Phytochemical Profiling of Coryphantha macromeris (Cactaceae) Growing in Greenhouse Conditions Using Ultra-High-Performance Liquid Chromatography–Tandem Mass Spectrometry

Chromatographic separation combined with mass spectrometry is a powerful tool for the characterization of plant metabolites because of its high sensitivity and selectivity. In this work, the phytochemical profile of aerial and radicular parts of Coryphantha macromeris (Engelm.) Britton & Rose growing under greenhouse conditions was qualitatively investigated for the first time by means of modern ultra-high-performance liquid chromatography–tandem mass spectrometry (UHPLC-PDA-HESI-Orbitrap-MS/MS). The UHPLC-PDA-HESI-Orbitrap-MS/MS analysis indicated a high complexity in phenolic metabolites. In our investigation, 69 compounds were detected and 60 of them were identified. Among detected compounds, several phenolic acids, phenolic glycosides, and organic acids were found. Within this diversity, 26 metabolites were exclusively detected in the aerial part, and 19 in the roots. Twenty-four metabolites occurred in both plant parts. According to the relative abundance of peaks in the chromatogram, ferulic and piscidic acids and their derivatives may correspond to one of the main phenolic compounds of C. macromeris. Our results contribute to the phytochemical knowledge regarding C. macromeris and its potential applications in the pharmaceutical and cosmetic industries. Besides, some metabolites and their fragmentation patterns are reported here for the first time for cacti species.


Introduction
The family Cactaceae is one of the most threatened within the plant kingdom [1], and is the most important plant family of the arid and semiarid regions of America [2], comprising around 1600 species [3]. Cacti are succulent plants which have been used for their functional properties and industrial utility [4][5][6]. Different cacti species obtained by cultivated or wild-collected methods have been used as food sources and fodder, for ornamental purposes, and for medicinal purposes (due to their antioxidant [7], antimutagenic [8], and bactericidal [9] properties), and have been cataloged as a source of bioactive compounds including alkaloids [10,11] and phenolics [12,13].

