Visualizing spatial distribution of small molecules in the rhubarb stalk (Rheum rhabarbarum) by surface-transfer mass spectrometry imaging

Phytochemistry 139 (2017) 72e80

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Visualizing spatial distribution of small molecules in the rhubarb stalk (Rheum rhabarbarum) by surface-transfer mass spectrometry imaging Joanna Nizioł*, Justyna Sekuła, Tomasz Ruman
w University of Technology, Faculty of Chemistry, 6 Powstan
co
w Warszawy Ave., 35-959, Rzeszo
w, Poland Rzeszo

a r t i c l e i n f o

a b s t r a c t

Article history: Received 15 October 2016 Received in revised form 7 April 2017 Accepted 11 April 2017 Available online 17 April 2017

Laser desorption/ionization mass spectrometry imaging (LDI-MSI) with gold nanoparticle-enhanced target (AuNPET) was used for visualization of small molecules in the rhubarb stalk (Rheum rhabarbarum L.). Analysis was focused on spatial distribution of biologically active compounds which are found in rhubarb species. Detected compounds belong to a very wide range of chemical compound classes such as anthraquinone derivatives and their glucosides, stilbenes, anthocyanins, flavonoids, polyphenols, organic acids, chromenes, chromanones, chromone glycosides and vitamins. The analysis of the spatial distribution of these compounds in rhubarb stalk with the nanoparticle-rich surface of AuNPET target plate has been made without additional matrix and with minimal sample preparation steps. © 2017 Elsevier Ltd. All rights reserved.

Keywords: Rheum rhabarbarum Polygonaceae Rhubarb Mass spectrometry imaging Low molecular weight compounds Gold nanoparticles

1. Introduction Rhubarb (Rheum rhabarbarum L.) is the common name for approximately 50 various kinds of Rheum species of plants in the family Polygonaceae, some of which have been domesticated as medicinal rhubarb (e.g., Rheum officinale B. and Rheum palmatum L.) and others are considered as vegetable rhubarb [Rheum rhabarbarum (syn. undulatum) L.]. Furthermore, some of them (e.g. Rheum rhaponticum L.) are used both as food and as raw material for medicinal purposes (Chin and Youngken, 1947). Rhubarb (R. rhabarbarum) is one of the oldest and most well-known traditional Chinese herbal medicine and has been widely used for more than thousands of years most frequently in China for the treatment of a variety of diseases including gastro-intestinal hemorrhage, constipation, inflammation, ulcers and jaundice (Xiao et al., 1984; Duke, 2002; Wu et al., 1995). The root and the dried rhizome are still being used as ingredients of some drugs. Recently, many research groups focused on the pharmaceutical applications of rhubarb which include not only purgation, analgesic, antibacterial, antitumor and antispasmodic effects, but its ingredients may be of use in case of renal disorders (Tang and

* Corresponding author. E-mail address: [email protected] (J. Nizioł). http://dx.doi.org/10.1016/j.phytochem.2017.04.006 0031-9422/© 2017 Elsevier Ltd. All rights reserved.

Eisenbrand, 1992; Huang, 1993). Till now, a large number of compounds have been isolated from Rheum species. The major biologically active compounds in rhubarb are anthraquinone derivatives including emodin (1,3,8-trihydroxy6-methylanthraquinone), aloe-emodin (1,8-dihydroxy-3-hydroxymethylanthraquinone), physcion (1,8-drihydroxy-3-methyl-6methoxyanthraquinone), chrysophanol (1,8-dihydroxy-3-methylanthraquinone), rhein (1,8-dihydroxy-3-carboxyanthraquinone), danthron (1,8-dihydroxy-9,10-anthraquinone) and their glucosides (Cai et al., 2004; Huang et al., 2007). What is interesting, biologically active compounds found in this plant belong into different classes of chemical compounds such as dianthrones, stilbenes, anthocyanins, flavonoids, polyphenols, organic acids, chromenes, chromone glycosides and vitamins classes (Agarwal et al., 2001). In recent years, many techniques have been reported for the separation and determination of active compounds in rhubarb. However, the analysis of the spatial distribution of molecules of interests in rhubarb tissue has not yet been done. The visualization of distribution of biomolecules in organs and organelles of biological samples has until recently mainly been done with conventional techniques, such as fluorescence microscopy with proper labelling or staining techniques. There is also a need to increase the knowledge regarding analysis of the patterns of temporal-spatial distribution of specific molecules in the plant metabolome that are induced by environmental stresses. To achieve mentioned aims,

