LC-MS characterization of bioactive metabolites from two Yemeni Aloe spp. with antioxidant and antidiabetic properties

2.1 Chemicals

4-Dimethylaminocinnamaldehyde (DMAC), Folin Ciocalteu reagent, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), phosphate buffer, iron(III) chloride hexahydrate, and catechin were purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA). Procyanidin B1 reference compounds were obtained from Chromadex (Irvine, CA, USA). Aloin A was purchased from MCE (MedChemExpress, NJ, USA). All organic solvents were of HPLC or LC-MS grades and obtained from VWR International (Suwanee, GA, USA).

2.2 Aloe samples

The resin obtained from Aloe perryi Baker (APR, local name Saber Socotri), and the gel obtained from Aloe barbadensis Mill (AVG, known as Aloe vera L gel, local name Saber Yamaniis) were obtained from local markets in Hadibu and Sanaa, respectively, Yemen, in 2017.

2.3 Preparation of Aloe extracts

APR and AVG dried powders were extracted with 100% methanol. Briefly, 5 g of the powdered plant material was added to 50 mL methanol, and the mixture was sonicated for 20 min at room temperature. The mixture was then centrifuged at 5000 rpm for 10 min (Sorvall RC-6 plus, Asheville, NC, USA). The plant residue was further extracted 4 more times, each with 50 mL methanol, and the combined supernatants was evaporated to dryness using rotary evaporator. The obtained extract was dissolved in methanol and filtered using 0.22 PTFE syringe filter (Fisher Scientific, Pittsburg, PA) before analysis for phenolics, HPLC, LC-MS, and spectrophotometric assays. Three replicate from each sample were tested.

2.4 LC-MS profiling of Aloe extracts

LC-MS was performed using the Shimadzu LC-IT-TOF-MS (Shimadzu, Tokyo, Japan) with a Shim-pack XR-ODS column (50 mm × 3.0 mm, 2.2 µm). Solvent gradient consisted of 0.1% formic acid in H₂O (A) and methanol (B). Compounds were eluted into the ESI ion source at the flow rate of 0.45 mL/min with a step gradient of 5–60% B (40 min), isocratic at 60% B (2 min), 5% B (2 min), then the column was re-equilibrated for 5 min at 5% B. Column was maintained at 40 °C during the run. The MS was programed to carry out a full scan over a range of m/z 100–1500 (MS1) and m/z 100–700 (MS2) in both positive and negative modes. The heat block and curved desolvation line (CDL) temperature was maintained at 200 °C; nitrogen was used as the nebulizing gas at a flow rate of 1.5 L/min, and as the drying gas at 75 kPa; the interface voltage was (+), 4.5 kV; (−), −3.5 kV; and the detector voltage was 1.8 kV. Compounds were characterized by their MS, MS/MS spectra, UV spectra, and in comparison with literature.

2.5 Quantitative analysis of major components by HPLC-PDA

HPLC was conducted on an Agilent 1200 HPLC (Agilent Technologies, Santa Clara, CA) with a photodiode array (PDA) detector, and an autosampler with Chemstation software. The HPLC was outfitted with a reversed phase Phenomenex Synergi 4 µm hydro-RP 80A column (250 mm × 4.6 mm × 5 µm, Torrance, CA.) equipped with a guard column (Phenomenex security guard cartridge, AQC 18.4 × 3.00 mm). The mobile phase consisted of 2% acetic acid in H₂O (A) and 0.5% acetic acid in acetonitrile-water 50:50 (B). The operating conditions were: autosampler tray at 10 °C; column oven at 30 °C; constant elution flow rate of 1 mL/min. Separation of Aloe compounds started with 20% B with a linear gradient to 40% B (0–10 min), 60%B (20 min), 65% B (25 min), 90% B (40 min), 90% B (42 min), and a post-run of 8 min with 20% B. Aloins and other anthrones were monitored at a wavelength of 355 nm, whereas chromones were monitored at 295 nm. A standard curve was constructed with aloin A reference standard, and anthrones were quantified as aloin A equivalent.

