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DOI: 10.1002/cbdv.202000820

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Metabolic Profiling of Vasorelaxant Extract from Malvaviscus arboreus by LC/QTOF-MS Sergio Rodríguez-Morales,a Blanca Ocampo-Medina,b Nancy Romero-Ceronio,b Cuauhtémoc Alvarado-Sánchez,b Miguel Ángel Vilchis-Reyes,b Luis Fernando Roa de la Fuente,b Rolffy Ortiz-Andrade,c and Oswaldo Hernández-Abreu*b a

Unidad de Química-Sisal, Facultad de Química, Universidad Nacional Autónoma de México, Puerto de abrigo S/N, 97356 Sisal Yucatán, Mexico b Centro de Investigación de Ciencia y Tecnología Aplicada de Tabasco, Universidad Juárez Autónoma de Tabasco, Carretera Cunduacán-Jalpa km. 1 Col. La Esmeralda, Cunduacán, 86690 Tabasco, Mexico, e-mail: [email protected] c Facultad de Química, Universidad Autónoma de Yucatán, Mérida, Calle 43 N. 613, Col. Inalámbrica, 97069 Mérida, Yucatán, Mexico

We aimed to develop a standardized methodology to determine the metabolic profile of organic extracts from Malvaviscus arboreus Cav. (Malvaceae), a Mexican plant used in traditional medicine for the treatment of hypertension and other illnesses. Also, we determined the vasorelaxant activity of these extracts by ex vivo rat thoracic aorta assay. Organic extracts of stems and leaves were prepared by a comprehensive maceration process. The vasorelaxant activity was determined by measuring the relaxant capability of the extract to decrease a contraction induced by noradrenaline (0.1 μM). The hexane extract induced a significant vasorelaxant effect in a concentration- and endothelium-dependent manner. Secondary metabolites, such as polyunsaturated fatty acids, terpenes and one flavonoid, were annotated by liquid chromatography/quadrupole time-of-flight mass spectrometry (LC/QTOF-MS) in positive ion mode. This exploratory study allowed us to identify bioactive secondary metabolites from Malvaviscus arboreus, as well as identify potentially-new vasorelaxant molecules and scaffolds for drug discovery. Keywords: bioactive, LC/QTOF-MS, Malvaviscus arboreus, metabolic profiling, pharmacognosy.

Introduction Ancient civilizations have used medicinal plant-based preparations to treat common diseases since long time ago. Traditional use is supported by thousands of years of experience allowing to recognize between medicinal and toxic species. Based on empirical observation, herbal preparations were the first available medicinal formulations to attend sick people.[1] Nowadays, modern medicine let us to explain how naturally-occurring molecules produces their biological effect and describe the structure-activity

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relationship.[2,3] Interestingly, advances in metabolomic technologies have been a powerful tool in molecular identification and characterization of plant-based secondary metabolites and potential drug targets.[4] In developing countries where the vast majority of population continues using traditional medicine due to healthcare access issues, natural product investigation represents a feasible strategy to assure effectiveness and safety of these preparations.[5] In this context, Malvaceae is a widely distributed family as weeds on pasture and wastelands from tropical and subtropical regions around the world.[6] Herbal preparations of species from this family have major contribution in the treatment of cough, throat infection, and further bronchial problems, as well as stomach and intestine irritation. Substances from © 2021 Wiley-VHCA AG, Zurich, Switzerland

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flowers and leaves are emollients and they are used for softening the skin. Also, they are applied as poultices to reduce swelling and eliminate toxins. The leaves help to reduce gut irritation and they have laxative effects, too.[7] Malvaceae family is the seventh botanical family most used by Chontal communities from Tabasco, Mexico. Particularly, M. arboreus is used for treating hypertension, diarrhea, stomachache, and other illnesses.[8] Active substances associated with its biological activity have not been characterized so far. So, the current lack of evidence on active metabolite identification from this plant might motivated this research. Therefore, we aimed to annotate the metabolic profile of an organic extract from M. arboreus by liquid chromatography/quadrupole time-of-flight mass spectrometry (LC/QTOF-MS) and determine its vasorelaxant activity on vascular smooth muscle by isolated thoracic aorta assay.