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laser desorption/ionization mass spectrometry imaging (LDI-MSI) methods are currently the best choice. LDI-MSI is a two-dimensional mass spectrometry based molecular imaging technique which allows visualization of the spatial distribution of many molecules in tissues sections without extraction, purification, separation, or labelling. Over the preceding decade, LDI-MSI has been widely adopted in several scientific fields including the medicinal, pharmaceutical, and botanical research communities (Svatos, 2010). Laser-based mass spectrometry imaging is a powerful analytical tool that allows for the analysis of hundreds to thousands of compounds in a single experiment. MSI produces important information concerning primary metabolism, natural products, plant defense, plant response to abiotic and biotic stress. In the recent years, several MS techniques have been reported for imaging of a wide range of materials. Among MSI techniques, matrix assisted laser-desorption ionization (MALDI) (Norris and Caprioli, 2013), secondary-ion mass spectrometry (SIMS) (Belu et al., 2003), and desorption electrospray ionization (DESI) (Heyman and Dubery, 2016; Thunig et al., 2011) are most commonly used. Spatial analysis of biological tissues by MSI is commonly performed using thin tissue sections performed in a cryostat with a microtome. The sections are then moved in two dimensions while the mass spectra are recorded (Norris and Caprioli, 2013). Alternatively, newer approaches applying variations of blotting or imprint techniques where the chemicals from biological samples are initially transferred to another surfaces were shown. This relatively new approach has been successfully applied in several MSI techniques including MALDI (Tucker et al., 2011), € vall et al., 2003), DESI (Watrous et al., 2010) and nanoSIMS (Sjo
et al., 2010). assisted laser desorption-ionization (Vidova LDI-MSI is expected to open a new frontier in plant science. In particular, to our best knowledge, the spatial distribution of metabolites in rhubarb stalk have never been examined previously. Herein, we present examples of imaging of biological tissue represented by cross-sections of rhubarb stalk, which was imprinted onto gold nanoparticle enhanced target (AuNPET) and followed by LDI-MSI analysis (Sekuła et al., 2015a, 2015b). 2. Results and discussion Gold nanoparticle-enhanced target (AuNPET) was used previously for LDI-MS analysis of low molecular weight (LMW) compounds in biological objects (Sekuła et al., 2015a). AuNPET target plate was shown in our recent work to be a promising alternative to traditional MALDI targets (Sekuła et al., 2015b). The list of compounds shown in Table 1 was created based on both MS imaging but also literature data. All of presented compounds were previously found in rhubarb tissue, usually in relatively large quantities. The identity of majority of compounds was confirmed with LIFT® MS/MS experiments (Supplementary Information). Figures presented within this work contain ion images generated for all compounds/ions listed in Table 1. Additional information regarding MS data is shown in Supplementary Information. LDI-MSI experiment data was performed by measuring series of high-resolution MS spectra with 40
40 mm lateral resolution of part of rhubarb stalk imprint of ca. 5
3 mm size made on AuNPET target plate. This methodology was previously shown to give excellent results (Nizioł et al., 2016). One of the most important classes of chemical compounds found in rhubarb species are anthraquinones and their glycosides due to their medical applications. They are phenolic-type compounds naturally occurring in all species of rhubarb (Tang and Eisenbrand, 1992; He and Luo, 1980). Compounds of this class were believed to be the active laxative agents used in Chinese

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traditional medicine. Purgative effect of rhubarb has been attributed to the significant amount of anthraquinone derivatives and their glycosides (Qu et al., 2008; Yang et al., 2011). The most important pharmaceutically relevant anthraquinone derivatives in rhubarb are emodin and aloe-emodin. These compounds have been determined to have a variety of additional therapeutic effects such as anti-inflammatory (Ghosh et al., 2010; Choi et al., 2013), antidiabetic (Wang et al., 2006; Zhao et al., 2009) and anti-tumor properties (Srinivas et al., 2007; Tabolacci et al., 2010; Wei et al., 2011). Our LDI-MSI studies on the constituents of rhubarb stalk have revealed the presence of a variety of anthraquinone derivatives i.e. emodin, aloe-emodin, chrysophanol, chrysophanic acid, 2(hydroxymethyl)anthraquinone and citreorosein (Table 1). All of mentioned compounds have previously been isolated from different species of rhubarb (Oshio, 1978; Tutin and Clewer, 1908; Chopra et al., 1978). Mass spectrometry imaging results of the rhubarb stalk imprint suggest, that the ions of m/z 271.0601 (Fig. 1D) corresponds to [MþH]þ form of ion of emodin and isomeric aloe-emodin. As noticeable on the ion image, these ions are located almost exclusively in ground tissue near epidermal cells. Moreover, high abundance of ions was observed from vascular bundle region of the plant situated near the epidermis. Image of ion distribution shown in Fig. 1E presents the lateral distribution of ion of m/z 255.0652 assigned to proton adduct of chrysophanol. In Chinese traditional medicine, the antraquinones have been used as a laxative, but pharmacological studies have credited antraquinones with hemostatic and bactericidal properties (Lu et al., 2010). Another ion image concerns the distributions of the potassium adduct (Fig. 1F) of 2-(hydroxymethyl)anthraquinone at m/z 277.0262 with highest abundance near epidermis cells. 2-(Hydroxymethyl)anthraquinone is one of the most important biologically active constituents found in rhubarb species, with antiinflammatory activity (Dzoyem et al., 2016) and strong activity against Helicobacter pylori (Park et al., 2006). In contrast to above-discussed ion localization, the ions at m/z 287.0550 (Fig. 1G) appears to be exclusively localized within the skin of the rhubarb stalk section. This has been tentatively identified as the citreorosein, which was found to have oestrogenic and tyrosinase-inhibitory activities (Lu et al., 2012). Anthraquinone glucosides were found almost in all species of rhubarb (Okabe et al., 1973). Spatial distribution of sodium adducts of emodin and aloe-emodin glucoside (m/z 455.0949, Fig. 1H), presents the highest abundance near the edge of the stalk. Similarly, ions assigned to protonated adducts of sennoside E and F (m/z 847.2080, Fig. 1I) were found almost exclusively near the epidermis. Mentioned glycosides of emodin and aloe-emodin were previously described as the important constituents of rhubarb (Wagner et al., 1963). The presence of isomeric sennosides E and F was also reported in rhubarb species (Oshio et al., 1972). Anthraglycosides, similarly to antraquinones, show purgative activity but it was concluded that anthraquinones are less active than their glycosides (Oshio et al., 1974). Rhubarb species produce a variety of stilbene derivatives. The most important stilbenes analyzed in this work include important anti-oxidant resveratrol, trans-stilbene, rhaponticin and piceatannol 4'-galloylglucoside. The presence of these compounds has been reported in various rhubarb species (Kashiwada et al., 1984; Matsuda et al., 2001). It can be judged from Fig. 1I that the highest concentration of resveratrol in form of sodium adduct at m/z 251.0679 is in ground tissue near epidermal cells. Resveratrol is a naturally occurring phytoalexin produced by some of higher plants. It was found to show cancer chemopreventive activity (Jang et al., 1997). Therapeutic potential of this

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Table 1 Assignment of selected peaks observed in the rhubarb stalk MS imaging experiment with references regarding shown compounds. Compound name

Ion formula

m/za

Figure

Ref.

