1. Introduction
The botanical name of
Aloe vera is
Aloe barbadensis Miller. It belongs to the Asphodelaceae (Liliaceae) family, and is a shrubby or arborescent, perennial, xerophytic, succulent pea-green colored plant [
1]. The origins of these plants are the dry regions of Africa, Asia, and Southern Europe, especially in the Mediterranean regions. In the Canary Islands,
Aloe vera plants naturally grow anywhere and everywhere and there is a considerable generally shared belief in the beneficial action of the gel among the population, estimated to be one of the few botanical medications in widespread domestic use. In folk medicine, the brown juice has been used traditionally for its purgative effects, and the fresh leaf gel in cosmetics and nutraceutical formulations [
2]. Among the purported benefits of
Aloe vera not supported by experimental or clinical data are the following: treatment of acne, haemorrhoids, psoriasis, anemia, glaucoma, petit ulcer, tuberculosis, blindness, seborrhoeic dermatitis, and fungal infections [
2]. Several reports have demonstrated the antioxidant, antinociceptive and anti-inflammatory activities of the aloe species [
3,
4]. In addition, recent studies have shown the anti-cancer effect of aloe-emodin, an anthraquinone compound present in the leaves of
Aloe vera [
5]. Studies of the
in vitro antimicrobial properties of the ethanolic extract of
Aloe vera leaf gel revealed that it was active against most of the studied pathogenic bacteria and fungi, even at very low doses [
6]. The list of different illnesses and conditions aided by the use of
Aloe vera is indeed impressive, covering everything from burns and slight infections to extremely serious medical conditions. Several reviews have focused on the main scientific discoveries on
Aloe vera reported over the last three decades [
2,
7,
8,
9,
10,
11,
12]. These reviews deal with the botany, the chemical properties, the gel stabilization technique, the biological functions, and the current uses and applications of
Aloe vera (mainly focusing on the exudate and gel of the
Aloe vera leaves).
Plant polyphenols have been implicated in diverse functional roles, including plant resistance against microbial pathogens and animal herbivores such as insects (antibiotic and antifeeding actions), protection against solar radiation, besides reproduction, nutrition, and growth [
13]. Phenolic compounds have also been reported to prevent diseases resulting from oxidative stress [
14,
15,
16].
Screening of the phytochemical (qualitative and quantitative) analysis of the
Aloe vera leaf (leaf skin and gel) showed that almost all of the chemical constituents are present: tannin, phlobatannins, saponin, flavonoids, steroids, terpenoids, and cardiac glycosides anthroquinones, which are used for medicinal purposes [
17]. Phenolic compounds are the second major substances found in
Aloe vera. The main active constituent of the
Aloe vera plant extract is aloine, an anthraquinone heteroside [
18]. Several papers have also been published that focus on the identification of the main phenolic compounds from the gel and leaf exudate of
Aloe. Okamura
et al. developed a procedure for determination of aloesin, 2'-
O-feruloylaloesin, aloeresin A, barbaloin, isobarbaloin, aloenin, aloe-emodin, 8-C-glucosyl-7-
O-methyl-(
S)-aloesol, isoaloeresin D and aloeresin E, which are phenolic constituents of aloe [
19]. Thirteen phenolic compounds from
Aloe barbadensis (syn. A. vera) and
A. arborescens were identified and quantified: aloesin, 8-
C-glucosyl-7-
O-methyl-(
S)-aloesol, neoaloesin A, 8-
O-methyl-7-hydroxyaloin A and B, 10-hydroxyaloin A, isoaloeresin D, aloin A and B, aloeresin E and aloe-emodin from
A. barbadensis; and aloenin, aloenin B, 10-hydroxyaloin A, aloin A and B, and aloe-emodin from
A. arborescens [
20]. A mixture of phenolic compounds, mainly anthrones (aloenin, aloenin B, isobarbaloin, barbaloin and other aloin derivatives from
Aloe secundiflora (Aloeaceae) has been determined from the leaf exudate [
21]. So far, little attention has been paid to the flowers or the leaf skin of the
Aloe. Previous studies have suggested using
Aloe vera flowers for phytotherapeutical purposes due to the presence of some polyphenols [
22].