Results and Discussion Yield and Biological Assay of Organic Extracts After successive maceration of stems and leaves, a yielding of 5.97 g, 8.21 g and 7.94 g were obtained from hexane (HEMa) dichloromethane (DEMa), and methanol (MEMa) extract, respectively. All organic extracts were evaluated to determine their relaxant activity. HEMa induced a significant vasorelaxant effect on endothelium-intact aortic rings (Emax = 60.91 � 2.03 %, IC50 = 50.56 � 17.4 μg/ml, Figure 1A). Also,

DEMa induced a vasorelaxation less effective than HEMa (Emax = 42.26 � 7.17 %, IC50 = 169.88 � 10.6 μg/ ml, Figure 1B). Both HEMa and DEMa showed less potency and efficacy than carbachol (Emax = 72.13 � 9.83 %, IC50 = 0.17 � 0.06 μg/ml), a muscarinic acetylcholine receptor agonist used as control[9] and nitrendipine (Emax = 83.98 � 3.92 %, IC50 = 0.012 � 0.007 μg/ml), a smooth muscle calcium channel blocker, control without endothelium.[10] MEMa did not show relaxant activity at all. Interestingly, when samples were assayed on endothelium-denuded aorta rings, there was no activity. This evidence suggests that all organic extracts did not act on aortic smooth muscle tissue. Metabolic Profiling by LC/QTOF-MS Since HEMa was the most active organic extract, it was analyzed by LC/QTOF-MS in order to characterize chemical composition. Liquid chromatography conditions were optimized by a gradient elution method for 30 min using a mobile phase A (0.1 % formic acidwater) and a mobile phase B (0.1 % formic acidacetonitrile). In these conditions, a positive mode base peak chromatogram (BPC) of the total content of HEMa was obtained (Figure 2). Such chromatogram was analyzed and compared in MELTIN database to get identity of metabolites contented in the organic extract. Most compounds were identified according to their retention times, chemical formula, ion type, MS/ MS fragments, and score (Table 1). Metabolites anno-

Figure 1. Concentration-response curves of relaxant effect on isolated rat aortic rings pre-contracted with NA (0.1 μM) of (A) HEMa and (B) DEMa. Results are expressed as the mean � S.E.M. of five experiments (*p < 0.05 vs. control).

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Figure 2. The base peak chromatograms (BPC) of the metabolites in hexane extract of Malvaviscus arboreus in positive mode.

tated included: polyunsaturated fatty acids: (1) (5Z,8Z,11Z,14Z)-octadecatetraenoic acid, (2) (9Z,12Z,15E)-octadecatrienoic acid, (5) (12R)-HETrE, and (9) (7Z,11Z,14E)-eicosatrienoic acid; vitamin: (3) 1α,25dihydroxy-2α-(3-hydroxypropyl)vitamin D3; sesquiterpenes: (4) emmotin A; diterpenes: (6) ( )-isoamijiol; steroidal and non-steroidal triterpenes: (7) longispinogenin, (8) theonellasterol B, (10) betulinic Acid, (12) 3α,12α-dihydroxy-5β-chol-8(14)-en-24-oic acid, and flavonoid: (11) artonin P (Figure 3). Exploratory chemical studies of compounds obtained from herbal extracts have an important role on determining both putative pharmacological or toxicological effects, as well as describe the impact in new drug development.[11] Commonly, bioactive compounds isolated from medicinal plants are identified, elucidated, and standardized by spectroscopic and spectrometric techniques. Other organic extracts with

Figure 3. Chemical structures of the metabolites annotated in hexane extract of Malvaviscus arboreus.

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14.313

15.843

16.576 18.646 18.654 19.852

19.859

20.217

20.600

24.149

26.426

2

3

4 5 6 7

8

9

10

11

12

tR: Retention time.

12.693

1

[a]

tR[a] (min)

Peak number

3α,12α-Dihydroxy-5βchol-8(14)-en-24-oic acid

Artonin P

(7Z,11Z,14E)Eicosatrienoic acid Betulinic acid

Theonellasterol B

(9Z,12Z,15E)Octadecatrienoic acid 1α,25-Dihydroxy-2α-(3hy-droxypropyl)vitamin D3 Emmotin A (12R)-HETrE ( )-Isoamijiol Longispinogenin

(5Z,8Z,11Z,14Z)Octadecatetraenoic acid

Identification 277.2162

279.2319 475.3782

301.1409 345.2399 305.2472 459.3834 423.3622 307.2631 457.3677

471.1049

413.2659

(M + H) + (M + H) + (M + H) + (M + Na) + (M + Na) + (M + H) + (M + H) + (M + H) + (M + H) + (M + H) + (M + Na) +

(M + Na) +

C30H50O4 C16H22O4 C20H34O3 C20H32O2 C30H50O3 C30H46O C20H34O2 C30H48O3 C25H20O8

C24H38O4

C18H30O2

C18H28O2

Experimental m/z

Ion type

Molecular formula

Table 1. Compounds annotated by LC/QTOF-MS in hexane extract of Malvaviscus arboreus.