Oxalic acidb Threoninec Lysineb Magnesium oxalatec Succinic acidb Phenylalaninec Paeonolb Carvacrolb Dehydroascorbic acidc p-Coumaric acidb 2-Methyl-5-acetyl-7-hydroxy-chromenec 2,5-dimethyl-7-hydroxychromoneb 2,5-Dimethyl-7-hydroxy-chromenec trans-Stilbeneb Pantothenic acidc 2-Methyl-5-carboxy methyl-7-hydroxy-chromenec Abscisic aldehydeb Resveratrolc Chrysophanolc Chrysophanic acid 2-Methyl-5-carboxymethyl-7-hydroxychromonec Aloe-emodinb 2-methyl-5-carboxymethyl-7-hydroxychromanonec 2-(Hydroxymethyl)anthraquinoneb Oleic Acid Citreoroseinc Quercetinc Hydroxycinnamoyl glucose estersc Sucroseb 7-Hydroxy-2-methyl-4-oxo-4H-1-benzopyran-5-carboxylic acid 7-glucosideb Aloe emodin glucoside Emodin glucosidec Rhaponticinc Isoquercitrinb Digalloyl glucose Gallic acid 4-O-(6-galloylglucoside)b Galloylsucroseb Resveratrol 40 -galloylglucosideb Piceatannol 40 -galloylglucosidec Daucosterolb Rutinc Galloyl-substituted procyanidin B1 and B2b (Procyanidin B1 3-gallate) Vitamin D1c Sennoside Ec Sennoside Fc Digalloyl-substituted procyanidin B2 and B5c

[C2H2O4þNa]þ [C4H9NO3þH]þ [C6H14N2O2þK]þ [C2MgO4þK]þ [C4H6O4þK]þ [C9H11NO2þH]þ [C9H10O3þNa]þ [C10H14O þ Na]þ [C6H6O6þH]þ [C9H8O3þK]þ [C12H12O3þH]þ [C11H10O3þNa]þ [C11H12O2þK]þ [C14H12þK]þ [C9H17NO5þH]þ [C12H12O4þH]þ [C15H20O3þH]þ [C14H12O3þNa]þ [C15H10O4þH]þ

112.9845 120.0655 147.1128 150.9279 156.9898 166.0863 167.0703 173.0937 175.0237 203.0105 205.0859 213.0522 215.0469 219.0571 220.1180 221.0808 249.1485 251.0679 255.0652

Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig.

2C 3G 3F 3N 2D 3H 2O 2G 2E 2F 2H 2K 2J 1M 3B 2I 3M 1J 1E

Chopra et al., 1978 Silber et al., 1960 Silber et al., 1960 Blundstone and Dickinson, 1964 Pucher and Vickery, 1941 Silber et al., 1960 Miyazawa et al., 1996 Agarwal et al., 2001 Parsons et al., 2011 Boz 2015 Kashiwada et al., 1990 Kashiwada et al., 1984 Kashiwada et al., 1984 Matsuda et al., 2001 Chopra et al., 1978 Kashiwada et al., 1984 (Sindhu et al., 1990) Matsuda et al., 2001 He and Luo, 1980

[C12H10O5þNa]þ [C15H10O5þH]þ [C12H12O5þK]þ [C15H10O3þK]þ [C18H34O2þH]þ [C15H10O6þH]þ [C15H10O7þH]þ [C15H18O8þK]þ [C12H22O11þK]þ [C17H18O10þH]þ [C21H20O10þNa]þ

257.0420 271.0601 275.0316 277.0262 283.2632 287.0550 303.0499 365.0633 381.0794 383.0973 455.0949

Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig. Fig.

2M 1D 2L 1F 3E 1G 1O 3O 3D 2N 1H

Kashiwada et al., 1984 (Tutin and Clewer, 1908) Kashiwada et al., 1984 Chopra et al., 1978 (Tutin and Clewer, 1908) Oshio, 1978 (Chumbalov and Nurgalieva, 1967) (Kashiwada et al., 1988a,b) (Loescher, 1987). Kashiwada et al., 1990 Okabe et al., 1973

[C21H24O9þK]þ [C21H20O12þH]þ [C20H20O14þK]þ

459.1052 465.1028 523.0485

Fig. 1N Fig. 2A Fig. 3L

Kashiwada et al., 1984 (Chumbalov and Nurgalieva, 1967) (Nonaka and Nishioka, 1983)

[C19H26O15þK]þ [C27H26O12þH]þ [C27H26O13þH]þ [C35H60O6þK]þ [C27H30O16þK]þ [C37H30O16þNa]þ

533.0903 543.1497 559.1446 615.4022 649.1165 753.1426

Fig. Fig. Fig. Fig. Fig. Fig.

(Kashiwada et al., 1988a,b) (Nonaka et al., 1977) (Singh, 2016) Li et al., 1998 (Krafczyk et al., 2008) (Kashiwada et al., 1984)

[C56H88O2þH]þ [C42H38O19þH]þ

793.6857 847.2080

Fig. 3C Fig. 1I

Chopra et al., 1978 Oshio et al., 1974

[C44H34O20þH]þ

883.1716

Fig. 3J

(Nonaka et al., 1981)

a b c

3I 1K 1L 3A 2B 3K

Calculated m/z values. Identity confirmed with LIFT MS/MS method. Putative identification.

