenzyl-2-pentyl-4,5-dimethylimidazilium (m/z 347) and 1, 3-dibenzyl-2-phenyl-4,5-dimethylimidazilium (m/z 353) are detected in the MALDI mass spectra of dry Maca. Compared the result of Fig. 4, it is reasonably speculated that these imidazole alkaloids may also be the products of Maca after drying process. For comparison, AuNPs and CHCA were used as matrices to analyze sucrose and alkaloids in dry Maca root in situ by MALDI-MSI in Fig. 5. The result suggests that imaging obtained from AuNPs matrix not only has stronger pixels and clearer spatial distribution in analysis of sucrose (m/z 381), but obtains a lower detection limit in analysis of alkaloids in situ. No signal of Lepidiline A (m/z 277), DPDI (1, 3-dibenzyl-2-propenyl-4, 5-dimethyl imidazilium, m/z 317), DIDI(1, 3-dibenzyl-2-isopropyl-4, 5-dimethyl imidazilium, m/z 319) and DBDI (1, 3-dibenzyl-2butyl-4, 5-dimethyl imidazilium, m/z 333) is detected in fresh Maca root using both CHCA and AuNPs as matrices. The reason that the imidazole alkaloids can be detected in dry Maca may be that they are formed during storage and drying process [9,20]. In conclusion, there were two advantages of AuNPs used as a matrix to analyze tissue
sample by MALDI-MSI: (1) devoid of matrix interfering ions in the low m/z region; (2) the clear distribution of imidazole alkaloids in the Maca root tissue. There are different chemical components in the three parts of the cell layer in fresh Maca root (Fig. A. 6 in Supporting Information). The localization of these compounds in tissues in situ can be visualized by MALDI-MSI using AuNPs as a matrix in Fig. 6. The upper part of Maca, closer to Maca stem, contains amino acids, amides alkaloids, imidazolium alkaloids, saccharide, and so forth. Compared to the component in the upper part of Maca, no signal of DBDI and DPDI was detected in the middle part of Maca. In addition, proline (m/z 116), 1-benzyl-5(methoxymethyl)-1H-pyrrole-2-carbaldehyde (m/z 252) and benzylnitrile (m/z 104) were not detected in the lower part of fresh Maca root. Arginine is mainly distributed in the xylem of the three parts of fresh Maca root. Leucine is distributed in the xylem and cortex in the upper part, but rarely in the medulla. Sucrose is distributed in the xylem in the upper part, in the cortex and xylem in the middle part, and uniformly in the whole tissue in the lower part. Lepidiline A is in the cortex of the upper part, a little in the cortex of the middle part, but uniformly in the lower part. The distribution of some component in the tissue of Maca fresh roots is shown in Fig. 6. In conclusion, the localization of components in Maca root tissue can be clearly observed in situ by MALDIMSI with AuNPs as matrix. The conclusion provides a meaningful reference to improve the extraction efficiency of active components and to further study the effect of active components in Maca root. AuNPs was successfully synthesized via a double solution method. Due to its characteristic of uniform size and interference-free background, AuNPs was used as a matrix to analyze small molecule and to visualize the localization of amino acids, amides alkaloids, imidazolium alkaloids and saccharide in situ in the upper, middle and lower parts of Maca root tissue. The result shows that the distribution of the components in the Maca root can be clearly visualized by MALDI-MSI using AuNPs as a matrix. The synthetic sites of active component and the 5
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localization of active component in Maca at different growth stages are currently in progress in our laboratories.
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Declaration of competing interest We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted. Acknowledgements This project is supported by grants from the National Natural Sciences Foundation of China (Grant No 21565033). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.microc.2019.104190. References [1] B.L. Zheng, K. He, C.H. Kim, L. Rogers, Y. Shao, Z.Y. Huang, Y. Lu, S.J. Yan, L.C. Qien, Q.Y. Zheng, Effect of a lipidic extract from Lepidium meyenii on sexual behavior in mice and rats, Urology 55 (2000) 598–602. [2] I. Muhammad, J. Zhao, D.C. Dunbar, I.A. Khan, Constituents of Lepidium meyenii ‘Maca’, Phytochemistry 59 (2002) 105–110. [3] J.P. Zhao, I. Muhammad, D.C. Dunbar, J. Mustafa, I.A. Khan, New alkamides from Maca (Lepidium meyenii), J. Agric. Food Chem. 53 (2005) 690–693. [4] S. Piacente, V. Carbone, A. Plaza, A. Zampelli, C. Pizza, Investigation of the tuber constituents of Maca (Lepidium meyenii Walp.), J. Agric. Food Chem. 50 (2002) 5621–5625. [5] B. Cui, B.L. Zheng, K. He, Q.Y. Zheng, Imidazole alkaloids from Lepidium meyenii, J. Nat. Prod. 66 (2003) 1101–1103. [6] M. Yu, X.-J. Qin, L.-D. Shao, X.-R. Peng, L. Li, H. Yang, M.-H. Qiu, Macahydantoins a and B, two new thiohydantoin derivatives from Maca (Lepidium meyenii): structural elucidation and concise synthesis of macahydantoin a, Tetrahedron Lett. 58 (2017) 1684–1686.
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