The aim of this study was to determine the differences in the phenolic profile of the methanol extracts derived from the leaf skin and flowers of
Aloe vera (L.) Burm. f. (syn.
A. barbadensis Mill.) from the Canary Islands to investigate the potential of the flowers and the leaf skin for uses in the health food industry, as well as in pharmaceuticals. As a result eighteen phenolic components were identified and quantified by reverse phase-high performance liquid chromatography (RP-HPLC). The antioxidant activities of the extracts were studied, as well as the preliminary
in vitro susceptibility of some mycoplasma strains. Mollicutes, trivially known as mycoplasmas, are phylogenetically related to the Gram-positive branch of the eubacteria and can be divided into five phylogenetic groupings, including the
anaeroplasma, asteroleplasma, spiroplasma, pneumoniae, and
hominis groups [
23]. Mycoplasmas are commensals or parasites on vertebrate, insect, and plant hosts, representing many significant pathogens in human and veterinary medicine. They are bacteria characterized by their lack of a cell wall and for their small genomes and highly structural and functional simplicity. Besides, they do not synthesize nucleotides or amino acids, express an unusual form of RNA polymerase, and certain species produce atypical ribosomes. All these characteristics make them naturally resistant to many antibiotics, reducing treatment options to tetracyclines, macrolides, and fluoroquinolones. Therefore, they represent magnificent targets for anti-microbe testing [
24].
3. Experimental
3.1. Chemicals
Methanol (of HPLC grade), ferric chloride (FeCl3·6H2O), ferrous sulphate (FeSO4·7H2O) and glacial acetic acid were obtained from Panreac (Barcelona, Spain) with formic acid and sodium acetate provided by Merck (Darmstadt, Germany) of analytical quality. The 1,1-Diphenyl-2-picrylhydrazyl (DPPH) and 2,4,6-tri(2-pyridyl)-1,3,5-triazine (TPTZ) were from Sigma-Aldrich Chemie (Steinheim, Germany).
The antimicrobial activity of the plant extracts was tested using susceptibility test disks (Oxoid, CT0998B, Ø = 5 mm). Polyphenol standards of gallic acid, protocatechuic acid, chlorogenic acid, (−) epicatechin, quercetin, myricetin, ferulic acid, p-coumaric acid, vanillic acid, syringic acid, (+) catechin, sinapic acid, quercitrin, kaempferol and apigenin were purchased from Sigma-Aldrich Chemie; rutin and gentisic and caffeic acids were supplied by Merck (Hohenbrunn, Germany).
3.2. Mycoplasmas
Four types of strains of mollicutes:
Mycoplasma mycoides subsp. Capri (Y-goat) (
M. mycoides Capri),
Mycoplasma agalactiae (PG2) (
M. agalactiae),
Acholeplasma (A.)
laidlawii (PG8) (
A. laidlawii) and
Mycoplasma gallisepticum (PG31) (
M. gallisepticum) were used in this study with the former two cultivated in PH medium [
37] and the latter two in SP4-II medium [
38]. The mycoplasmas were cultured under aerobic conditions at 37 °C.
3.3. Plant Material
The Aloe vera leaves and flowers were collected fresh in February 2010. The plant was identified in the Herbarium of the Viera y Clavijo Botanical Gardens in Gran Canaria where a voucher specimen was deposited (LPA: 27058-27060). A voucher specimen was deposited in the Herbarium of the Viera y Clavijo Botanical Gardens in Gran Canaria (LPA: 27058-27060). Soon after collection, the skin of the leaves and the flowers were separated, shaken and frozen. The leaf skin was separated and cleaned with a knife. The frozen samples were then freeze-dried and pulverized into powder using a blender (Moulinex, 600 W, Ecully Cedex, France) and were subsequently kept in the dark at −20 °C under nitrogen.
