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Chemical composition and antioxidant, α-glucosidase inhibitory and antibacterial activities of three Echeveria DC. species from Mexico
⁎Corresponding author at: Biotechnology Graduate Program, Faculty of Chemical and Biological Sciences (FCQB) of the Autonomous University of Sinaloa (UAS), University City 80010, Culiacan, Sinaloa, Mexico. Tel./fax: +52 667 713 6615. fdelgado@uas.edu.mx (Francisco Delgado-Vargas) fcodelgadovargas@gmail.com (Francisco Delgado-Vargas)
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Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Peer review under responsibility of King Saud University.
Abstract
Three Echeveria species from Sinaloa, Mexico (Echeveria craigiana, Echeveria kimnachii and Echeveria subrigida) were analyzed for their content of antioxidant compounds (β-carotene, ascorbic acid, α-tocopherol, total phenolics and flavonoids) and the in vitro antioxidant (DPPH, ABTS, ORAC and β-carotene bleaching [β-CBM]), α-glucosidase inhibitory and antibacterial activities. The studied Echeveria species showed high α-tocopherol content (2.9–9.0 mg/100 g f.w.) and total phenolics as Gallic Acid Equivalents (GAE) (152.2–400.5 mg GAE/100 g f.w.). Antioxidant activities of the three Echeveria methanol extracts (ME) were higher than those of other well-known plants with this property; the activities of E. craigiana (ABTS, 65.91 μmol ET/g f.w.) and E. subrigida (β-CBM, 79.3%) were remarkable. The Echeveria ME showed stronger α-glucosidase inhibition (IC50 25.21–50.57 μg/mL) than acarbose (IC50 3.59 mg/mL) as well as high antibacterial activity (Minimal Inhibitory Concentrations, MICs ⩽ 1 mg/mL), mainly against Gram positive bacteria. The results showed the three Echeveria species had components/biological activities with high potential for food/pharmacological uses and could be exploited by sustainable management schemes.
Keywords
Echeveria
Crassulaceae
Phenolics
Antioxidant
α-Glucosidase inhibition
Antibacterial
- asl
-
above sea level
- ABTS
-
2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt
- AAPH
-
2,2-azobis(2-amidinopropane) dihydrochloride
- BHA
-
butylated hydroxyanisole
- BHT
-
butylated hydroxytoluene
- β-CBM
-
β-carotene bleaching method
- CDD
-
chronic degenerative diseases
- CFU
-
colony forming unit
- DPPH
-
2,2-diphenyl-1-picrylhydrazyl
- f.w.
-
fresh weight basis
- GAE
-
gallic acid equivalents
- Trolox
-
6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid
- LSD
-
least significant difference
- ME
-
methanol extract
- MBC
-
minimal bactericide concentration
- MIC
-
minimal inhibitory concentration
- pNPG
-
4-nitrophenyl-α-d-glucopyranoside
- ORAC
-
oxygen radicals absorbance capacity
- PBS
-
phosphate buffer solution
- QE
-
quercetin equivalents
- TFA
-
trifluoroacetic acid
Abbreviations
1 Introduction
All over the world, plants are the main source of food, bioactive compounds (e.g. antimicrobial, antioxidant), and those used in traditional medicine are a basic source of new drugs (Conforti et al., 2008; Kennedy, 2012). Nowadays, 25% of the commercial drugs are obtained from a natural product and many of them have been used to treat chronic degenerative diseases (CDD) and infectious diseases (De Luca et al., 2012; Gulati et al., 2012); some of these pathologies are associated with oxidative stress (Ozsoy et al., 2009) and are within the main causes of mortality in the world (e.g. CDD 59.7%; diabetes 2.0%; infectious diseases 16.2%) (WHO, 2010). Up to date, treatments against these maladies are not 100% effective and bacterial resistance is aggravating the infectious diseases prognosis. Thus, new drugs are required and plants are still the main reservoir (De Luca et al., 2012).