390.2767

448.1157

456.3609

306.2559

422.3550

278.1517 322.2506 304.2401 458.3761

474.3710

278.2246

276.2088

Theorical m/z

0.93

0.22

1.29

0.03

0.33

0.3 0.49 0.53 0.15

0.22

0.22

0.45

Error (ppm) 259.2059, 217.1045, 149.1323 261.2214, 149.0232 457.3681, 423.3690, 249.1847 261.1307 305.2473 261.1306 441.3727, 423.3623 391.3574, 282.2793 288.2897, 261.2214 438.4117, 425.3792, 207.1857 453.4128, 438.4125, 429.3726, 397.3465 391.2838, 316.3208, 225.1959

MS/MS fragments

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phenolic compounds and pure phenolic compounds have been reported with interesting vasorelaxant bioactivity in rat aorta preparations.[12,13] In this sense, pentacyclic triterpenes, such as betulinic acid, are considered interesting compounds due to their vasorelaxant activity.[14] Previous reports regarding biological activity of secondary metabolites from M. arboreus have showed a variety of biological effects. For instance, there are studies where volatile compounds exhibited moderate antifungal activity,[15] phenolic compounds showed antioxidant capacity,[16,17] and antimicrobial activity.[18,19] Also, further studies have showed identification of kaempferol, cyanidin and pelargonidin.[20] Recently, β-resorcylic, caffeic, protocatechuic, and 4-hydroxyphenylacetic acids, as well as flavonoids, such as trifolin and astragalin, were identified in M. arboreus extracts with potential hepatoprotective activity.[21] Other flavonoids, anthocyanins, and phenolic acids have showed notable antioxidant, antitrypanosomal, and anti-protease properties.[22] It should be noted that identified fatty acid molecules in in M. arboreus flowers are very similar to the (5Z,9Z)-5,9-nonadecadienoic acid reported by Carballeira.[ 23] Figure 2 shows non-identified peaks that could be derived from primary-secondary metabolism or artifacts formed from pollution. Certainly, environmental substances released into air, soil and water could be absorbed by plants through root and leaves and they might change their metabolism. Moreover, some factors, such as season, soil nutrients, and ecosystem may influence in plant secondary metabolism.[24,25] Secondary metabolites identified on organic extract are related with medicinal action of M. arboreus. So, it is necessary to make more investigation to identify the active molecules in order to purify them. In addition, regarding outcomes showed in isolated aorta ring assay, vasorelaxant effect induced by HEMa is probably mediated by endothelium-dependent signaling pathway, such as activation of endothelium-dependent hyperpolarizing factor (EDHF), regulation of K + channels opening, nitric oxide (NO) releasing or activation of muscarinic receptors.[26] However, further experimentation is necessary to corroborate and describe most comprehensively the mechanism of action of single or combined compounds.

Conclusions

molecules. This report provides evidence to scientifically corroborate the vasorelaxant effect of HEMa preparation and the association with its antihypertensive effect described in folk medicine. In addition, secondary metabolites identified represent interesting molecules for further studies and drug discovery.

Experimental Section Chemical Reagents Chromatographic supplies as methanol, acetonitrile, water, and formic acid (LC/MS grade) were supplied by Tedia Company, Inc. Quality Systems. Noradrenaline bitartrate (NA), carbamylcholine chloride (carbachol) and nitrendipine were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Organic solvents for maceration were of technical grade. Other reagents were analytical grade and purchased from local sources. Plant Material and Extraction A M. arboreus specimen was collect in February 2017 (Teapa, Tabasco. Latitude 17°30’48’’, longitude 92°55’18’’). The botanical identification was carried out by Guadarrama-Olivera María, M.Sc. A voucher specimen (No. 35344) was deposited at the Herbarium of Academic Division of Biological Sciences of Universidad Juárez Autónoma de Tabasco (UJAT). Organic extracts were obtained by extraction methods previously described.[27] Briefly, dried plant material of stems and leaves were pulverized (yield 657.52 g) and successive maceration process was performed. Material was exposed to different solvents three times for 72 h at room temperature. Hexane, methylene chloride and methanol were the solvent used on this extraction process. After filtration, extracts were concentrated in vacuo. Animals Male Wistar rats (250–300 g) were supplied by Academic Division of Health Sciences (UJAT). All animals were maintained under standard laboratory conditions with free access to food and water. All animal procedures were conducted in accordance with our Federal Regulations for Animal Experimentation and Care (SAGARPA, NOM-062-ZOO-1999, México) and approved by the Institutional Animal Care and Use Committee Recommendation.