2.6 Total phenolics content

The total polyphenol concentrations were measured spectrophotometrically using a microplate-adapted Folin–Ciocalteu assay (Grace et al., 2016) at a wavelength of 765 nm. Results were standardized against a gallic acid standard curve, and results were expressed as gallic acid equivalent (mg GAE per g Aloe extract). All determinations were carried out in triplicate.

2.7 Total flavonoid content

The content of total flavonoid in the Aloe extracts was determined by using a colorimetric method that depends on the formation of flavonoid-aluminium complex (Roshanak et al., 2016). The color intensity was read at 510 nm using the spectrophotometer (Shimadzu UV–VIS 2450, Japan). The flavonoid content was calculated from a calibration curve constructed with catechin, and results were expressed as mg/g catechin equivalent. Determinations were carried out in triplicate.

2.8 Total proanthocyanidin content

Total proanthocyanidins were quantified colorimetrically using the DMAC method, in a 96-well plate against a standard curve constructed with procyanidin B1 reference standard, as previously described (Prior et al., 2010). Concentrations were calculated as procyanidin B1 equivalents (mg /g extract). Determinations were carried out in triplicate.

2.9 DPPH radical scavenging assay

The radical scavenging activity was measured using the stable DPPH and Trolox as reference substances according to a modified method (Grace et al., 2016). An aliquot (20 μL) of each sample was added to 180 μL of DPPH solution (150 μM in 95% ethanol) to initiate the reaction. After a reaction time of 3 h at ambient temperature, the reaction had reached completion. The decrease in absorbance of DPPH free radicals was read at 515 nm against ethanol as a blank using a Molecular Devices M3 microplate reader. Trolox (0, 100, 200, 300, 400, and 500 μM) was used as a standard antioxidant compound. Analysis was performed in triplicate for each sample and each concentration of standard. The antioxidant activity was reported in mmole of Trolox equivalents per gram extract (mM TE/g).

2.10 ABTS radical scavenging assay

The ABTS assay was performed as described earlier (Re et al., 1999), and is based on the reduction of ABTS^•+ radicals by antioxidants in the Aloe extracts. ABTS radical cation (ABTS^•+) was produced by reacting ABTS solution (7 mM) with 2.4 mM potassium persulfate (final concentration) and allowing the mixture to stand in the dark at room temperature for 12–16 h before use. For the assay, the ABTS^•+ solution was diluted in deionized water or ethanol to an absorbance of 0.7 (±0.02) at 734 nm. After the addition of 9 μL of the extract solutions to 271 μL of ABTS^•+ solution, the absorbance reading was taken at 30 °C 10 min after initial mixing. All determinations were carried out in triplicate. The antioxidant activity was reported in mmole of Trolox equivalents per g extract (mM TE/g).

2.11 Ferric reducing antioxidant power (FRAP) assay.

The antioxidant capacity of Aloe extracts was determined using the FRAP assay. The reduction of the colorless ferric complex (Fe³⁺ tripyridyltriazine) to a blue-colored ferrous complex (Fe²⁺−tripyridyltriazine) performed by the action of electron-donating antioxidants. The assay was performed in a 96-well microplate according the standard method (Grace et al., 2016), with minor modifications. The working FRAP reagent was freshly prepared by mixing 10 volumes of 30 mM acetate buffer, pH 3.6, with 1 vol of 10 mM TPTZ in 40 mM hydrochloric acid and with one volume of 20 mM FeCl₃. A standard curve was constructed using various concentrations of FeSO₄·7H₂O. FRAP solution (175 μL), warmed at 37 °C was added to three replicates of the sample (25 μL), whereas the same volume of acetate buffer was added to the fourth one (blank). The reaction mixture was incubated for 30 min at 37 °C, and then the absorbance was measured at 593 nm. The absorbance of the blank was subtracted from the absorbance of the samples. In this assay, the reducing capacity of the extracts tested was calculated with reference to the reaction signal given by a Fe²⁺ solution. FRAP values were expressed as mmole Fe²⁺ per g extract (mM Fe⁺²/g).