Recently, the Crassulaceae has emerged as an interesting plant family. Plants of the genera Sedum (English common names, e.g., cat’s claw and great stonecrop plant), Kalanchoe (English common names, e.g., never die and dog’s liver plant) and Rhodiola (English common name, life plant) show several biological activities (e.g. antibacterial, antioxidant, adaptogenic, anti-inflammatory, antidiabetic) (Dimo et al., 2006; Gupta et al., 2010; Szewczyk et al., 2012; Tatsimo et al., 2012). By contrast, the genus Echeveria has been poorly studied in spite of its high number of species (>130) and its common use as a source of ornate plants. This genus is distributed in the American continent and Mexico is considered its center of diversity and endemism with 95% of the registered species (Vázquez-García et al., 2013).
Echeveria plants are traditionally used to treat a number of diseases/symptoms such as diarrhea, oral herpes, inflammation, pain, stomach aches and fever (Instituto Nacional Indigenista, 2009). Only few of these uses are supported with scientific data; it has been demonstrated the contraceptive effect of Echeveria gibbiflora (English common name, donkey ear plant) and the antimicrobial activity of Echeveria leucotricha (English common name, chenille plant) (Martínez-Ruiz et al., 2012; Reyes et al., 2005).
Our research group is engaged in the study of the agro-industrial potential of wild plants from Sinaloa/Mexico by characterizing their chemicals, nutrients and biological activities. This paper shows the content of antioxidant compounds and the in vitro biological activities (antioxidant, α-glucosidase inhibitory and antibacterial) of three Echeveria species: Echeveria kimnachii, Echeveria craigiana and Echeveria subrigida (Syn. Cotyledon subrigida Robinson & Seaton and Echeveria angusta Poelln). These plants are potential sources of active principles for food/pharmaceutical uses.
2 Materials and methods
2.1 Plant material
Echeveria plants were collected from Sinaloa, Mexico; for each species description, we show in parentheses the coordinates, the name of the collector, and the assigned number in the herbarium of the Agronomy Faculty of the Autonomous University of Sinaloa (UAS). E. craigiana E. Walther is distributed in the states of Chihuahua, Sonora and Sinaloa; it was collected in “El Zapote” community, Choix, Sinaloa (1050 m above sea level, asl; 26°46′03″N, 108°08′34″O; Vega-Aviña R.; 10816). E. kimnachii J. Meyrán & R. Vega is endemic of Sinaloa, specifically of the municipality of Culiacan; it was collected from the South of the “Estancia de los García”, Culiacan, Sinaloa (450 m asl; 24°21′45″N, 107°01′05″O; Vega-Aviña R.; 9206). E. subrigida (B. L. Rob. & Seaton) Rose is distributed in the Mexico State, Guanajuato, Queretaro and Sinaloa; it was collected near “El Palmito” town, Concordia, Sinaloa (2000 m asl; 23°34′06″N, 105°50′53″O; Vega-Aviña R.; 11742).
Leaves of the same day of collection were used for the analysis in fresh. For the other analyses, leaves were freeze dried (VirTis 25EL, VirTis Co. USA) and milled to obtain a fine flour that passed through a 40 mesh (sample moisture (%) of 95.4 ± 0.0 for E. craigiana; 96.5 ± 0.7, E. kimnachii; and 93.6 ± 0.1, E. subrigida). Dried flours of leaves were stored at −20 °C/in darkness until their use.
2.2 Bacteria
Seven human pathogen bacterial strains were used for the antibacterial assay: two ATCC (Staphylococcus aureus 29213 and Escherichia coli 25922) (DIFCO Laboratories, MI, USA) and five clinical isolates (Streptococcus group A-4, S. aureus 3, E. coli AO11, Salmonella enterica serovar Typhi and Shigella dysenteriae) provided by the Laboratory of Bacteriology of the National Institute of Pediatrics, D.F., Mexico.