compound has been reviewed few years ago (Baur and Sinclair, 2006). What is interesting, stilbenes isolated from the rhizome were investigated due to their anti-allergic and antioxidant activity (Matsuda et al., 2000, 2001, 2004). The images of two protonated adducts of stilbene glycosides - resveratrol 4'-galloylglucoside (m/z 543.497) and piceatannol 4'-galloylglucoside (m/z 559.1446) can be found in Fig. 1K and L respectively. Both ions were found to be originated from the area near the edge of the rhubarb stalk. Several stilbene derivatives including trans-stilbene and rhaponticin have been found in green vegetables including commercially-available rhubarbs (Rheum species) (Matsuda et al., 2001; Nonaka et al., 1977). MS imaging proved that trans-stilbene (m/z 219.0571, Fig. 1M) is located in all ground tissue with relatively high abundance. The distribution of the rhaponticin (m/z 459.052, Fig. 1N) greatly differs when compared to other stilbenes. Most of its ions were found in form of one spot near epidermis. Ions based on compounds belonging to other very important group - flavonoids, which are plant pigments and important

antioxidants, were also analyzed. Rhubarb stalks are rich sources of flavonoids, among which a quercetin and its derivatives are the most important ones. Quercetin and isoquercetin have been already isolated from rhubarb (Chumbalov and Nurgalieva, 1967). The ions assigned as protonated adducts of quercetin (m/z 303.0499) and isoquercetin (m/z 465.1028, Fig. 2A) and also potassium adduct of rutin (m/z 649.1165, Fig. 2B) were found to be in highest abundances in the area of vascular bundles. Rhubarb contain relatively large quantities of organic acids such as oxalic acid, succinic acid, carvacrol, dehydroascorbic acid and pcoumaric acid. The ions of m/z 112.9845 were assigned to sodium adduct of oxalic acid. As can be seen in Fig. 2C there is low abundance of this compound near the epidermis. Rhubarb species contain oxalic acid which is a toxic substance that in high concentrations can cause stomach irritation and kidney problems (Chopra et al., 1978). Oxalic acid is the simplest but a relatively strong dicarboxylic acid found in many plants and also vegetables. Potassium adduct of succinic acid at m/z 156.9898 (Fig. 2D) and

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Fig. 1. LDI-MSI analysis of the cross-sectional surface of the rhubarb stalk (40 mm pixel size) on AuNPET. (A) Optical image of rhubarb stalk (Rheum rhabarbarum) cross-section. Grey dashed-line rectangle in this image marks the region of analysis. (B) Optical image of the AuNPET surface with rhubarb imprint and (C) magnified analyzed region. Images D-O are graphical representations (TIC normalization) of ions of m/z: 271.0601 (D), 255.0652 (E), 277.0262 (F), 287.0550 (G), 455.0949 (H), 847.2080 (I), 251.0679 (J), 543.1497 (K), 559.1446 (L), 219.0571 (M), 459.1052 (N), 303.0499 (O). All representations are within ±0.05 m/z. Bottom part (P) shows AuNPET LDI-MS spectrum of rhubarb extract.

protonated adduct of dehydroascorbic acid at m/z 175.0937 (Fig. 2E) were located in the area of vascular bundles near the cortex. Succinic acid and dehydroascorbic acid are metabolites present in plant tissues (Pucher and Vickery, 1941; Parsons et al., 2011). In addition, another carboxylic acid-based ion at m/z 203.0105 [MþK]þ was assigned to p-coumaric acid was located in whole ground tissue (Fig. 2F) area. p-Coumaric acid is an antioxidant and antimicrobial naturally occurs in many plants (Boz, 2015). A number of polyphenols is also present in rhubarb. Compound belonging to this group is carvacrol previously found in discussed plant (Agarwal et al., 2001). Its sodium adduct at m/z 173.0937 was located mainly in the ground tissue except the area corresponding to the location of vascular bundles (Fig. 2G). Literature describes that in a variety of rhubarb species, derivatives of chromene, chromanones and chromone (Kashiwada et al., 1984, 1990) are present. LDI-MSI analysis of rhubarb crosssection allowed again to localize compounds and their ions. Fig. 2H proves that the highest abundance of protonated adduct of 2-methyl-5-acetyl-7-hydroxy-chromene (m/z 205.0859) and protonated 2-methyl-5-carboxymethyl-7-hydroxy-chromene (m/z 221.0808, Fig. 2I) is near epidermis. Ions of similar compounds - and potassium adduct of 2,5-dimethyl-7-hydroxy-chromene (m/z 215.0469, Fig. 2J), sodium adducts of 2,5-dimethyl-7hydroxychromone (m/z 213.0522, Fig. 2K) and 2-methyl-5carboxymethyl-7-hydroxychromanone (m/z 275.0316, Fig. 2L) are located mainly along the edge of the stalk. In contrast to above-discussed compounds, 2-methyl-5carboxymethyl-7-hydroxychromone sodium adduct showed that the highest abundance of these ion was observed in the most outer

part of the rhubarb stalk (Fig. 2M). The ion at m/z 383.0973 [MþH]þ assigned to 7-hydroxy-2methyl-4-oxo-4H-1-benzopyran-5-carboxylic acid 7-glucoside was found at very high intensities in vascular bundles (Fig. 2N). It is a chromone glycoside commonly present in rhubarb species (Kashiwada et al., 1990). Compounds belonging to essential oil group were also isolated from rhubarb. This group includes paeonol, which protonated ion can be found at m/z 167.0703. As MSI data suggests (Fig. 2O), this compound is localized in whole studied cross-section with highest concentration in the near-edge ca. 0.1 mm-wide strip. The phenolic paeonol having a warm aromatic odour, was one of the main volatile components obtained from Rhei rhizoma of Rheum palmatum L. (Miyazawa et al., 1996). Another phenolic-type compound, daucosterol, that was confirmed as an ingredient of rhubarb (Li et al., 1998) was found to be localized in form of potassium adduct at m/z 615.4022 (Fig. 3A) relatively uniformly in the cross-section. Vitamin B5 and D1 have also been identified in several plant species including rhubarb (Chopra et al., 1978). Ion image for m/z 220.1180 was assigned to proton adduct of pantothenic acid, also called vitamin B5, which was located in the ground tissue (Fig. 3B), with a high abundance near collenchyma, except the area of vascular bundles. Another ion of m/z 793.6857 was found to have the same distribution pattern as B5 proton adduct. This ion (Fig. 3C) was assigned to the potassium adduct of vitamin D1. Sucrose is a non-reducing disaccharide, widely occurring in plants, which function is mainly as fast-accessible energy storage (Loescher, 1987). Ions, that were assigned to potassium adduct of lactose, maltose or sucrose at m/z 381.0794 were detected only in

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Fig. 2. Ion images (AO) of the same area of the rhubarb stalk imprint as shown in Fig. 1B and C for m/z values of 465.1028 (A), 649.1165 (B), 112.9845 (C), 156.9898 (D), 175.0237 (E), 203.0105 (F), 173.0937 (G), 205,0859 (H), 221.0808 (I), 215.0469 (J), 213.0522 (K), 275.0316 (L), 257.0420 (M), 383.0973 (N), 167.0703 (O) respectively. Spatial resolution 40
40 mm.