2.12 α-Glucosidase inhibitory activity

The inhibition of α-glucosidase enzyme activity was determined using the standard method (Lordan et al., 2013). Briefly, one mg of α-glucosidase from Saccharomyces cerevisiae (Sigma-Aldrich, St. Louis, MO, USA) was dissolved in 100 mL of 100 mM phosphate buffer (PB, pH 6.8). The Aloe extracts were prepared in 80% methanol (serial concentrations 10–0.08 mg/mL). To each well of a 96-well plate, 10 uL of Aloe extract or solvent (control) was mixed with 100 uL α-glucosidase working solution, and incubated at 30 °C for 5 min. 100 μL of the substrate, p-nitrophrnyl-α-D-glucopyranoside (PNPG; 1 mL in PB), was charged to each well, and absorbance was measured at 405 nm for 30 min using Molecular Devices M3 microplate reader at 30 °C (Molecular Devices Inc., Sunnyvale, CA, USA). Blank wells (sample + PB + PNPG) were subtracted from sample wells (sample + enzyme + PNPG), and results were compared to no inhibition control (enzyme + PNPG). Acarbose was used as a positive control. For each sample, the percentage inhibitory activity was calculated using the following equation: $$\%\text{inhibitory}\text{activity}=\left[{\text{A}}_{\text{control}}-\left({\text{A}}_{\text{sample}}-{\text{A}}_{\text{blank}}\right)/{\text{A}}_{\text{control}}\right]\times 100$$ where A_control is the absorbance of the uninhibited enzyme, A_sample is the absorbance of the enzyme treated with samples, and A_blank is the absorbance of the extract with no enzyme. Concentration of inhibitor that inhibited 50% of the enzyme activity (IC₅₀) was determined from the curve constructed with log concentration of sample against the percentage inhibition.

Half maximal inhibitory concentrations (IC₅₀) data was calculated after logarithmic transformation and expressed as the geometric mean with 95% confidence intervals .using GraphPad Prism V8 (GraphPAD Software, Inc., La Jolla, CA).

3.1 Total phenolics, flavonoids and proanthocyanidins

The beneficial health-promoting properties present in Aloe leaves are attributed to their phytochemical composition and in particular to characteristic polyphenols, which are one of the major groups of compounds acting as free radical terminators or primary antioxidants (Miliauskas et al., 2004).

The Aloe leaves produce two exudates; the first one is the resin or latex which comes from the cells in the outer green rind of the leaf, and the second is the transparent sap/mucilage coming from the inner parenchyma zone of the leaf, which is called the gel. Polyphenols accumulate in high amounts in the leaf rind, while polysaccharides are the major components in the leaf gel (Joseph and Raj, 2010).

The methanol extracts from APR and AVG accounted for 87.5% and 80%w/w of the dried powders, respectively. Results presented in Table 1 indicate that APR extract contained total phenolics of 205 ± 14.2 mg/g GAE (20.5 mg/100 g). The obtained value is much higher than all Aloe species investigated recently (Cardarelli et al., 2017), where the highest value reported was 14.35 g/100 g. Total flavonoids measured 24.02 ± 0.97 mg/g (2.4 mg/100 g) as catechin equivalent, and the proanthocyanidin group was the lowest phenolic class measuring only 0.95 ± 0.05 (0.09 mg/100 g) as procyanidin B1 equivalent.

The methanol extract from the Yemeni AVG contained total phenolics of 97.2 ± 12.0 mg/g; total flavonoids 9.27 ± 1.07 mg/g, and total proanthocyanidins 0.55 ± 0.005 mg/g. Several factors can affect the quality and quantity of a particular phytochemical. Phytochemical composition of plants is influenced by a variety of environmental factors including the geography, climate, soil type, sun exposure, grazing stress, seasonal changes, age of the plant, etc. (Nduche and Otaka, 2019).