Results and Discussion
Methanolic extracts prepared with aerial and radicular parts of C. macromeris cultivated under greenhouse conditions were analyzed by ultra-high-performance liquid chromatography-tandem mass spectrometry and a photodiode array detector (UHPLC-PDA-HESI-Orbitrap-MS/MS). This technique has been used for the characterization of metabolites in complex mixtures [31], and the chromatographic conditions used in this work have shown efficiency for the separation and identification of different secondary metabolites [32][33][34]. For C. macromeris extracts, UHPLC conditions allowed a good separation of many sample components.
The phytochemical characterization of C. macromeris was achieved by comparing the obtained information by UHPLC-PDA-HESI-Orbitrap-MS/MS with spectroscopic evidences existing in the literature or by structure searching and studying the fragmentation pattern of molecules. Additionally, retention time and UV (λ max ) spectra were used for peak characterization. As far as we know, metabolite profiling of C. macromeris is reported for the first time in this work. Elution profile ( Figure 1) and mass spectra obtained in negative ion mode indicated a highly complex phenolic composition. Peak characteristics (i.e., retention time, theoretical and measured mass, UV (λ max ), and fragmentation pattern) and tentative identification of each compound are summarized in Table 1. All detected compounds exhibited an accuracy smaller than 5 ppm. Under the proposed UHPLC-PDA-HESI-Orbitrap-MS/MS method, 69 compounds were separated, and 60 of them were tentatively identified in C. macromeris greenhouse plants (Table 1). Within this complexity, 26 compounds occurred in the aerial part, 19 in occurred in the roots, and 24 occurred in both plant parts, suggesting a translocation mechanism from actively photosynthetic tissues to metabolite consuming/storage areas. a translocation mechanism from actively photosynthetic tissues to metabolite consuming/storage areas. Figure 1. Ultra-high-performance liquid chromatography (UHPLC) chromatogram of Coryphantha macromeris methanolic extracts prepared with aerial (a) and radicular (b) parts. Peak numbers refer to the metabolites indicated in Table 1; repeated numbers in (a) or (b) indicate that the metabolite is found in both plant parts.  Table 1; repeated numbers in (a) or (b) indicate that the metabolite is found in both plant parts.   Compounds 1-5 were detected in both parts of C. macromeris. Compound 1 was assigned as vaccihein A (Figure 2a). This antioxidant metabolite was isolated for the first time in Vaccinium ashei (Ericaceae) by Ono, et al. [35] and this is the first time that is reported for cacti species. Compounds 2-5 were assigned as carboxylic acids: compounds 2 and 3 were proposed as dihydroxy methoxy butanoic acid and malic acid [36], respectively, and compounds 4 and 5 as isocitric acid isomers [13]. Citric acid occurs as one of the main hydrophilic constituents in Opuntia species (Cactaceae) [13,37], and its presence may be also related to the crassulacean acid metabolism of cacti species [12].
Additionally, compound 13 was only detected in the aerial part and was assigned as lucuminic acid, since the pseudomolecular ion yielded fragments at m/z: 163.03947, 119.04939 and 107.04942 (see Table 1). Lucuminic acid has been reported for Calocarpum sapota (Sapotaceae) by Takeda, et al. [41] and this is the first time that is reported for cacti species. In our investigation, sinapic acid (compound 20) and two of its derivatives (compounds 19 and 25) were also detected and characterized as reported previously [42]. Sinapic acid is synthesized from ferulic acid through enzymatic mechanisms [43] and it has been proposed to have anti-inflammatory activities [44].
In the same way, other phenolic metabolites such as two syringic acid derivatives (compounds 21 and 23), two caffeic acid isomers [45] (compounds 24 and 45), and cinnamic and gallic acid derivatives (compound 31 and 59, respectively) were also found (see Table 1). In addition, piscidic and ferulic acid and/or their derivatives were detected as the most recurrent phenolic acids in C. macromeris extracts: six isomers of piscidic acid (compounds 7, 8, 10, 11, 12, and 15) and four if its derivatives (compounds 17, 18, 22, and 37) were detected and assigned as reported previously [46,47]. Piscidic acid has been previously identified in juices prepared with Opuntia ficus-indica fruits [13] and according to the number of peaks detected and the relative abundance shown in the chromatogram, piscidic acid may be one of the main constituents of the C. macromeris aerial part (see Figure 1a, Table 1); further studies are required to confirm the structure of this metabolite and its total concentration in each part of the plant. On the other hand, compounds 28, 33, 34, and 36 were assigned as ferulic acid isomers [48], and compounds 29, 35, 38, 39, and 41 as ferulic acid derivatives, since they showed characteristic fragment ions of ferulic acid (see Table 1). Our results indicate that ferulic acid is present in both parts of the plant but with a higher relative abundance in the shoots (Figure 1a). Ferulic acid has been detected in different cacti species [18,37,49,50] and its antiproliferative effect in human colon carcinoma HT29 cell line [51] as well its therapeutic potential against cardiovascular and neurodegenerative disorders [52] has been proposed. Our results suggest that C. macromeris could represent a potential source for the isolation of this compound or its derivatives; further studies are required to isolate and characterize the structure of these compounds (isomers) and to know their concentration, elicitation, and production in biotechnological systems. 2-5, compounds 26, 27, 32, 40, 44, 47, 51-53, 55, and 63 were also assigned as carboxylic acids. The fragmentation pattern of these metabolites was mainly characterized by the loss of water (see Table 1). Among these metabolites, compounds 51 and 55 were detected in both parts of the plant and assigned as corchorifatty acid F isomers. This metabolite has been previously identified in leaves of Corchorus olitorius (Tiliaceae) by Yoshikawa, et al. [53] and in aerial parts of Chaenomeles sinensis (Rosaceae) [54]. It also confers activity against pathogenic fungus Pyricularia oryzae in resistant rice cultivars [55]. To our knowledge, this is the first time that is reported for cacti species. On the other hand, compound 40 was identified as azelaic acid, according to Liu, et al. [56]. This dicarboxylic acid has been used for the treatment of acne and skin disorders [57,58] and skin hyperpigmentation [59]. Compound 52 was assigned as tianshic acid due to the presence of fragments at m/z: 165.12788 and 127.11205 (see Table 1). The occurrence of this metabolite has been previously reported for Sambucus williamsii (Adoxaceae) by Yang, et al. [60] and no information exists for cacti species.

