Hindawi Journal of Chemistry Volume 2018, Article ID 6123850, 12 pages https://doi.org/10.1155/2018/6123850

Research Article Chemical Composition and Antioxidant Activity of Aloe vera from the Pica Oasis (Tarapaca´, ) by UHPLC-Q/Orbitrap/MS/MS

Cristina Quispe ,1 Michael Villalobos,1 Jorge Bo´rquez,2 and Mario Simirgiotis 3,4

1Instituto de EtnoFarmacolog´ıa, Facultad de Ciencias de la Salud, Universidad , Avda. Arturo Prat 2120, 1110939, Chile 2Laboratorio de Productos Naturales, Depto. de Qu´ımica, Facultad de Ciencias Ba´sicas, Universidad de Antofagasta, Antofagasta 124000, Chile 3Instituto de Farmacia, Facultad de Ciencias, Universidad Austral de Chile, Campus Isla Teja, 5090000, Chile 4Center for Interdisciplinary Studies on the Nervous System (CISNe), Universidad Austral de Chile, Valdivia 5090000, Chile

Correspondence should be addressed to Cristina Quispe; [email protected] and Mario Simirgiotis; [email protected]

Received 16 May 2018; Revised 21 September 2018; Accepted 13 November 2018; Published 20 December 2018

Academic Editor: Jose A. Pereira

Copyright © 2018 Cristina Quispe et al. *is is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. *e chemical composition of Aloe vera growing in the small town of San Andre´s de Pica, an oasis of warm waters and typical fruits, located in Tamarugal in the Northern Chilean region of Tarapaca´ is reported. *e chemical characterization was performed using liquid chromatography (UHPLC) coupled to PDA and high-resolution mass spectrometry (HESI-Q- Orbitrap -MS) in four different plant parts of Aloe (peel, flowers, gel, and roots). Twenty-five phenolic compounds were identified,® including cinnamic acids and other derivatives (e.g., caffeic and chlorogenic acids), chromones (e.g., aloesin and isoaloeresin D), anthracene compounds and derivatives (e.g., aloin A/B and emodin), and several C-flavonoids (e.g., orientin and isovitexin), among others. Total antioxidant activity of the ethanolic extracts of the peels, flowers, gel, and roots was measured as the capturing of the DPPH• and ABTS •+ radicals, while the iron-reducing antioxidant power (FRAP) was measured by spectroscopic methods. *e peel had the highest antioxidant activity with values of 2.43 mM ET/g MF (DPPH•), 34.32 mM ET/g MF (ABTS •+), and 3.82 mM ET/g MF (FRAP). According to our results, the peel is the best part of the plant for the production of nutraceuticals or cosmetics products for its greatest number of bioactive compounds. *is is a new and innovative finding since the only part used in traditional medicine is the gel of Aloe, and the peel is generally considered waste and discarded.