3.5. Free Radical Scavenging Activity on DPPH
The reducing ability of the antioxidants on the DPPH radical was evaluated by measuring the loss of 1,1-diphenyl-2-picrylhydrazyl (DPPH) colour at 515 nm after reaction with test extracts [
41]. The sample solution (100 μL of extracts) was rapidly mixed with 1 mL of a solution of 0.2 mM DPPH. After 30 min incubation in darkness at ambient temperature (23 °C), the reduction of the DPPH radical was measured by monitoring the decline in absorbance (Abs) against a methanol blank at 515 nm using a Shimadzu 1700 UV-Vis spectrophotometer. The percentage inhibition was calculated by application of the equation: RSA = 100 (1 − Abs in the presence of sample/Abs in the absence of sample).
3.6. Ferric Reducing Antioxidant Power Assay (FRAP Assay)
Reducing power was determined according to [
42]. This method is based on the reduction of Fe
3+ to Fe
2+, which is recorded by measuring the formation of a blue coloured Fe
2+-tripyridyltriazine compound from the colourless oxidized Fe
3+ form via the action of electron-donating antioxidants. The FRAP reagent consists of 300 mM acetate buffer (3.1 g sodium acetate + 16 mL glacial acetic acid, made up to 1 litre with distilled water; pH = 3.6), 10 mM TPTZ in 40 mM HCl and 20 mM FeCl
3·6H
2O in a ratio of 10:1:1.
The extract (50 μL) was added to 1.5 mL freshly prepared and pre-warmed (37 °C) FRAP reagent. The mixture was incubated at 37 °C for 10 min and the absorbance was measured against a reagent blank (1.5 mL FRAP reagent + 50 μL distilled water) at 593 nm. A standard curve of Fe(II) was constructed over the concentration range of 0.1 mM to 2.0 mM. The results were determined by the regression equation of the curve (Abs = 0.00221[Fe(II)] + 0.02464, r = 0.9998) and expressed as µmol ferric ions reduced to ferrous form.
3.7. Determination of the Phenolic Profile by RP-HPLC
To prepare the samples for the HPLC quantification, the freeze-dried plant material (50 mg) and 2 mL of methanol were mixed and homogenized using a vortex for 30 s. The mixture was stirred in a rotator (SB 3, Stuart, Staffordshire, UK) for 60 min at room temperature in darkness. After centrifugation at 7,000 × g for 20 min at 4 °C, the supernatant was collected and evaporated. The dry residue and 0.5 mL of water were mixed and filtered through a 45 μm nylon syringe filter prior to injection.
Chromatographic analysis was performed on a Varian ProStar 210 system, equipped with a vacuum degasser, a binary pump, a thermostat column compartment and a diode array detector (DAD), connected to ChemStation software. The separation was performed with a reverse-phase Pursuit XRs C18 (250 mm × 4.6 mm, 5 micrometers (μm)) column and a Pursuit XRs C18 (10 mm × 4.6 mm, 5 μm) guard column (Varian, Barcelona, Spain). A gradient system, involving two mobile phases, was used. Eluent A was water with 0.1% formic acid and eluent B, methanol. The flow rate was 1.0 mL/min, and the injection volume was 60 μL of crude extracts (rheodyne injector). The system operated at 27 °C. The elution conditions applied were: 0–4 min, linear gradient from 20% to 30% B; 4–10 min, 30% B isocratic; 10–13 min, linear gradient from 30% to 50% B; 13–15 min, linear gradient from 50% to 80% B and finally, washing and re-conditioning of the column. Monitoring was set at 254 nm for quantification.
Method 1: to quantify the compounds sinapic acid, quercitrin, kaempferol and apigenin in the extracts, five different concentrations of the analytes were injected in triplicate. The calibration curves were constructed by plotting the peak areas
versus the concentration of each analyte. The linearity was assessed by linear regression analysis, which was calculated by the least squares method. Each point on the calibration plot was the mean of two area measurements. All correlation coefficients were over 0.9976 (
Table 2). The wavelength was fixed at 254 nm for quantification. The selectivity of the method was determined by analysis of standard compounds and samples. The peaks of polyphenols were identified by comparing their retention times (RT) and overlaying of UV spectra with those of standard compounds.