In addition to compounds
In the same way, other polar compounds were characterized in C. macromeris methanolic extracts. Compound 14 was assigned as hyrtioerectine C (alkaloid). Youssef [61] isolated this alkaloid and proposed its cytotoxic activity against HeLa cells. In our research group, studies are being carried out to elucidate the presence of other alkaloids in, and the potential applications of, C. macromeris extracts. Signals revealed for compound 30 indicated the presence of a glucopyranoside derivative [62] in the aerial part of C. macromeris. Similarly, compound 42 was tentatively identified as 2-phenylethyl β-D-glucopyranoside. This phenolic glycoside has been previously reported in Pachysandra terminalis (Buxaceae) [63] and in Lactuca indica (Asteraceae) [64]. To our knowledge, this is the first time that it is reported for cacti species. Compound 43 was tentatively identified as dalbergioidin (see Figure 2d); this antifungal isoflavone was reported in Vigna angularis (Fabaceae) as one of the mechanisms against Phytophthora vignae zoospores germination [65], suggesting that its presence in the C. macromeris aerial part may correspond to a constitutive adaptation mechanism since no pathogenic conditions were given to the plants; further studies are required to assess this hypothesis. Compounds 46, 48, and 50 were also assigned as metabolites of glycosidic nature (see Table 1). On the other hand, the pseudomolecular ion and fragmentation pattern of compound 49 indicated the presence of buteine (chalcone; see Figure 2e) in the radicular part of C. macromeris. Buteine exists in Rhus verniciflua (Anacardiaceae), a traditional herb used for cancer treatment [66], and its antioxidant activity [67] and potential as a chemotherapeutic agent [68] has been proposed. Compounds 57 and 60 were tentatively identified as nordihidrocapsiate isomers (see Figure 2f) and compound 58 as plastoquinone 3.
Finally, compounds 54, 56, 61, 62, and 65-69 were not identified since molecular information obtained did not match with theoretical information existing in the literature. Absorption spectrum of these compounds suggest that they may contain benzoic acid within its structure (UV λ max : 280 nm). Further studies are required for structural elucidation of these metabolites. characterized by the loss of water (see Table 1). Among these metabolites, compounds 51 and 55 were detected in both parts of the plant and assigned as corchorifatty acid F isomers. This metabolite has been previously identified in leaves of Corchorus olitorius (Tiliaceae) by Yoshikawa, et al. [53] and in aerial parts of Chaenomeles sinensis (Rosaceae) [54]. It also confers activity against pathogenic fungus Pyricularia oryzae in resistant rice cultivars [55]. To our knowledge, this is the first time that is reported for cacti species. On the other hand, compound 40 was identified as azelaic acid, according to Liu, et al. [56]. This dicarboxylic acid has been used for the treatment of acne and skin disorders [57,58] and skin hyperpigmentation [59]. Compound 52 was assigned as tianshic acid due to the presence of fragments at m/z: 165.12788 and 127.11205 (see Table 1). The occurrence of this metabolite has been previously reported for Sambucus williamsii (Adoxaceae) by Yang, et al. [60] and no information exists for cacti species.
In the same way, other polar compounds were characterized in C. macromeris methanolic extracts. Compound 14 was assigned as hyrtioerectine C (alkaloid). Youssef [61] isolated this alkaloid and proposed its cytotoxic activity against HeLa cells. In our research group, studies are being carried out to elucidate the presence of other alkaloids in, and the potential applications of, C. macromeris extracts. Signals revealed for compound 30 indicated the presence of a glucopyranoside derivative [62] in the aerial part of C. macromeris. Similarly, compound 42 was tentatively identified as 2phenylethyl β-D-glucopyranoside. This phenolic glycoside has been previously reported in Pachysandra terminalis (Buxaceae) [63] and in Lactuca indica (Asteraceae) [64]. To our knowledge, this is the first time that it is reported for cacti species. Compound 43 was tentatively identified as dalbergioidin (see Figure 2d); this antifungal isoflavone was reported in Vigna angularis (Fabaceae) as one of the mechanisms against Phytophthora vignae zoospores germination [65], suggesting that its presence in the C. macromeris aerial part may correspond to a constitutive adaptation mechanism since no pathogenic conditions were given to the plants; further studies are required to assess this hypothesis. Compounds 46, 48, and 50 were also assigned as metabolites of glycosidic nature (see Table 1). On the other hand, the pseudomolecular ion and fragmentation pattern of compound 49 indicated the presence of buteine (chalcone; see Figure 2e) in the radicular part of C. macromeris. Buteine exists in Rhus verniciflua (Anacardiaceae), a traditional herb used for cancer treatment [66], and its antioxidant activity [67] and potential as a chemotherapeutic agent [68] has been proposed. Compounds 57 and 60 were tentatively identified as nordihidrocapsiate isomers (see Figure 2f) and compound 58 as plastoquinone 3.
Finally, compounds 54, 56, 61, 62, and 65-69 were not identified since molecular information obtained did not match with theoretical information existing in the literature. Absorption spectrum of these compounds suggest that they may contain benzoic acid within its structure (UV max: 280 nm). Further studies are required for structural elucidation of these metabolites.   Table 1.