1. Introduction *e different secondary metabolites produced by the plants are influenced by environmental conditions such as In Tarapaca´ Region (I region, Northern Chile), a remote part extreme light, water, soils, salts, chemicals, temperature, and of the Atacama Desert, there is a small town and oasis called geographical variations [3]. San Jose´ de Pica. Pica has a lush greenery and thriving Environmental factors such as light intensity, temper- agriculture due to underground water sources surfacing in ature, water availability, type, and soil composition among the middle of the desert [1]. *is desert is one of the driest others, have a substantial influence on the quality and places on Earth resulting in extreme environmental con- productivity of medicinal plants. Plants of the same species ditions. *ese varying abiotic conditions, as seasonal fluc- occurring in different condition environments may differ tuations of chemical and physical water composition significantly in their content of secondary metabolites [4]. (e.g., nutrients, temperature, and salinity), are challenging Furthermore, the chemical composition of any plant for the biota and affect the species assemblages and eco- depends upon the local geographical condition, type of soil, system stability [2]. and its composition. For example, it has been reported that 2 Journal of Chemistry the chemical composition and yield of the essential oil of microbiological stabilization of A. vera gel using the Mentha piperita var., grown under different agroecological treatment of HHP [18]. locations in Egypt, vary significantly according to the climate Moreover, other studies about the influence of tem- conditions. Plants growing in high temperatures showed perature on drying kinetics, physicochemical properties, and high menthone/menthol contents and high antioxidant antioxidant capacity of Aloe gel [19], plus the effect of activity that could be attributed to their high number of temperature in the structural properties were also published phenolic compounds and flavonoids compared to other [20]; however, studies covering the chemical composition of locations [5]. A. vera growing in extreme climatic conditions like the one Aloe vera (Aloe barbadensis Miller) is a native species to growing in the region of Tarapaca´ have never been reported. South Africa, which has been widely distributed in the Several studies have shown that climatic conditions continent of Europe from where they have spread to almost cause plants to develop metabolites that help in their sur- the entire world [6]. *is plant is also extensively distributed vival; thus, species growing in extreme conditions, such as in South America [6, 7], where it is known for its therapeutic the Atacama Desert, can develop interesting metabolites to effects. A. vera has been studied for its clinical effectiveness be studied. In this scenario, the use of state-of-the-art tools against a great variety of affections and disorders of the skin such as metabolomic mass fingerprinting can help to study [8], for example, wounds and burns [6, 9–11], for its effect as the metabolomic processes in extreme systems like the one anti-inflammatory, antioxidant, healing, and antibacterial; occurring among the “biodiversity of the Atacama Desert.” these actions are biologically attributed to its chemical For a complete chemical characterization of A. vera from components [8]. *e part of the plant that has been usually Tarapaca Region, we used high-resolution hyphenated LC- used for therapeutic purposes is the gel [6, 9]. Traditionally, MS (UHPLC-MS) techniques whose advantage is the rapid A. vera gel has been used both externally for the treatment of separation of compounds and the most accurate de- wounds, minor burns, and irritations of the skin, and in- termination of the masses [21]. *is technique is considered ternally in different formats to treat constipation, cough, gold standard for the analysis of phenolic compounds, due to ulcers, diabetes, and headaches among others [12]. Re- its versatility, precision, and relatively low cost [22]. *e garding its chemical composition, A. vera gel consists mainly UHPLC machine can be coupled to several mass spec- of water (>98%) and polysaccharides, including pectins, trometers, such as time-of-flight (TOF or Q-TOF), cellulose, hemicellulose, glucomannan, and acemannan, the quadrupole-Orbitrap (Q-OT), or triple quadrupole (TQ) latter being considered as the main functional component of mass spectrometers. *e Orbitrap is an ion trap mass an- A. vera gel, formed from a long chain of acetylated mannose alyzer that consists of a high-resolution hybrid mass spec- [12]. Aloe latex, also known as Aloe juice, is a bitter yellow trometer, which has recently been published as an innovative exudate of the pericyclic tubules in the outer skin of the leaf. technology that offers high-resolution MS/MS fragments, *e main active component of Aloe juice are hydroxyan- for metabolomic analysis of a variety of metabolites, in- thracenic derivatives, which represent between 15 and 40% cluding toxins, pesticides, antibiotics, peptides, and several of the total components, and among them are anthraqui- small organic molecules up to 2000 Daltons [21]. none glycosides aloin A and B (also called barbaloin) along Based on this background, we have studied the chemical with Aloe emodin [12]. A. vera flowers have received little composition of A. vera from Tarapaca´ Region, given the attention, although there are some studies that suggest the geographic conditions and the possible influence on the use of these flowers for phytotherapeutic purposes, due to secondary metabolites present in the species. *e phenolic the presence of several phenolic compounds such as caffeic compounds of each part of A. vera were characterized using acid, chlorogenic acid, and ferulic acid among others. *e UHPLC-Q/Orbitrap/MS/MS, and the chemical composition compound mannose-6 phosphate, which is a constituent of was related with the antioxidant activity. the sugar of A. vera gel, has been shown to have wound- healing properties as well. In addition, some glycoproteins 2. Materials and Methods present in the A. vera gel have antitumor and antiulcer effects and may increase the proliferation of normal human 2.1. Chemicals and Plant Material. Folin-Ciocalteu phenol skin cells [12]. In the case of the root, some phenolic reagent (2 N), reagent-grade Na2CO3, HCl, NaNO2, NaOH, compounds, especially naphthoquinones and anthraqui- FeCl3, AlCl3, quercetin, trichloroacetic acid, sodium acetate, nones, have also been identified [13–15]. HPLC-grade water, lichrosolv HPLC-grade acetonitrile, It is well known that the types and levels of the chemical MeOH, reagent-grade MeOH, formic acid, CH3COOH, components present in the plants can vary according to the CH3COONa, potassium persulfate, and ABTS [2,2′-azino- geographical origin or variety; although there are some bis(3-ethylbenzothiazoline-6-sulfonic acid)] were obtained chemical characterization studies of A. vera from other from Merck (Darmstadt, Germany). Gallic acid, 2,4,6-tri(2- countries, we were not able to find reports on the chemical pyridyl)-s-triazine (TPTZ), trolox, tert-butyl hydroperoxide, composition of A. vera from the Chilean region of Tar- nitroblue tetrazolium, xanthine oxidase, and DPPH (1,1- apaca. In Chile, the studies carried out in A. vera have been diphenyl-2-picrylhydrazyl radical) were purchased from scarce, focusing mainly on the farming conditions, the Sigma-Aldrich Chemical Co. effect of high hydrostatic pressures (HHPs) on rheological A. vera plant was collected in March 2016, from the properties [16], the effect of HHPs on functional properties sector La Concova in Pica, Tarapaca Region, Chile (latitude: and characteristics of the quality of Aloe gel [17], and the −20.48612; longitude: −69.318967; 1379 metres above the sea Journal of Chemistry 3

(a) (b) (c)