Method 2: the phenolic compounds gallic acid, protocatechuic acid, catechin, vanillic acid, epicatechin and syringic acid, chlorogenic acid, gentisic acid, caffeic acid,
p-coumaric acid and ferulic acid, rutin, myricetin, and quercetin were quantified in line with a previously reported method [
27]. Briefly, eluent A was Milli-Q water with 0.1% formic acid and eluent B was methanol. The elution conditions applied were: 0–5 min, 20% B isocratic; 5–30 min, linear gradient from 20% to 60% B; 30–35 min, 60% B isocratic; 35–40, linear gradient from 60% to 20% B and finally, washing and reconditioning of the column. Each standard was individually tested to determine its retention times (RT) as follows: gallic acid (RT: 5.3 min), protocatechuic (RT: 10.0 min), catechin (RT: 12.7 min), chlorogenic acid (RT: 14.9 min), gentisic acid (RT: 17.1 min), vanillic acid (RT: 17.7 min), epicatechin (RT: 17.9 min), caffeic acid (17.9 min), syringic acid (RT: 18.9 min), coumaric acid (RT: 23.4 min), rutin (RT: 28.1 min), ferulic acid (RT: 24.3 min), myricetin (RT: 30.6 min), and quercetin (RT: 34.6 min) were well resolved. Simultaneous monitoring was set at 270 nm (gallic acid, protocatechuic acid, (+) catechin, vanillic acid, (−) epicatechin and syringic acid), 324 nm (chlorogenic acid, gentisic acid, caffeic acid,
p-coumaric acid and ferulic acid), and 373 nm (rutin, myricetin, and quercetin) for quantification. The limits of detection (LOD) and limits of quantification (LOQ) were estimated from the signal-to noise-ratio of the individual peaks, assuming a minimum detectable signal-to-noise level of 3 and 10, respectively. The LODs were found to be in the range of 0.0003–0.1230 μg·mL
−1 and the LOQs were observed in the range of 0.0008–0.4100 μg·mL
−1. This indicated that the proposed method was suitably sensitive for the quantification of polyphenols. The linearity was assessed by linear regression analysis, which was calculated by the least square method. Each point on the calibration plot was the mean from two area measurements. All the correlation coefficients were no less than 0.9982. Reproducibility, expressed as the relative standard deviation (RSD), was obtained by analyzing six replicate sample RSDs values ranging from 1.91% to 5.81%. The accuracy was expressed as the recovery of standard compounds added to the pre-analyzed sample. The recovery was found to be in the range of 87.97%–115.79%.
3.8. Evaluation of the Antimycoplasmic Activity
The antimycoplasmic activity of all plant extracts was determined using a modified disc diffusion method as described for growth inhibition tests elsewhere [
43]. A total of 25 μL of each extract was tested in the disc diffusion assay against four strains of mycoplasmas. The plates were subsequently incubated and examined daily for colonies (1–2 days) under the conditions described above. Organisms were considered resistant when their growth was not inhibited by the 25 µL extract-impregnated wafers (5-mm sterilized filter paper discs). The presence of a zone of inhibition, as well as any changes in the colony morphology or in the colony concentration, was considered to be indicative of antimycoplasmic activity. The inhibition zones were measured in µm using an optical microscope Olympus CKX41 (Olympus, Hamburg, Germany), with a digital camera ProgRes C12 plus (Jenoptik, Jena, Germany) inserted, and using ProgRes® Image Capture Software for the measurements. Each antimycoplasmal assay was performed at least in triplicate and inhibition zones were measured at least three times per well, at perpendicular angles. Mean values and standard deviations (SD) were registered and calculated as mean ± SD to the effects of this study. Filter discs impregnated with 25 μL of Tilmicosin (0.4 μg/mL) were used as positive control for antimicrobial activity and impregnated with 25 μL of distilled sterilised water were used as negative controls.
Aroa López 1, Miguel Suárez De Tangil 1, 