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Fig. 3. Ion images (AO) of the same area of the rhubarb stalk imprint as shown in Fig. 1B and C for m/z values of 615.4022 (A), 220.1180 (B), 793,6857 (C), 381.0794 (D), 283.2632 (E), 147.1128 (F), 120.0655 (G), 166.0863 (H), 533.0903 (I), 883.1716 (J), 753.1426 (K), 523.0485 (L), 249.1485 (M), 150.9279 (N), 365.0633 (O) respectively. Spatial resolution 40
40 mm.

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specific regions of the vascular bundles (Fig. 3D). The distribution of the proton adducts of oleic acid at m/z 283.2632 was within the skin (Fig. 2E). Oleic acid, is an unsaturated 18-carbon fatty acid, and is often found as an ingredient of plant membrane lipids. Oleic acid occurs naturally in various vegetable oils including rhubarb species (Tutin and Clewer, 1908). Another biologically important compounds e basic building blocks - amino acids threonine, lysine and phenylalanine were also found in MS spectra and localized by MS imaging. The ion at m/z 147.1128 (Fig. 3F) assigned to hydrogen adduct of lysine was localized mainly in areas complementary to the vascular bundles located near epidermis. What is interesting, its levels were very low in the center sections of the stalk. In contrast to above-discussed location, protonated adduct of threonine at m/z 120.0655 (Fig. 3G) was found in regions near the edge of the stalk. The ions at m/z 166.0863 (Fig. 3H) with the highest concentration at the center of the stalk correspond to phenylalanine (Silber et al., 1960). Ion of derivative of gallic acid studied in this work - potassium adduct of galloylsucrose at m/z 533.0903 was found to originate from the most outer part of the skin of the plant (Fig. 3I). Galloylsucroses are a tannins found in green vegetables isolated from various Rheum species (Kashiwada et al., 1988a,b). Similar compounds - digalloylglucose, gallotanins were found in green vegetables including rhubarb species. Compound of similar type - gallic acid 4-O-(6-galloylglucoside) has also been isolated from commercial rhubarb (Nonaka and Nishioka, 1983). Digalloyl-substituted procyanidin B2 and B5 (3,3'-di-O-galloylprocyanidin B2 and B5) belong to the class of organic compounds known as proanthocyanidins. These compounds are known constituents of commercial rhubarb (Rhei Rhizoma) (Nonaka et al., 1981). Galloyl-substituted procyanidin B1 and B2 (3-O-galloylprocyanidin B1 and B2) have been isolated from rhubarb (Kashiwada et al., 1984). MS imaging on AuNPET proved that digalloylsubstituted procyanidin B2 and B5 (m/z 883.1716, Fig. 3J) and galloyl-substituted procyanidin B1 and B2 (m/z 753.1426, Fig. 3K) are located in the ground tissue while digalloylglucose (m/z 523.0485, Fig. 3L) is located exclusively close to epidermis. Fig. 3M contains the ion image of m/z 249.1485 assigned to proton adduct of abscisic aldehyde which is an intermediate in the biosynthesis of the plant hormone abscisic acid. It was found that m/z 249.1485 ion was generated in ionization process mainly near epidermal cells except regions of vascular bundles (Sindhu et al., 1990). Magnesium oxalate is a salt of a relatively strong organic, dicarboxylic acid occurring in many plants and vegetables including rhubarb species (Blundstone and Dickinson, 1964). The magnesium oxalate assigned to the ion at m/z 150.9279 [MþK]þ is present in ground tissue except the area corresponding to the localization of vascular bundles (Fig. 3N). In contrast to above-discussed localization of two ions, highest abundance of ion of potassium adduct of 6-O-p-coumaroyl-Dglucose (m/z 365.0633, Fig. 3O) was observed at the area corresponding to the vascular bundles. This type of glucose esters was isolated from green vegetables including various commercial rhubarbs (Rheum sp.) (Kashiwada et al., 1988a,b). 3. Conclusion In this study, laser desorption/ionization mass spectrometry technique with the use of nanoparticle-enhanced SALDI-type AuNPET target was used for imaging of rhubarb stalk (Rheum rhabarbarum) cross-section. Ion images produced for a few dozens of compounds of interest presented attention-grabbing differentiation of intensities. The most important compounds having bioactive properties and pharmacological applicability were discussed in detail.