Figure 1: Map of Chile showing the location of Tarapaca Region: commune of Pica (a), La Concova sector (b), and the A. vera crops (c). level) (Figure 1). *e plant was identified by the botanist spectrometer, caffeine, N-butylamine, buspirone hydro- Alicia Marticorena, and voucher herbarium specimens are chloride, sodium dodecyl sulfate and taurocholic acid kept at the Natural Products Laboratory of the University of (Sigma-Aldrich, St Louis, Missouri, USA) besides Ultramark La Serena under reference number 14014. 1621 (Alfa Aesar, Heysam, UK), were used as standards. *e softwares Q Exactive 2.0 SP 2, XCalibur 2.3, and Trace Finder 3.2 (*ermo Fisher Scientific and Dionex Softron 2.2. Instrumentation. An ultrasonic bath Branson 3510, a GmbH) were used to control the mass spectrometer and for vacuum filtration equipment Medi-Pump model N° 1136- data processing. For UHPLC-mass spectrometer control and THOMAS, a Rotavapor Laborota 4001, a −86°C Ultralow data processing, Q Exactive 2.0 SP 2, XCalibur 2.3, and Trace Temperature Freezer-Gene Xpress, a Labconco-Freezone 6- Finder 3.2 software (*ermo Fisher Scientific and Dionex Plus Liophylizer, a Merck Spectroquant Pharo 300 spec- Softron GmbH Part of *ermo Fisher Scientific) were used, trophotometer, a Agilent 1260 Infinity LC Quaternary high- respectively [21]. performance liquid chromatography (HPLC) system, and a *ermo Scientific Dionex 3000 Ultrahigh-performance 2.3. LC Parameters. A portion of each extract (2.5 mg) liquid chromatography (UHPLC, *ermo Fisher Scien- obtained as explained above was dissolved in 1 mL of 1% tific, Bremen, Germany) system with a modern PDA de- formic acid in MeOH, filtered through a 0.45 µm micropore tector and quadrupole hybrid high-resolution mass membrane (PTFE, Waters Milford, MA, USA) before use. spectrometer-Orbitrap Q Exactive Focus were employed, Later, it was injected into the UHPLC-PDA and ESI- using a quaternary Series RS pump® and TCC-3000RS col- Orbitrap-MS equipment. *e parameters of liquid chro- umn compartments with a WPS-3000RS autosampler plus a matography were those previously published by our research rapid separations PDA detector controlled by Chromeleon group [21]. 7.2 and Xcalibur 2.3. *e chromatographic system was coupled to the MS with a heated electrospray ionization 2.4. MS Parameters. *e HESI parameters were optimized source II (HESI II). Nitrogen (purity >99.999%) obtained as reported previously [21]. Detection was based on cal- from a Genius NM32LA nitrogen generator (Peak Scientific, culated exact mass and on retention time of target com- Billerica, MA, USA) was employed as both the collision and pounds, presented in Table 1. damping gas. Mass calibration for the Orbitrap was per- formed in both negative and positive modes, to ensure a working mass accuracy lower than or equal to 5 ppm. *e 2.5. Sample Preparation. *e Aloe leaves were carefully cut calibration was done once a week. For calibration of the mass and washed, leaving the leaf upright to drain the exudate and 4 Journal of Chemistry

Table 1: Tentative identification of phenolic compounds in peel, flower, gel, and root of A. vera from Tarapaca Region detected by UHPLC- Q-OT-MS. Elemental Retention *eoretical Measured Peak Error Other ions Classification of Tentative identification composition time mass mass no. (ppm) (m/z) the compound [M-H]− (min) (m/z) (m/z) 203.07106 Chromone (C- (C H O −) 1 Aloesin or aloeresin B C H O − 9.23 393.11911 393.11917 −0.15263 12 11 3 glycosylated 19 21 9 245.08168 − chromone) (C14H13O4 ) − 191.05534 2 Chlorogenic acid C16H17O9 9.54 353.08781 353.08752 0.82133 − Cinnamic acid (C7H11O6 ) − 135.04451 3 Caffeic acid C9H7O4 9.92 179.03498 179.03464 1.89907 − Cinnamic acid (C8H7O2 ) Anthracene − 4 Aloe emodin-diglucoside C27H29O15 9.98 593.15119 593.15063 0.94411 — compound (anthrone) Flavonoid (O- 301.55185 5 Isoquercitrin C H O − 9.98 463.08820 463.08752 1.46840 glycosylated 21 19 12 (M-glucose) flavonoid) Flavonoid (O- Kaempferol-3-O-hexosyl-O- 6 C H O − 10.29 579.13554 579.13513 0.70795 — glycosylated pentoside 26 27 15 flavonoid) 6-Methyl-1,3,8- 225.05438 Anthracene − − 7 trihydroxyanthraquinone C15H9O5 10.40 269.04555 269.04538 0.63186 (C14H9O3 ) compound (emodin) 241.73468 (anthrone) 327.05048 Flavonoid (C- Luteolin-8-C-glucoside (C H O −) 8 C H O − 10.41 447.09329 447.09293 0.80520 17 11 7 glycosylated (orientin) 21 19 11 299.05582 − flavonoid) (C16H11O6 ) − 191.05547 9 Feruloylquinic acid C17H19O9 10.46 367.10346 367.10309 1.00789 − Cinnamic acid (C7H11O6 ) Anthracene 270.05280 10 10-Hydroxyaloin A C H O − 10.90 433.11402 433.11392 0.23089 compound 21 21 10 (C H O −) 15 10 5 (anthrone) 311.05588 Flavonoid (C- (C H O −) 11 Isovitexin C H O − 10.80 431.09837 431.09833 0.09279 17 11 6 glycosylated 21 19 10 283.06058 − flavonoid) (C16H11O5 ) Flavonoid (O- − 12 Chrysoeriol-7-O-glucuronide C22H19O12 11.11 475.08820 475.08856 −0.75775 — glycosylated flavonoid) − 13 Caffeoyl ester of aloesin C29H29O12 11.74 569.16645 569.16626 0.33382 — Chromone 297.07687 Anthracene (C H O −) 14 Aloin A C H O − 12.18 417.11911 417.11908 0.07192 17 13 5 compound 21 21 9 268.07318 − (anthrone) (C16H12O4 ) − 15 Isoaloeresin D C29H31O11 11.85 555.18719 555.18677 0.75650 — Chromone 2′-p- Anthracene − 16 Methoxycoumaroylaloeresin C29H29O11 12.29 553.17154 553.17041 2.04277 — compound B (anthrone) Anthracene 4,5-Dimethyl ether of Aloe 17 C H O − 12.29 297.07685 297.07669 0.53858 — compound emodin 17 13 5 (anthrone) 297.07669 Anthracene (C H O −) 18 Aloin B C H O − 12.36 417.11911 417.11893 0.43153 17 13 5 compound 21 21 9 268.07419 − (anthrone) (C16H12O4 ) Anthracene − 459.12842 compound (C- 19 6′-Malonylnataloin C24H23O12 12.69 503.11950 503.11911 0.77516 − (C23H23O10 ) glycosylated anthrone) Journal of Chemistry 5