3.2 HPLC quantification of anthrones

Anthrones could be distinguished from other compounds based on their UV spectra, which were further confirmed by LC-MS. HPLC of Aloe extracts was conducted at three different wavelengths, 355, 295 and 280 nm, to select the best profile showing all peaks. The HPLC profile recorded at 355 nm revealed only the anthrone peaks, while the 295 nm showed both the anthrones and chromone compounds (see Fig. 1 for chemical structures, Fig. 2 for HPLC). This enabled the distinction between anthrones from other phenolics. Quantification was performed based on peak area measurement against a standard curve constructed with the commercially available aloin A, and results were expressed as mg/g aloin A equivalent (Table 2). Total anthrones measured 677 mg/g (67.7% w/w) of APR extract. Major metabolites were the aloinosides B & A (317 mg/g), microdontins B & A (150 mg/g), homonataloin B (120 mg/g) and aloin B (88.6 mg/g). AVG extract contained total anthrones 194.9 mg/g (19.5% w/w). Aloins B & A, homonataloins B &A, aloinosides B & A, and microdontins B & A measured 64.7, 59.2, 48.7, and 23.3 mg/g, respectively (Table 2).

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Fig. 1 Structures of the main components identified in Aloe perryi resin and A. vera gel.

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Fig. 2 HPLC profiles of the methanol extracts from the Aloe perryi Baker resin and A. vera L. gel, recorded at 355 nm.

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3.3 Liquid chromatography-mass spectrometry profiling

The extracts from APR and AVG were subjected to untargeted screening by liquid chromatography-ion trap-top of flight-mass spectrometer (LC-IT-TOF-MS). All compounds were detected in the positive ion mode as [M + H]⁺. A typical total ion chromatograms (TIC) of the two Aloe extracts is shown in Fig. 3. MS² studies of the molecular ion of each compound were performed for compound characterization (see Supplimentary data). Compounds were identified based on their accurate masses, fragment masses, UV spectra, elution sequence and with the aid of literature. A total of 20 compounds with their MS, MS² and UV spectral data are presented in Table 3, listed according to their elution order.

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Fig. 3 Total Ion Chromatograms (TIC) of LC-MS of Aloe perryi resin (a) and A. vera gel (b). Refer to Table 3 for compound identification.

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Anthrones were the main group of phenolics in the investigated Aloe products. The preliminary identification of anthrones was their characteristic UV maximum absorption at 270, 300 and 355 nm. A characteristic pattern of aloin (often referred to as barbaloin) (12, 13), aloinosides (17, 18), and microdontoside (19, 20) was found in the two Aloe species with varying concentrations (see Table 2 for quantitative analysis). The presence of aloin, as a major bioactive ingredient, is known for A. barbadensis (A. vera) and is considered a taxonomic marker for this species (Zhong et al., 2015). The diglucosides aloinosides B & A, and microdontins B & A were the major metabolites in APR. Aloin B (12) was also present in APR but not aloin A (13), while both isomers were present in AVG. The occurrence of this taxonomic distribution in Aloe was found in 95% of the Aloe plants (36 species) screened in North-East Africa (Viljoen et al., 2001). This could provide a link between a species in south Yemen and its counterparts from the in African continent. The same pattern of anthrones was also identified in the latex of the Ethiopian A. sinana Reynolds (Minale et al., 2014), that supports the link between the Ethiopian Aloe species with A. perryi and A. vera from Yemen. Homonataloins B & A are another major anthrone compounds characterised and quantified in Aloe species. These two isomers were detected in only few species indigenous to south Yemen and east Africa (Viljoen et al., 2001). Dihydroisocoumarin glucoside was detected as twin peak in the extracted ion chromatogram of m/z 507 [M + H]⁺ (7, 8). It was characterized in both APR and AVG extracts. The same compound had been previously reported in A. hildebrandtii, native to north Somalia (Viljoen et al., 2001). The absolute configuration at C-3 of the compound has not been determined. In our analysis, the two conformational structures were present in AVG. The structure is closely related to the corresponding aglycone feralolide that was reported in A. vera (Lucini et al., 2015). It appears that this compound is a useful taxonomic marker among Aloe species.

Another group of phenolic compounds characteristic for Aloe is the chromones (1,4-benzopyrone derivatives, Fig. 1). Chromones were reported as the main phenolics in A. barbadences (Joseph and Raj, 2010). Chromones were present at very low levels in APR, while they were prominent in AVG extract. The most abundant chromones were aloresin A (6), isoaloresin D (11), and aloresin E (15). These results agree with the reported literature on A. vera (A. barbadenses) (Joseph and Raj, 2010).