4. Experimental methods 4.1. Materials and equipment AuNPET target plate was prepared as previously described in our recent work (Sekuła et al., 2015a,b). All solvents used in this work were of HPLC quality. Optical photographs of plant material were made with the use of an Olympus SZ10 microscope equipped with an 8 MPix Olympus digital camera. Rhubarb (Rheum rhabarbarum L., family: Polygonaceae) stalk of typical size was collected from the yard located in the south part of Poland (near Rzeszow, 50 000 19.400 N 21570 06.700 E) on 6th May 2016 and used in imaging experiment after maximum 10 min after harvesting. Stalk from 20years-old plant were used in experiment. LIFT® MS/MS measurements were made with LIFT lowmass method at default settings. Mentioned MS/MS were made by analysis of water-acetone extracts of rhubarb stalk fragment (100 mg of frozen stalk was homogenized with 3 mL of acetone followed by centrifugation and 100-fold dilution) placed on AuNPET (0.5 mL). 4.2. LDI-MS imaging experiments Laser desorption ionization mass spectrometry imaging (LDIMSI) experiments were performed using a Bruker Autoflex Speed time-of-flight mass spectrometer in positive-ion reflectron mode. FlexImaging 4.0 software was used for data processing and analysis. The apparatus was equipped with a SmartBeam II 1000 Hz 355 nm laser. Laser impulse energy was approximately 100e190 mJ, laser repetition rate was 1000 Hz, and deflection was set on m/z lower than 100 Da. The m/z range was 100e2000 Da, spatial resolution 40
40 mm. The experiments were made with 500 laser shots per individual spot with a default random walk applied. All spectra were calibrated with the use of gold ions (Auþ to Auþ 7 , 7 calibration points). The first accelerating voltage was held at 19 kV, and the second ion source voltage was held at 16.7 kV. Reflector voltages used were 21 kV (the first) and 9.55 kV (the second). All of the analyzed imaging m/z values were within ±0.05%. TIC normalization was used with all results shown. 4.3. Imaging sample preparation Before MSI analysis the sample was washed two times with deionized water. Cross-section of ca. 8
9 mm size was made approx. 20 cm from the end of stalk with disposable razor blade. Rhubarb stalk cross-section was then immediately touched to the cellulose filter paper (3 times) to remove excess of liquid material (although this operation removes some part of liquid, it is important as too thick layer of material is unfavorable for gold nanoparticles-catalyzed ionization) and touched to AuNPET plate with light pressure for 3 s and removed. Part of the cross-section (Fig. 1B and C) was then directly analyzed with an MS apparatus. Imaging experiment was made at 40
40 mm superficial resolution. Acknowledgements Research was supported by the National Science Center (NCN Poland; PRELUDIUM project no. UMO-2015/19/N/ST4/00379). We also thank German and Polish Bruker-Daltonics for FlexImaging 4.0. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.phytochem.2017.04.006.

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References Agarwal, S.K., Singh, S.S., Lakshmi, V., Verma, S., Kumar, S., 2001. Chemistry and pharmacology of rhubarb (Rheum species)d a review. J. Sci. Ind. Res. (India) 60, 1e9. Baur, J.A., Sinclair, D.A., 2006. Therapeutic potential of resveratrol: the in vivo evidence. Nat. Rev. Drug Discov. 5, 493e506. Belu, A.M., Graham, D.J., Castner, D.G., 2003. Time-of-flight secondary ion mass spectrometry: techniques and applications for the characterization of biomaterial surfaces. Biomaterials 24, 3635e3653. Blundstone, H.A.W., Dickinson, D., 1964. The chemistry of edible rhubarb. J. Sci. Food Agr. 15, 94e101. Boz, H., 2015. p-Coumaric acid in cereals: presence, antioxidant and antimicrobial effects. Int. J. Food Sci. Tech. 50, 2323e2328. Cai, Y., Sun, M., Xing, J., Corke, H., 2004. Antioxidant phenolic constituents in roots of Rheum officinale and Rubia cordifolia: structure-radical scavenging activity relationships. J. Agric. Food Chem. 52, 7884e7890. Chin, T.C., Youngken, H.W., 1947. The cytotaxonomy of Rheum. Am. J. Bot. 34, 401e407. Choi, R.J., Ngoc, T.M., Bae, K., Cho, H.J., Kim, D.D., Chun, J., Khan, S., Kim, Y.S., 2013. Anti-inflammatory properties of anthraquinones and their relationship with the regulation of P-glycoprotein function and expression. Eur. J. Pharm. Sci. 48, 272e281. Chopra, R.N., Nayar, S.L., Chopra, I.C., 1978. Glossary of Indian medicinal plants. Counc. Sci. Ind. Res. Bull. (New Delhi). Chumbalov, T.K., Nurgalieva, G.M., 1967. Flavonoids of Rheum tataricum. Khim. Prir. Soedin. 