Table 1: Continued. Elemental Retention *eoretical Measured Peak Error Other ions Classification of Tentative identification composition time mass mass no. (ppm) (m/z) the compound [M-H]− (min) (m/z) (m/z) Naringenin-4′-methoxy-7- 20 C H O − 14.39 445.11402 445.11401 0.02247 — Flavonoid O-glucuronide 22 21 10 7-Methylether of 2′- 21 C H O − 13.40 583.18210 583.18146 1.09743 — Chromone feruloylaloesin 30 31 12 Anthracene 269.04495 22 Aloe emodin-8-O-glucoside C H O − 14.52 431.09837 431.09793 1.02065 compound 21 19 10 (C H O −) 15 9 5 (anthrone) 5,3′-Dihydroxy-6,7,4′- − 23 trimethoxyflavone C18H15O7 14.81 343.08233 343.08231 0.05830 — Flavonoid (eupatorin) − 24 Trihydroxy octadecenoic acid C18H33O5 18.28 329.23335 329.23328 0.21262 — Oxilipin − 25 3,4-Di-O-caffeoylquinic acid C25H23O12 19.04 515.11950 515.11847 1.99954 — Cinnamic acid soaking it in distilled water overnight. Later, the peeled leaf method of Sogi et al. [23]. A portion of the DPPH stock and the gel inside was cut into pieces, soaked in water for half solution (0.24 g/100 mL methanol) was diluted into 10 an hour, then passed through a strainer, and then liquefied. parts methanol at 80% (4 :1 ratio of methanol and water, A. vera peel was also cut into small pieces and washed with respectively) so that the working solution obtained an distilled water. *e roots were carefully cut into small pieces absorbance of 1.10 ± 0.02 at 515 nm. 3 mL of the working and washed with distilled water. Flowers were provided by solution of DPPH was mixed with 0.6 mL of blank, stan- the company “Mundo Aloe vera” from Pica, Tarapaca Re- dard, or sample, kept in the dark for 20 minutes, and the gion, and dried at room temperature (25–30°C). absorbance was recorded at 515 nm. Methanol at 80% (control) was used to calculate the radical decoloration activity of a standard curve, which was prepared with trolox 2.5.1. Phenolic Compounds Extraction. Each part of Aloe solution (50–250 μM, R2: 0.9905). Samples were analyzed in obtained (peels, gel, roots, and flowers; previously weighed) triplicate, and the results are expressed in units equivalent were macerated with methanol for 48 hours (sample: to trolox (ET), mM ET/g fresh weight (FW). methanol; 1 : 2 (w : w)) then sonicated for 30 minutes in a Branson 3510 ultrasonic apparatus. *e extracts from each •+ sample were combined, filtered, and evaporated in vacuo in 2.6.2. ABTS Assay. *e ABTS antioxidant activity of the •+ the dark (40°C) in a rotavapor (Laborota 4001-efficient). *e extracts was carried out using the ABTS radical cation methanolic extracts were maintained at −86°C in an ultralow discoloration test as described in Reference [23], with some freezer for 24 hours and then freeze-dried in a Labconco- modifications. *e solution of 7 mM ABTS and 2.45 mM Freezone 6-Plus equipment. *e extracts were suspended in potassium persulfate was mixed in a 1 :1 ratio, and the 20 mL ultrapure water and loaded onto an XAD-7 (100 g) solution was allowed to stand in the dark for 12–16 hours to •+ column. *e column was rinsed with water (100 mL), and produce the ABTS cation radical solution. *e stock so- phenolic compounds were eluted with 100 mL of MeOH lution was then diluted ten times, with an 80% methanol acidified with 0.1% HCl. *e solutions were combined and solution, to reach the absorbance of 0.700 ± 0.020 at 734 nm. •+ evaporated to dryness under reduced pressure (40°C) to give 3 mL of the ABTS stock solution was mixed with 30 μL of dark-brown extracts from peels, gel, roots, and flowers. blank, standard, or sample, and after 6 min, the absorbance Samples were then analyzed by HPLC using the Agilent 1260 at 734 nm was measured using a spectrophotometer. As a Infinity and by UHPLC using the *ermo machine coupled blank, 80% methanol was used, and the quantification was to the PDA detector *ermo Q Exactive Focus mass performed using a standard calibration curve of trolox 2 spectrometer. antioxidant (0.30–1.5 mM, R : 0.9886). *e samples were analyzed in triplicate, and the results were expressed in mM ET/grams of fresh mass (FM). 2.6.AntioxidantAssays. *e antioxidant activity of the peels, flowers, gel, and roots was determined by the following methods: DPPH (1,1-diphenyl-2-picrylhydrazyl), ABTS 2.6.3. Ferric Reducing Antioxidant Power (FRAP) Assay. [2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)] *e determination of ferric reducing antioxidant power or and FRAP (antioxidant potential of iron reduction), which ferric reducing ability (FRAP assay) of the extracts was are detailed below. performed as described by Sogi et al. [23], with some modifications. *e stock solutions prepared were 10 mM TPTZ (2,4,6-tri (2-pyridyl)-s-triazine) solution in 40 mM 2.6.1. DPPH Assay. *e DPPH• radical decoloration ac- HCl, 300 mM acetate buffer (pH 3.6), and 20 mM tivity of the A. vera extracts was determined using the FeCl3·6H2O solution. *e solution using in this assay was DPPH solution methanol, following the modification prepared with mixed buffer acetate, TPTZ solution, and 6 Journal of Chemistry ferric chloride solution at a proportion of 10 :1 :1 (v : v : v), root (2.67 ± 0.16), flowers (2.01 ± 0.10), and in the last place, respectively. Plant extracts, standards, or methanolic trolox the gel (0.38 ± 0.01 ET/g FM). *ere was a statistical cor- solutions (300 µL) were incubated at 37°C with 3 mL of the relation between the three antioxidant assays (p < 0.05). FRAP solution (prepared by mixing 25 mL acetate buffer, Although the total phenolic content was not determined, 5 mL TPTZ solution, and 10 mL FeCl3·6H2O solution) for when comparing the chromatographic profiles of the dif- 30 min in the dark. Absorbance of the blue ferrous tripyr- ferent parts of Aloe, under the same chromatographic idyltriazine complex formed was then read at 595 nm. conditions and sample concentration, we noticed that the Quantification was performed using a standard calibration greater variety, quantity, and abundance of the chemical curve of antioxidant trolox (from 50 to 250 µM, R2: 0.995). compounds were in the peel, and these results correlate to Samples were analyzed in triplicate and results are expressed the greater antioxidant activity. in mM ET/g fresh weight (FW). 