Plant Material
The plants of Coryphantha macromeris were obtained from the in vitro germplasm bank of the Autonomous University of Aguascalientes, México. Coryphantha macromeris was maintained on semisolid Murashige and Skoog medium [69] (3% sucrose, 8 g·L −1 agar, and pH 5.7) and incubated at 25 °C with fluorescent light (40 μmol·m 2 ·s −1 ) and 16/8 (light/dark) photoperiod. Each plant was subcultured for three months during one year with the described conditions, and then in vitro plants were acclimated to greenhouse conditions as reported previously [26] and kept for one year until harvesting for phytochemical analysis. The plant material was botanically identified by Professor Miguel Alvarado Rodríguez. A voucher specimen was deposited at the herbarium of the Autonomous University of Aguascalientes (HUAA; Voucher No. 6386).

Sample Preparation
For analysis, one-year-old plants growing under greenhouse conditions were collected and separated into aerial and radicular parts. Each part of the plant was sliced and dried in an oven (40 °C) during 1 week in dark conditions. Dried material was finally pulverized in a mortar and then extracted three times with methanol in an ultrasonic bath (30 min each time). The resultant extract was filtered and evaporated under reduced pressure at 40 °C and freeze-dried (Labconco Freeze Dryer; Labconco Corporation, Kansas City, MO, USA). Each freeze-dried sample was resuspended (2.5 mg·mL −1 ) in HPLC-MS-grade methanol and sonicated over 10 min. All samples were filtered (0.22 μm) and injected in an UHPLC system hyphenated with a mass spectrometer, as given below.  Table 1.

Plant Material
The plants of Coryphantha macromeris were obtained from the in vitro germplasm bank of the Autonomous University of Aguascalientes, México. Coryphantha macromeris was maintained on semisolid Murashige and Skoog medium [69] (3% sucrose, 8 g·L −1 agar, and pH 5.7) and incubated at 25 • C with fluorescent light (40 µmol·m 2 ·s −1 ) and 16/8 (light/dark) photoperiod. Each plant was subcultured for three months during one year with the described conditions, and then in vitro plants were acclimated to greenhouse conditions as reported previously [26] and kept for one year until harvesting for phytochemical analysis. The plant material was botanically identified by Professor Miguel Alvarado Rodríguez. A voucher specimen was deposited at the herbarium of the Autonomous University of Aguascalientes (HUAA; Voucher No. 6386).

Sample Preparation
For analysis, one-year-old plants growing under greenhouse conditions were collected and separated into aerial and radicular parts. Each part of the plant was sliced and dried in an oven (40 • C) during 1 week in dark conditions. Dried material was finally pulverized in a mortar and then extracted three times with methanol in an ultrasonic bath (30 min each time). The resultant extract was filtered and evaporated under reduced pressure at 40 • C and freeze-dried (Labconco Freeze Dryer; Labconco Corporation, Kansas City, MO, USA). Each freeze-dried sample was resuspended (2.5 mg·mL −1 ) in HPLC-MS-grade methanol and sonicated over 10 min. All samples were filtered (0.22 µm) and injected in an UHPLC system hyphenated with a mass spectrometer, as given below.