3, 345e346. Duke, J.A., 2002. Handbook of Medicinal Herbs, second ed. CRC Press, Boca Raton, FL. Dzoyem, J.P., Donfack, A.R.N., Tane, P., McGaw, L.J., Eloff, J.N., 2016. Inhibition of nitric oxide production in LPS-stimulated RAW 264.7 macrophages and 15-LOX activity by anthraquinones from Pentas schimperi. Planta Med. 82, 1246e1251. Ghosh, S., Sarma, M., Patra, A., Hazra, B., 2010. Anti-inflammatory and anticancer compounds isolated from Ventilago madraspatana Gaertn., Rubia cordifolia linn. And Lantana camara linn. J. Pharm. Pharmacol. 62, 1158e1166. He, L.Y., Luo, S.R., 1980. Studies on the analysis of anthraquinone derivatives of Chinese medicinal herbs. I. Separation and determination of constituents of Chinese rhubarb. Yao Xue Xue Bao 15, 555e562. Heyman, H.M., Dubery, I.A., 2016. The potential of mass spectrometry imaging in plant metabolomics: a review. Phytochem. Rev. 15, 297e316. Huang, K.C., 1993. The Pharmacology of Chinese Herbs. CRC Press, Boca Raton, FL. Huang, Q., Lu, G., Shen, H.-M., Chung, M.C.M., Ong, C.N., 2007. Anti-cancer properties of anthraquinones from rhubarb. Med. Res. Rev. 27, 609e630. Jang, M.S., Cai, E.N., Udeani, G.O., Slowing, K.V., Thomas, C.F., Beecher, C.W.W., Fong, H.H.S., Farnsworth, N.R., Kinghorn, A.D., Mehta, R.G., Moon, R.C., Pezzuto, J.M., 1997. Cancer chemopreventive activity of resveratrol, a natural product derived from grapes. Science 275, 218e220. Kashiwada, Y., Nonaka, G.I., Nishioka, I., 1984. Studies on rhubarb (Rhei rhizoma). V. Isolation and characterization of chromone and chromanone derivatives. Chem. Pharm. Bull. 32, 3493e3500. Kashiwada, Y., Nonaka, G.I., Nishioka, I., Yamagishi, T., 1988a. Galloyl and hydroxycinnamoylglucoses from rhubarb. Phytochemistry 27, 1473e1477. Kashiwada, Y., Nonaka, G.I., Nishioka, I., 1988b. Galloylsucroses from rhubarbs. Phytochemistry 27, 1469e1472. Kashiwada, Y., Nonaka, G.-I., Nishioka, I., 1990. Chromone glucosides from rhubarb. Phytochemistry 29, 1007e1009. Krafczyk, N., Koetke, M.K., Lehnert, N., Glomb, M.A., 2008. Phenolic composition of rhubarb. Eur. Food Res. Technol. 228, 187e196. Li, J.L., Li, J.S., Wang, A.Q., 1998. Studies on the non-anthraquinones of hotao rhubarb (Rheum hotaoense). Chin. Tradit. Herb. Drugs 29, 721e723. Loescher, W.H., 1987. Physiology and metabolism of sugar alcohols in higher plants. Physiol. Plant. 70, 553e557. Lu, C.C., Yang, J.S., Huang, A.C., Hsia, T.C., Chou, S.T., Kuo, C.L., Lu, H.F., Lee, T.H., Wood, W.G., Chung, J.G., 2010. Chrysophanol induces necrosis through the production of ROS and alteration of ATP levels in J5 human liver cancer cells. Mol. Nutr. Food Res. 54, 967e976. Lu, Y., Suh, S.J., Li, X., Liang, J.L., Chi, M., Hwangbo, K., Kwon, O., Chung, T.W., Kwak, C.H., Kwon, K.M., Murakami, M., Jahng, Y., Kim, C.H., Son, J.K., Chang, H.W., 2012. Citreorosein inhibits production of proinflammatory cytokines by blocking mitogen activated protein kinases, nuclear factor-kB and activator protein-1 activation in mouse bone marrow-derived mast cells. Biol. Pharm. Bull. 35, 938e945. Matsuda, H., Kageura, T., Morikawa, T., Toguchida, I., Harima, S., Yoshikawa, M., 2000. Effects of stilbene constituents from rhubarb on nitric oxide production in lipopolysaccharide-activated macrophages. Bioorg. Med. Chem. Lett. 10, 323e327. Matsuda, H., Morikawa, T., Toguchida, I., Park, J.Y., Harima, S., Yoshikawa, M., 2001. Antioxidant constituents from Rhubarb: structural requirements of stilbenes for the activity and structures of two new anthraquinone glucosides. Bioorg. Med. Chem. 9, 41e50. Matsuda, H., Tewtrakul, S., Morikawa, T., Yoshikawa, M., 2004. Anti-allergic activity of stilbenes from Korean rhubarb (Rheum undulatum L.): structure requirements for inhibition of antigen-induced degranulation and their effects on the release of TNF-a and IL-4 in RBL-2H3 cells. Bioorg. Med. Chem. 12,