3.3. Identification of Phenolic Compound to A. vera from Pica, 2.7. Analysis of Data. *e statistical analysis was carried out Tarapaca Region using the SPSS program version 20. *e determination was repeated at least three times for each sample solution. 3.3.1. Fingerprinting from Phenolic Compounds. *e phe- Analysis of variance was performed using ANOVA. Sig- nolic profiles of PEE were assessed by UHPLC-PDA-QOT/ nificant differences between means were determined by MS (ultrahigh-performance liquid chromatography photo Tukey’s comparison test (p values < 0.005 were regarded as diode array quadrupole Orbitrap mass spectrometry); using significant). the negative heated HESI mass detection mode, phenolic compounds were tentatively identified in the different ex- 3. Results and Discussion tracts of A. vera. Comparative UHPLC-TIC (total ion current) chromatograms of A. vera parts are showed in 3.1. Yield Percentage. *e peels, flowers, gel, and roots were Figure 2. *e retention time (Rt), UV spectral maxima, MS extracted three times (n � 3) with MeOH, and phenolics fragmentation, and tentative identification of the com- were retained on Amberlite XAD-7 to obtain the phenolic- pounds are summarized in Table 1. enriched extract (PEE). *e highest PEE was obtained from *e methanolic extracts of the peel, flower, gel, and root the peels (16.2%), while the extraction yields for flowers, gel, of A. vera retained in XAD-7 was analyzed by HPLC-PDA, and roots were 12.6, 12.3, and 8.5%, respectively. to obtain the fingerprint chromatograms for each of the parts. Figure 2 indicates that the peel and the flowers had the greatest abundance of compounds, followed by the 3.2. Antioxidant Activity Quantification. Table 2 shows roots. *e composition of the gel is scarce in phenolic antioxidant activity of the four methanolic extracts from compounds; thus, we could state differences in the com- several parts of A. vera using different methods for the position of the organic compounds in each of the parts of antioxidant capacity. *ree antioxidant assays were used to the plant. evaluate the antioxidant capacity of the samples, based on Twenty-five compounds (Table 1) were tentatively chemical aspects for the measurements of radical scavenging • •+ identified in different parts of A. vera. *e highlighted activity (DPPH and ABTS ) assays and a method based on compounds determined include various cinnamic acids and metals reduction (FRAP). • their derivatives, chromones, anthracene compounds, and In the DPPH radical trapping capacity assay (Table 2), flavonoids, some of which have been reported previously in the extract of the peels showed the greatest antioxidant Aloe species. *e identification was performed based on its capacity (2.43 ± 0.14 mM ET/g FM), followed by the extract total mass compared to the theoretical mass (<5 ppm) and of the roots (1.43 ± 0.08 mM ET/g MF), then flowers (1.25 ± the characteristic fragments for each compound, finding 0.03 mM ET/g FM), and finally with less antioxidant ca- differences and similarities between the samples analyzed. pacity, the gel extract (0.34 ± 0.01 mM ET/g FM), with a Peaks 1–6, 8, 10–21, and 24–25 were detected in the peel, statistically significant difference between them (p < 0.05), peaks 1–6, 8–12, 14, 15, 19, and 22–24 in the flower, peaks 1, except between the flower extract and the Aloe roots. •+ 14, 15, 17, 18, 20, 21, 23, and 24 in the gel, and peaks 1–4, 6, *e ABTS assay (Table 2, column (b)) showed that the 7, 10–15, 23, and 24 in the root of A. vera, shown in Figure 2. peel has the highest antioxidant activity with average values A detailed explanation of the characterization of these of 34.32 ± 2.60 mM ET/g FM, followed by the roots (17.54 ± compounds, grouped based on their chemical characteris- 0.77 mM ET/g FM), then the flowers with 16.55 ± 2.30 mM tics, is given below. ET/g FM, and finally the Aloe gel with 2.06 ± 0.06 mM ET/g FM. *ere is a statistically significant difference between peel and gel extract (p < 0.05). *is study allowed the measuring 3.3.2. Cinnamic Acids and Derivatives. Four cinnamic acids of the antioxidant capacity of polyphenols through the and their derivatives were tentatively identified in the capture of free radicals. *e antioxidant activity values negative mode. Peaks 2 and 9 were identified as chlorogenic − obtained by the DPPH method were carried out using the acid (5-caffeoylquinic acid, C16H17O9 ) and feruloylquinic − − trolox reagent as a standard. In the column (c), the same acid (C17H19O9 ), respectively. Peak 2 shows an [M-H] sample presented the highest activity in the FRAP assay, with ion at m/z of 353.08752 amu with retention times of values of 3.82 ± 0.23 mM ET/g FM for peel, followed by the 9.54 min, and peak 5 shows an ion [M-H]− at m/z around Journal of Chemistry 7