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Isolated Rat Aorta Ring Assay By sodium pentobarbital injection (60 mg/kg, ip) all animals were anaesthetized and sacrificed by cervical dislocation. Thoracic aorta was cleaned of adhering connective tissue and cut it into 3 –5 mm length rings. To obtain denuded-endothelium rings, inner vascular tissue was mechanically rubbed using forceps. All aorta ring preparations were placed in an incubation chamber using stainless steel hooks. Tissues were drawn under an optimal tension of 3 g. A Krebs solution (composition, mM: NaCl, 118; KCl, 4.7; CaCl2, 2.5; MgSO4, 1.2; KH2PO4, 1.2; NaHCO3, 25.0; EDTA, 0.026 and glucose, 11.1, pH 7.4) at 37 °C and bubbled with oxygen (O2/CO2, 19 : 1) was used for incubation. Isometric tension was recorded using TSD125 C force transducers accompanying DA100 C amplifier (Astromed®, West Warwick, RI, USA) connected to MP160 analyzer (Biopac® Instruments, Santa Barbara, CA, USA) as previously described.[27] After equilibration time, denuded- and intact-aortic rings were precontracted with NA (0.1 μM) and washed every 30 min. The presence of endothelium was confirmed by lack of relaxing response after carbachol (1 μM) administration. Once aorta rings maintained a plateau contraction, increasing concentrations (3 –1000 μg/ml) of the test samples (HEMa, DEMa, MEMa) or positive control were added to the bath chamber. The relaxant effect was calculated by comparing maximum vascular contraction before and after samples. LC/MS/MS Analytical Conditions Liquid chromatography analysis was performed using a LC system (1290 Infinity II, Agilent Technologies), equipped with a quaternary pump, and coupled to an electrospray ionization with quadrupole time-of-flight (Q-TOF) mass spectrometer Agilent 6545 Agilent Technologies, Santa Clara, CA, USA. The analyte (HEMa, 1 mg) was dissolved in 1 ml acetonitrile and vortexed for few seconds. After this, the sample was filtered through a 0.45 μm nylon filter and diluted 1 : 100. Chromatographic separation was performed on a bioZen™ XB C18 LC column (100 × 2.1 mm × 1.7 μm) by use of the mobile phase A (0.1 % formic acid-water) and mobile phase B (0.1 % formic acid-acetonitrile) in a gradient elution method (50 : 50, v/v, 0– 5 min; 25 : 75, v/v, 5–15 min; 0 : 100, v/v 15–25 min and 50 : 50 25– 30 min), injection 5.00 μL and flow rate was set at 0.2 ml/min. Accurate mass measurements were obtained by ion mass corrections using reference masses www.cb.wiley.com

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of protonated purine (121.050873 m/z) and protonated hexakis(1H,1H,3H-tetrafluoropropoxy)phosphazine (HP-921; 922.009798 m/z). Targeted MS/MS experiments were performed to annotate metabolic profiling, as well as to recognize fragmentation patterns by acquisition mode Targeted MS2 and fixed collision energy of 20 eV. Reference solution was added into the AJS ESI source in each analysis. Data Analysis Vasorelaxant activity is expressed as mean of five separated experiments � S.E.M. Concentration –response curves were plotted and adjusted by the nonlinear curve fitting program ORIGIN® 6.0. The statistical significance (P < 0.05) was established by one way-ANOVA and post-hoc Tukey’s test. From extracted ion chromatograms, the accurate mass of each metabolite peak was obtained by means of calibrant solution. Chromatographic data were processed using MassHunter Qualitative Analysis Software (Version B.07.00). Compounds were extracted from the raw data using the Molecular Feature Extraction (MFE) algorithm, and compound identification was performed using the METLIN Metabolite Database (www.metlin.scripps.edu) and Molecular Formula Generation software (Agilent Technologies).[28]

Acknowledgements Authors thank Dr. Juan Carlos Sánchez-Salgado for his contribution on style correction and edition of this manuscript. This study was financed by a grant from ‘Apoyo a la Incorporación de NPTC’, Folio: UJAT-PTC263.

Author Contribution Statement Blanca Ocampo-Medina: Designed the project and performed the biological experiments. Rolffy OrtizAndrade: Performed the biological experiments, analyzed the biological data, and drafted the manuscript. Nancy Romero-Ceronio: Technical assistance and revised the manuscript. Cuauhtémoc Alvarado-Sánchez: Analyzed the data and approved the final manuscript. Miguel Ángel Vilchis-Reyes: Designed the project, performed the liquid chromatography experiments, and drafted the manuscript. Luis Fernando Roa de la Fuente: Performed the liquid chromatography experiments. Sergio Rodríguez-Morales: Analyzed the LC/ © 2021 Wiley-VHCA AG, Zurich, Switzerland

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QTOF-MS data. Oswaldo Hernández-Abreu: Designed the project, supervised the experiments, analyzed the data, drafted, and revised the final manuscript.

[15]

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Received October 3, 2020 Accepted February 5, 2021

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