79

4871e4876. Miyazawa, M., Minamino, Y., Kameoka, H., 1996. Volatile components of the rhizomes of Rheum palmatum L. Flavour Frag. J. 11, 57e60. Nizioł, J., Ossolinski, K., Ossolinski, T., Ossolinska, A., Bonifay, V., Sekuła, J., Dobrowolski, Z., Sunner, J., Beech, I., Ruman, T., 2016. Surface-transfer mass spectrometry imaging of renal tissue on gold nanoparticle enhanced target. Anal. Chem. 88, 7365e7371. Nonaka, G., Nishioka, I., 1983. Tannins and related compounds. X. Rhubarb (2): isolation and structures of a glycerol gallate, gallic acid glucoside gallates, galloylglucose and isolindleyin. Chem. Pharm. Bull. 31, 1652e1658. Nonaka, G., Minami, M., Nishioka, I., 1977. Studies on rhubarb (Rhei rhizoma). III. Stilbene glycosides. Chem. Pharm. Bull. 25, 2300e2305. Nonaka, G., Nishioka, I., Nagasawa, T., Oura, H., 1981. Tannins and related compounds. I. Rhubarb. I. Chem. Pharm. Bull. 29, 2862e2870. Norris, J.L., Caprioli, R.M., 2013. Analysis of tissue specimens by matrix-assisted laser Desorption/Ionization Imaging mass spectrometry in biological and clinicalresearch. Chem. Rev. 113, 2309e2342. Okabe, H., Matsuo, K., Nishioka, I., 1973. Studies on rhubarb (Rhei rhizoma). II. Anthraquinone glycosides. Chem. Pharm. Bull. 2, 1254e1260. Oshio, H., 1978. Investigation of rhubarbs. IV. Isolation of sennoside D, citreorosein and laccaic acid D. Shoyakugaku Zasshi 32, 19e23. Oshio, H., Imai, S., Fujioka, S., Sugawara, T., Miyamoto, M., Tsukui, M., 1972. Investigation of rhubarbs. II. Isolation of sennoside E, a new purgative compound. Chem. Pharm. Bull. 20, 621e622. Oshio, H., Imai, S., Fujioka, S., Sugawara, T., Miyamoto, M., Tsukui, M., 1974. Investigation of rhubarbs. III. New purgative constituents, sennosides E and F. Chem. Pharm. Bull. 22, 823e831. Park, B.S., Lee, H.K., Lee, S.E., Piao, X.L., Takeoka, G.R., Wong, R.Y., Ahn, Y.J., Kim, J.H., 2006. Antibacterial activity of Tabebuia impetiginosa martius ex DC (Taheebo) ́ ́ 105, 255e262. against Helicobacter pylori. J. ́ Ethnopharmacol. Parsons, H.T., Yasmin, T., Fry, S.C., 2011. Alternative pathways of dehydroascorbic acid degradation in vitro and in plant cell cultures: novel insights into vitamin C catabolism. Biochem. J. 440, 375e383. Pucher, G.W., Vickery, H.B., 1941. Succinic acid as a metabolite in plant tissues. Plant Physiol. 16, 771e783. Qu, Y., Wang, J.B., Li, H.F., Wnag, Q., Xiao, X.H., He, Y.Z., 2008. Study on relationship of laxative potency and anthraquinones content traditional Chinese drugs. Zhongguo Zhong Yao Za Zhi 33, 806e808. Sekuła, J., Nizioł, J., Rode, W., Ruman, T., 2015a. Gold nanoparticle-enhanced target (AuNPET) as universal solution for laser desorption/ionization mass spectrometry analysis and imaging of low molecular weight compounds. Anal. Chim. Acta 875, 61e72. Sekuła, J., Nizioł, J., Misiorek, M., Dec, P., Wrona, A., Arendowski, A., Ruman, T., 2015b. Gold nanoparticle-enhanced target for MS analysis and imaging of harmful compounds in plant, animal tissue and on fingerprint. Anal. Chim. Acta 895, 45e53. Silber, R.L., Becker, M., Cooper, M., Evans, P., Fehder, P., Gray, R., Gresham, P., Rechsteiner, J., Searles, M.A., 1960. Paper chromatographic identification and estimation of the free amino acids in thirty-two fruits. J. Food Sci. 25, 675e680. Sindhu, R., Griffin, D.H., Walton, D.C., 1990. Abscisic aldehyde is an intermediate in the enzymatic conversion of xanthoxin to abscisic acid in Phaseolus vulgaris L. leaves. Plant Physiol. 93, 689e694. Singh, P., nee Pant, G.J., Rawat, M.S.M., 2016. Phytochemistry and biological activity perspectives of Rheum species. Nat. Prod. J. 6, 84e93. €vall, P., Lausmaa, J., Nygren, H., Carlsson, L., Malmberg, P., 2003. Imaging of Sjo membrane lipids in single cells by imprint-imaging time-of-flight secondary ion mass spectrometry. Anal. Chem. 75, 3429e3434. Srinivas, G., Babykutty, S., Sathiadevan, P.P., Srinivas, P., 2007. Molecular mechanism of emodin action: transition from laxative ingredient to an antitumor agent. Med. Res. Rev. 27, 591e608. Svatos, A., 2010. Mass spectrometric imaging of small molecules. Trends Biotechnol. 28, 425e434. Tabolacci, C., Lentini, A., Mattioli, P., Provenzano, B., Oliverio, S., Carlomosti, F., Beninati, S., 2010. Antitumor properties of aloe-emodin and induction of transglutaminase 2 activity in B16eF10 melanoma cells. Life Sci. 87, 316e324. Tang, W., Eisenbrand, G., 1992. Rheum spp. In: Tang, W., Eisenbrand, G. (Eds.), Chinese Drugs of Plant Origin. Springer Berlin Heidelberg, pp. 855e875. Thunig, J., Hansen, S.H., Janfelt, C., 2011. Analysis of secondary plant metabolites by indirect desorption electrospray ionization imaging mass spectrometry. Anal. Chem. 83, 3256e3259. Tucker, K.R., Serebryannyy, L.A., Zimmerman, T.A., Rubakhin, S.S., Sweedler, J.V., 2011. The modified-bead stretched sample method: development and application to MALDI-MS imaging of protein localization in the spinal cord. Chem. Sci. 2, 785e795. Tutin, F., Clewer, H.W.B., 1908. XCIX.-The constituents of rhubarb. J. Chem. Soc. Trans. 99, 946e967.
l, J., Havlí Vidov
a, V., Nov
ak, P., Strohalm, M., Po cek, V., Volný, M., 2010. Laser desorption-ionization of lipid transfers: tissue mass spectrometry imaging without MALDI matrix. Anal. Chem. 82, 4994e4997. Wagner, H., Hӧrhammer, L., Farkas, L., 1963. Genuine Anthrachinonglykoside aus dem Rhizom von Rheum palmatum var. tangut. Z. Naturforsch. B 18, 89-89. Wang, J.P., Huang, H.Q., Liu, P.Q., Tang, F.T., Qin, J., Huang, W.G., Chen, F.Y., Guo, F.F., Liu, W.H., Yang, B., 2006. Inhibition of phosphorylation of p38 MAPK involved in the protection of nephropathy by emodin in diabetic rats. Eur. J. Pharmacol. 553, 297e303.

80

J. Nizioł et al. / Phytochemistry 139 (2017) 72e80

Watrous, J., Hendricks, N., Meehan, M., Dorrestein, P.C., 2010. Capturing bacterial metabolic exchange using thin film desorption electrospray ionization-imaging mass spectrometry. Anal. Chem. 82, 1598e1600. Wei, W.T., Chen, H., Ni, Z.L., Liu, H.B., Tong, H.F., Fan, L., Liu, A., Qiu, M.X., Liu, D.L., Guo, H.C., Wang, Z.H., Lin, S.Z., 2011. Antitumor and apoptosis-promoting properties of emodin, an anthraquinone derivative from Rheum officinale Baill, against pancreatic cancer in mice via inhibition of Akt activation. Int. J. Oncol. 39, 1381e1390. Wu, X.Y., Wu, Q.T., Liu, L.J., 1995. Study of pharmacology and application of rhubarb.

Acta Chin. Med. Pharmacol. 2, 54e55. Xiao, P.G., He, L.Y., Wang, L.W., 1984. Ethnopharmacologic study of Chinese rhubarb. J. Ethnopharmacol. 10, 275e293. Yang, H., Xu, L.N., He, C.Y., Liu, X., Fang, R.Y., Ma, T.H., 2011. CFTR chloride channel as a molecular target of anthraquinone compounds in herbal laxatives. Acta Pharmacol. Sin. 32, 834e839. Zhao, X.Y., Qiao, G.F., Li, B.X., Chai, L.M., Li, Z., Lu, Y.J., Yang, B.F., 2009. Hypoglycaemic and hypolipidaemic effects of emodin and its effect on L-type calcium channels in dyslipidaemicediabetic rats. Clin. Exp. Pharmacol. Physiol. 36, 29e34.