Table 2: Scavenging of the 1,1-diphenyl-2-picrylhydrazyl radical (DPPH•), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS_+), ferric-reducing antioxidant power (FRAP), and extraction yield of four plant parts of A. vera. Parts from A. vera PEE (%)a DPPH•b ABTS•+c FRAPd Peel 16.2 2.43 ± 0.14 34.32 ± 2.60 3.82 ± 0.23 Flower 12.6 1.25 ± 0.03f 16.55 ± 2.30e 2.01 ± 0.10 Gel 12.3 0.34 ± 0.01 2.06 ± 0.06 0.38 ± 0.01 Root 8.5 1.43 ± 0.08f 17.54 ± 0.77e 2.67 ± 0.16 aPhenolic-enriched extract expressed in percentage; bantiradical DPPH activities are expressed as mM equivalent of trolox/g fresh weight, ; cABTS expressed as mM equivalent of trolox/g fresh weight; dFRAP expressed as mM equivalent of trolox/g fresh weight. e,fValues are not significantly different (at p < 0.05). All measurements were repeated three times (n � 3).

RT: 0.00–35.01 100 17 18 90 16 80 15 20 70 60 1 8 14 50 19 10 12 40 21 6 11 13 30 3 4 Relative abundance Relative 2 5 20 24 25 10 0 0 2 4 6 8 10 12 14 16 18 20 22 Time (min) (a) RT: 0.00–35.01 100 3 4 90 24 80 8 11 70 9 60 1 6 14 15 50 5 10 12 40 2 19 30 22 23 Relative abundance Relative 20 10 0 0 2 4 6 8 10121416182022 Time (min)

(b) RT: 0.00–35.01 100 17 18 90 80 70 1 15 60 21 50 20 14 40 30

Relative abundance Relative 23 20 24 10 0 0 2 4 6 8 10121416182022 Time (min)

(c) Figure 2: Continued. 8 Journal of Chemistry

RT: 0.00–35.01 100 17 18 90 15 80 14 1 70 60 10 50 11 40 12 30 19 Relative abundance Relative 3 20 7 2 4 13 20 10 6 23 24 0 0 2 4 6 8 10121416182022 Time (min) (d)

Figure 2: UHPLC-chromatograms of (a) peel, (b) flower, (c) gel, and (d) root from Northern Chilean A. vera.

367.10309 amu (Rt of 10.46 min). *ese compounds were respectively) with respect to the theoretical mass ion [26]. tentatively identified by their difference in mass of Isoaloeresin D has been reported in A. eru, A. grandi- 0.82133 ppm and 1.00789 ppm with respect to the theoretical dentata, A. perfoliata, A. brevifolia [26], and A. barbadensis mass ion for each of the peaks. Both peaks showed a Miller [29]. *e compound 7-methylether of 2′-fer- n − characteristic ion MS at m/z 191.05534 amu (C7H11O6 ) uloylaloesin was described in A. eru, A. grandidentata, and [24, 25]. Peak 3 with a molecular anion at m/z: A. saponaria [26], and caffeoyl ester of aloesin was de- − 179.03464 amu was identified as caffeic acid (C9H7O4 ) by scribed in A. broomii [30]. its difference in mass of 1.89907 ppm with respect to the theoretical mass ion and confirmed by producing an ion n − MS at m/z 135.04451 amu (C8H7O2 ) due to the loss of a 3.3.4. Anthracene Compounds. Nine anthracene com- CO2 molecule from the original ion [24]. Finally, peak 25 pounds corresponding to peaks 4, 7, 10, 14, 16, 17, 18, with an [M-H]− ion at m/z: 515.11847 amu was identified as 19, and 22 were identified using UHPLC-ESI-MS-MS anal- − 3,4-di-O-caffeoylquinic acid (C25H23O12 ), compound ysis. Peak 4 was identified as Aloe emodin-diglucoside − − identified by its difference in mass of 1.99954 ppm respect to (C27H29O15 ), which showed an [M-H] ion at m/z the theoretical mass ion [26]. *e presence of chlorogenic 593.15063 amu (Rt 9.98 min). *is compound was tentatively acid has been reported in A. brevifolia leaves [26] and caffeic identified by its difference in mass of 0.94411 ppm with respect acid was reported in leaves of A. barbadensis Miller and A. to the theoretical mass ion. *is compound was reported in A. arborescens Miller [27]. In the case of 3,4-di-O-caffeoyl- arborences, A. grandidentata, and A. ferox [26]. *e compound − − quinic acid, its presence was reported in A. saponaria [26], emodin (C15H9O5 ) was assigned to peak 7 with an [M-H] ion and the feruloylquinic acid had not been reported in any at m/z 269.04538 amu (Rt 10.40 min), identified by its differ- Aloe species until this study. ence in mass of 0.63186 ppm with respect to the theoretical mass ion and by the identification of the typical fragments MSn, − 225.05438 amu (C14H9O3 ) and 241.73468 amu [31]. Peak 10 − 3.3.3. Chromones. Four chromones were tentatively iden- was identified as 10-hydroxyaloin A, (C21H21O10 , m/z tified (peaks 1, 13, 15, and 21) using UHPLC-ESI-MS-MS 433.11392 amu). *e major diagnostic daughter MSn ion of this − − analysis. Peak 1, with an [M-H] ion at m/z: 393.11917 (Rt compound was at an m/z of 270.05280 amu (C15H10O5 ) [29]. 9.23 min) was described as aloesin or aloeresin B *e 10-hydroxyaloin A was reported in A. barbadensis Miller, − (C19H21O9 ), recognized by their difference in mass of A. grandidentata, and A. perfoliata [26, 29]. − −0.15263 ppm with respect to the theoretical mass ion Peak 14 was aloin A (C21H21O9 ), which showed an and by the identification of two typical fragments of MSn; [M-H]− ion at m/z 417.11908 amu (Rt 12.18 min). *is peak − n m/z: 203.07106 amu (C12H11O3 ) and 245.08168 amu was identified by its major diagnostic daughters, MS ions at − − (C14H13O4 ) [26]. *e aloesin was reported in A. grandi- m/z 297.07687 amu (C17H13O5 ) and 268.07318 amu − dentata, A. perfoliata [26], A. ferox Miller [28], and A. (C16H12O4 ) [29]. Figure 3 shows TIC (total ion current, barbadensis Miller [29]. Peaks 13, 15, and 21 showed [M- negative mode) and full high-resolution mass spectra showing H]− ions at m/z 569.16626 amu (Rt 11.74 min), the UHPLC chromatograms of [M-H]− ion and proposed 555.18677 amu (Rt 11.85 min), and 583.18146 amu (Rt structure of aloin A. 13.40 min) and were tentatively identified as a caffeoyl ester In the same manner, Peak 18 with an [M-H]− ion at m/z − of aloesin [30], isoaloeresin D, and 7-methylether of 2′- 417.11893 amu was identified as aloin B (C21H21O9 ) isomer feruloylaloesin [26], respectively. For these peaks, a great to aloin A. *is peak was identified by the difference in accuracy was observed demonstrated by their small mass of 0.43153 ppm with respect to the theoretical mass ion differences in ppm (0.33382, 0.75650, and 1.09743, and by the identification of two typical ions MSn; at Journal of Chemistry 9