3.4 Antioxidant activity

Aloe has developed a high reputation as a health promoting dietary component, with most activity ascribed to its antioxidant activity. Antioxidant activity was measured by DPPH, ABTS and FRAP assays. The DPPH and ABTS methods evaluate the free radical scavenging capacity of a sample, while the FRAP method evaluates the content of electron-donating species with a certain redox potential (Prior, 2015). The results of the three assays are shown in Table 1. The radical scavenging activity measured 0.09 ± 0.002 mM, and 0.06 ± 0.01 mM Trolox equivalent (APR), 0.05 mM and 0.03 mM TE (AVG), for DPPH and ABTS assays, respectively. The FRAP assay results were 20.5 ± 0.3 and 15.5 ± 0.29 mM/g FeSO₄ equivalent for APR and AVG, respectively. The antioxidant results agree with the reported literature for A. vera (Kim et al., 2014). It was previously reported that A. perryi methanolic extract had 94% radical scavenging activity when tested at 1 mg/mL, which was comparable to the activity ascorbic acid in the same experiment (Mothana et al., 2009). The antioxidant activity of anthrone compound (200 ppm) was found to inhibit the peroxidation of linoleic acid with comparable activity to the known synthetic compound BHA (butylated hydroxyanisole) as indicated previously (Yen et al., 2000).

3.5 Potential antidiabetic activity (inhibition of α-glucosidase activity)

The α-glucosidase has been recognized as a therapeutic target for the modulation of postprandial hyperglycemia, which is the earliest metabolic symptom occurring in type II diabetes. The enzyme cleaves glycosidic bonds in complex carbohydrates yielding absorbable monosaccharides. Hence, the inhibition of α-glucosidase can slow down the glucose liberation from dietary carbohydrates and delay its absorption resulting in reduced postprandial plasma glucose levels, and suppression of postprandial hyperglycemia (Kumar et al., 2011). Numerous studies have reported on the antidiabetic properties of various plant extracts through inhibition of carbohydrate-hydrolysing enzymes, as evaluated in this study (Alam et al., 2019).

The inhibitory effect of Aloe extracts against α-glucosidase was determined using p-nitrophenyl-α-D-glucopyranoside (PNPG) as a colorimetric indicators. Results indicated that the Aloin A standard reference compound showed the highest inhibitory activity (IC₅₀ 0.34 ± 0.05 mg/mL), and was higher than the positive control acarbose (IC₅₀ 0.54 ± 0.14 mg/mL), with no significant differences (P < 0.05). APR extracts showed also high inhibitory activity with IC₅₀ 0.67 ± 0.25 mg/mL, whereas, AVG was less active measuring at IC₅₀ 1.09 ± 0.14 mg/mL (Table 4 and Fig. 4). Aloin has been reported as a potent inhibitor of α-glucosidase enzyme (Ghamari et al., 2013). Therefore, the α-glucosidase inhibitory activity APR and AVG can be referred to the presence of large concentrations of aloin and its glycosides in the extracts. Moreover, the in vivo hypoglycemic property of A. perryi was investigated on diabetic albino rats through intraperitoneal injection of Aloe extract at 2 mg/Kg, which resulted in 22.5% reduction of the fasting glucose level after 54 h (Ibegbulem and Chikezie, 2013). It was also found that polysaccharides play a role in the antidiabetic activities of Aloe species by increasing the insulin level and hence showing hypoglycemic properties (Minjares-Fuentes and Femenia, 2019). Polysaccharides isolated from eight Aloe species growing in Egypt, including A. vera had been tested for their α-glucosidase inhibitory activity. Their results indicated that polysaccharides had moderate α-glucosidase inhibitory activity (El Sayed et al., 2016).

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Fig. 4 Inhibitory activity of Aloe perryi resin, A. vera gel extracts, and aloin reference standard vs acarbose as a positive control. Samples were evaluated at final concentration range of 0.8–1000 μg/mL.

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