UHPLC-PDA-HESI-Orbitrap-MS/MS Conditions
Phytochemical analysis was performed as reported previously [32][33][34], using a Dionex Ultimate 3000 UHPLC system (Thermo Fisher Scientific, Bremen, Germany) with a C18 column (ID: 150 × 4.6 mm, 5 µm; Restek Corporation, Bellefonte, PA, USA) and equipped with a Quaternary Series RS pump and a Dionex Ultimate 3000 Series TCC-3000RS column compartments with an Ultimate 3000 Series WPS-3000RS autosampler (Thermo Fisher Scientific) and a rapid separations PDA detector. The detection wavelengths were 254, 280, 320, and 440 nm, and PDA was recorded from 200 to 800 nm for peak characterization. The separation was performed in a gradient elution mode composed by 1% formic aqueous solution (A) and acetonitrile (B). The flow rate was 1.0 mL·min −1 and the injection volume 10 µL. The gradient program [time (min), %B] was: (0.00, 5), (5.00, 5), (10.00, 30), (15.00, 30), (20.00, 70), (25.00, 70), (35.00, 5), and 12 min for column equilibration before each injection. The system was controlled by Chromeleon 7.2 Software (Thermo Fisher Scientific, Waltham, MA, USA and Dionex Softron GmbH division of Thermo Fisher Scientific) and hyphenated with a Thermo high resolution Q Exactive focus mass spectrometer (Thermo Fisher Scientific). The chromatographic system was coupled to the mass spectrometer with a heated electrospray ionization source II (HESI II). Nitrogen (purity > 99.999%) was employed as both the collision and damping gas. Nitrogen was obtained from a Genius NM32LA nitrogen generator (Peak Scientific, Billerica, MA, USA). Mass calibration for Orbitrap was performed once a week, in both negative and positive modes. Caffeine and N-butylamine (Sigma-Aldrich, Saint Louis, MO, USA) were the calibration standards for positive ions and buspirone hydrochloride, sodium dodecyl sulfate, and taurocholic acid sodium salt were used to calibrate the mass spectrometer. These compounds were dissolved in a mixture of acetic acid, acetonitrile, water, and methanol (Merck Darmstadt, Hesse, Germany) and infused using a Chemyx Fusion 100 syringe pump. XCalibur 2.3 software and Trace Finder 3.2 (Thermo Fisher Scientific, San Jose, CA, USA) were used for UHPLC control and data processing, respectively. Q Exactive 2.0 SP 2 (Thermo Fisher Scientific, Waltham, MA, USA) was used to control the mass spectrometer.

MS Parameters
The HESI parameters were optimized as follows: sheath gas flow rate 75 units; auxiliary gas flow rate 20 units; capillary temperature 400 • C; auxiliary gas heater temperature 500 • C; spray voltage 2500 V (for ESI-); and S lens RF level 30. Full scan data in negative mode was acquired at a resolving power of 70,000 full width half maximum (FWHM) at m/z 200. For the compounds of interest, a scan range of m/z 100-1000 was chosen; the automatic gain control (AGC) was set at 3 × 10 6 and the injection time was set to 200 ms. Scan rate was set at 2 scans s −1 . External calibration was performed using a calibration solution in positive and negative modes before each sample series. In addition to the full scan acquisition method, for confirmation purposes, a targeted MS/MS analysis was performed using the mass inclusion list and expected retention times of the target analytes, with a 30 s time window, with the Orbitrap spectrometer operating both in positive and negative mode at 17,500 FWHM (m/z 200). The AGC target was set to 2 × 10 5 , with the maximum injection time of 20 ms. The precursor ions were filtered by the quadrupole operating at an isolation window of m/z 2. The fore vacuum, high vacuum, and ultra-high vacuum were maintained at approximately 2 mbar, from 105 to below 1010 mbar, respectively. Collision energy (HCD cell) was operated at 30 eV. Detection was based on calculated exact mass and on retention time of target compounds presented in Table 1. The mass tolerance window was set to 5 ppm.

Conclusions
The utilized procedures allowed the separation of 69 metabolites present in methanolic extracts of aerial and radicular parts of C. macromeris, and the identification of most of them. The aerial part showed higher diversity of metabolites compared with roots, and 24 metabolites occurred in both plant parts. Among detected compounds, ferulic and piscidic acid and their derivatives were the most recurrent phenolic metabolites in C. macromeris. In addition, different classes of compounds with reported functional properties were detected. To our knowledge, the phytochemical profile of aerial and root parts of C. macromeris is reported here for the first time by means of modern ultra-high-performance liquid chromatography-tandem mass spectrometry. Based on the detected compounds, C. macromeris may find potential applications in the pharmaceutical and cosmetic industries. The obtained information could be also useful as chemotaxonomic markers for Coryphantha species.