RT: 0.00–35.01 12.36 100 12.11 90 13.66 80 13.01 70 60 9.18 14.41 50 10.44 17.98 10.65 14.83 40 1.31 1.51 10.30 17.45 30 15.46

Relative abundance Relative 1.66 15.58 18.58 20 1.14 1.83 19.37 19.70 20.84 22.67 10 0.07 2.98 4.01 4.92 5.67 6.26 7.76 8.79 0 0 2 4 6 8 10121416182022 Time (min) (a) T: FTMS - p ESI full ms [100.00–1500.00] 417.11844 100 OHO OH

80

O– 60 OH 40 118.02869 395.09747 453.09491 O HO

Relative abundance Relative 20 OH 297.07611 OH 160.84108 231.06548 357.08542 497.08459 555.18573 657.15698 821.28284 871.21594 988 0 100 200 300 400 500 600 700 800 900 m (z)

(b) T: FTMS - p ESI full ms2 [email protected] [100.00–750.00] 297.07614 100 OH OH O– OH O OH 80

60 O–

40 HO HO 268.07355 298.07874 Relative abundance Relative 20 279.06567 0 265 270 275 280 285 290 295 300 305 m (z)

(c)

Figure 3: UHPLC chromatograms of A. vera peel: (a) TIC (total ion current, negative mode), (b) full high-resolution mass spectra showing the ion aloin A, and (c) fragmentation ions.

− 297.07669 amu (C17H13O5 ) and 268.07419 amu human consumption of barbaloin present in derived − (C16H12O4 ) [24]; both isomers were confirmed by their products of Aloe is 10 mg/L [33]. characteristic UV max at retention time of 12.18 min for Peaks 16 and 17 were 2′-p-methoxycoumaroylaloeresin − aloin A and 12.36 min for aloin B [32]. B (C29H29O11 ) and 4,5-dimethyl ether of Aloe emodin − − Aloin A has been reported in A. barbadensis Miller, A. (C17H13O5 ), which showed an [M-H] ions at m/z arborences, and A. grandidentata, while aloin B was reported 553.17041 amu and 297.07669 amu, respectively. *ese peaks in A. barbadensis Miller and A. grandidentata [26]; both aloins were identified by their difference in mass of 2.04277 ppm (A and B) have also been reported in A. ferox Miller [28] and 0.53858 ppm with respect to the theoretical mass ion. and in A. barbadensis Miller [29]. Aloin is a mixture *e 2′-p-methoxy coumaroyl aloeresin B was reported in A. of aloin A (also called barbaloin) and aloin B (or iso- eru, A. perfoliata, and A. saponaria [26]. Mass spectra of barbaloin), corresponding to an anthraquinone glycoside peak 19 (Rt 1.69 min) showed [M-H]− ion at m/z to which attributed a characteristic of purgative effects, 503.11911 amu, and it was identified as 6′-malonylnataloin present in the Aloe leaf [26]. According to the International (nataloin). *is compound was identified by the difference Aloe Science Council, the maximum concentration for in mass of 0.77516 ppm with respect to the theoretical mass 10 Journal of Chemistry ion and detection of an MSn ion at m/z 459.12842 amu Table 3: Distribution of the phenolic compounds identified ten- − (C23H23O10 ) product of the loss of a CO2 molecule [34]. tatively in methanolic extract of peel, flower, gel, and root from *is peak was detected in A. barbadensis Miller, A. arbor- Chilean A. vera. ences, A. eru, A. grandidentata, A. brevifolia, A. ferox [26], Peak Tentative identification Peel Flower Gel Root and A. ellenbeckii [34]. no. Aloe emodin-8-O-glucoside was detected as peak 22 (Rt 1 Aloesin and aloeresin B X X X X − 14.52 min) showing [M-H] at m/z 431.09793 amu 2 Chlorogenic acid X X — X − (C21H19O10 ). *is peak was identified by the difference in 3 Caffeic acid XX—X mass of 1.02065 ppm with respect to the theoretical mass ion 4 Aloe emodin-diglucoside X X — X n − and its daughters MS ion at m/z 269.04495 amu (C15H9O5 ) 5 Isoquercitrin XX—— Kaempferol-3-O-hexosyl-O- [26]. Other studies reported the presence of this compound 6 XX—X in A. barbadensis Miller, A. arborences, A. eru, A. perfoliata, pentoside A. saponaria, and A. ferox, both metabolites being consid- 6-Methyl-1,3,8- ered as chemotaxonomic markers of the Aloe species in one 7 trihydroxyanthraquinone ———X (emodin) study [26]. Luteolin-8-C-glucoside 8 XX—— (orientin) 9 Feruloylquinic acid — X — — 3.3.5. Flavonoids. Seven flavonoids were tentatively iden- 10 10-Hydroxyaloin A X X — X tified (peaks 5, 6, 8, 11, 12, 20, and 23) using UHPLC-ESI- 11 Isovitexin XX—X MS-MS analyses in the negative mode [M-H]−. Peak 5, with 12 Chrysoeriol-7-O-glucuronide X X — X an [M-H]− ion at m/z 463.08752 amu, was tentatively 13 Caffeoyl ester of aloesin X — — X − classified as isoquercitrin (C21H19O12 ) with Rt 9.98 min 14 Aloin A XXXX and their difference in mass of 1.46840 ppm with respect to 15 Isoaloeresin D X X X X n 2′-p- the theoretical mass ion and its characteristic MS ion at m/ 16 X——— z 301.55185 amu [24, 35]. *is compound was reported in Methoxycoumaroylaloeresin B 4,5-Dimethyl ether of Aloe A. arborences and A. eru [26]. Peak 6 was recognized as 17 X—XX − emodin kaempferol-3-O-hexosyl-O-pentoside (C26H27O15 ) with − 18 Aloin B X—XX an [M-H] ion at m/z 579.13513 amu (Rt 10.29 min) with a 19 6′-Malonylnataloin X X — X difference in mass of 0.70795 ppm with respect to the Naringenin-4′-methoxy-7-O- 20 X—XX theoretical mass ion. *is compound was reported in A. glucuronide 7-Methylether of 2′- arborences and A. grandidentata [26]. *e retention time 21 X—X— 10.41 min showed for peak 8, identified it as luteolin-8-C- feruloylaloesin − − 22 Aloe emodin-8-O-glucoside — X — — glucoside or Orientin (C21H19O11 ) with an [M-H] ion at 5,3′-Dihydroxy-6,7,4′- m/z 447.09293. Peak 8 was characterized according to the 23 —XXX small error of 0.80520 ppm with respect to the theoretical trimethoxyflavone (eupatorin) 24 Trihydroxy octadecenoic acid X X X X mass ion and its characteristic two ions at MSn at m/z − 25 3,4-Di-O-caffeoylquinic acid X — — — 327.04977 amu (C17H11O7 ) and 299.05603 amu − X: presence of compounds; —: absence of compounds. (C16H11O6 ) [35]. *is compound was reported in A. barbadensis Miller, A. arborences, A. grandidentata, A. perfoliata, and A. ferox [26]. *e negative mode ESI-MS naringenin-4′-methoxy-7-O-glucuronide had not been re- spectrum of peak 11 (Rt 10.80 min) showed a strong [M- ported in any Aloe species until this study. H]− parent ion at m/z 431.09833 amu which yielded − daughters ions at m/z 311.05588 amu (C17H11O6 ) and 3.3.6. Oxylipins. An oxylipin corresponding to peak 24, − − 283.06058 amu (C16H11O5 ) [35]. Peak 11 was determined was identified as trihydroxy octadecenoic acid (C18H33O5 ) as isovitexin, and this compound was only reported in A. with a m/z 329.23328 amu (Rt 18.28 min), determined by its perfoliata [26]. small difference in ppm (0.21262) with the theoretical mass Peaks 12 (Rt 11.11 min), 20 (Rt 14.39 min), and 23 ion; this compound has been previously reported in A. (14.81 min) showed [M-H]− at m/z 475.08856 amu, saponaria [26]. 445.11401 amu (Rt 14.39 min) and 343.08231 amu. Peaks 12, *e distribution of phenolic compounds identified in 20, and 23 were identified tentatively as chrysoeriol-7-O- this study can be observed in Table 3, allowing a more − glucuronide (C22H19O12 ), naringenin-4′-methoxy-7-O- graphical demonstration of the differences or similarities in − glucuronide (C22H21O10 ), and 5,3′-dihydroxy-6,7,4′- the different plant parts. UHPLC-Q/Orbitrap/MS/MS − trimethoxy-flavone (eupatorin) (C18H15O7 ), respectively, analysis of the methanol extract of the peel, flower, gel, due to its great accuracy demonstrated by their small dif- and root showed that the highest number of phenolic ferences in mass −0.75775, 0.02247, and 0.05830 with respect compounds is found in peel, flowers, and roots of Aloe. to the theoretical mass ion, respectively. Chrysoeriol-7-O- Peaks 1, 14, 15, and 24 were detected in the peel, flowers, gel, glucuronide was reported in A. grandidentata and 5,3′- and roots of the methanolic extract. Among the twenty-five dihydroxy-6,7,4′-trimethoxy-flavone in A. arborences, compounds detected, only nine compounds were detected in A. eru, A. grandidentata, and A. brevifolia [26], while the gel of Aloe. Journal of Chemistry 11

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