2.3 Reagents and solvents
The bacteriological culture media were trypticase soy agar and Mueller Hinton broth (Becton Dickinson). The following reagents were purchased from Sigma/Aldrich (St. Louis, MO, USA): β-carotene, ascorbic acid, 4-nitrophenyl-α-d-glucopyranoside (pNPG), α-glucosidase from Saccharomyces cerevisiae, 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), trifluoroacetic acid, 6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox), Folin–Ciocalteu reagent, disodium fluorescein, 2,2-azobis(2-amidinopropane) dihydrochloride (AAPH). HPLC grade organic reagents were from Baker Inc. (Phillipsburg, NJ, USA). All reagents were of analytical grade.
2.4 Preparation of the methanol extracts
Methanol extracts (ME) of the three Echeveria species were obtained by maceration. Flours of Echeveria leaves (10 g) were extracted with methanol (1:20 w/v) for three days; the solvent was exchanged every 24 h and the methanol phases were mixed. The solvent was eliminated under vacuum (40 °C) with a rotary evaporator (BÜCHI Labortechnick AG, Switzerland), followed by removal of any residual solvent in a vacuum oven at 40 °C. The obtained ME were stored at −20 °C in darkness until their use.
2.5 Phytochemical characterization
The presence or absence of phytochemicals in the ME of the three Echeveria species were determined by tube or thin layer chromatography assays as follows: the Salkowski reaction for terpenes/sterols; the Shinoda test for flavonoids; reaction with 1.0% gelatin solution and quinine sulfate solution with FeCl3 for tannins; lather formation for saponins; yellow fluorescence by reaction with NaOH for coumarins; the Bornträger reaction for free anthracenics; the reagents of Dragendorff and of Mayer and Wagner for alkaloids; and the reagents of Baljet, Raymond–Marthoud, Keller–Killiani, Lieberman–Burchard, and Salkowski for cardiotonics (Harborne, 1973).
2.6 Quantitation of antioxidant components
2.6.1 β-Carotene
β-Carotene was assayed as reported by Valverde-Juárez et al. (2009). Echeveria flour (0.3 g de E. craigiana/E. kimnachii or 0.5 g de E. subrigida) was mixed with 2 mL of cold acetone and the mixture was sonicated (Ultrasonic FS30H, Fisher Scientific, USA) for 10 min; the solids were removed by filtration and re-extracted with cold acetone until they were uncolored. The recovered acetone extracts were concentrated under a nitrogen stream, the residue was re-suspended in 1 mL of acetone, passed through syringe filters (17 mm × 0.45 μm, PVDF, Titan), and 100 μL was analyzed with an HPLC-DAD 1100 (Agilent Technologies, USA). The separation was carried out using a YMC carotenoid column (250 mm × 4.6 mm i.d. × 5 μm) (YMC, Wilmington, USA) at 30 °C. The mobile phase contained methanol (A), methyl t-butyl ether (B) and water (C). A lineal gradient from 81% A/15% B/4% C to 18.5% A/77.5% C/4% C in 70 min was used. The flow rate was 0.7 mL/min and the eluted components were detected at 447 nm. Quantitation was carried out using a calibration curve prepared with pure β-carotene.
2.6.2 Ascorbic acid
Ascorbic acid was determined as described by Pío-León et al. (2012). Briefly, fresh Echeveria leaves were cut and 10 g was extracted with 25 mL cold water; the mixture was homogenized with a waring blender for 20 s, filtered through cheesecloth and the volume of the recovered liquid was measured. Liquid was sequentially passed through a syringe filter (17 mm, 0.45 μm PVDF, Titan) and Sep-Pak C18 cartridge. A 10 μL aliquot of the filtrate was injected to an HPLC-DAD Agilent 1100 and separated by a SPHERECLONE ODS column (250 mm × 4.6 mm i.d. × 5 μm) (Phenomenex, Torrance, CA). The mobile phase used was an aqueous solution of TFA (0.1% v/v) with a flow rate of 0.7 mL/min and detection at 254 nm. Quantitation was carried out using a calibration curve prepared with pure ascorbic acid.
2.6.3 α-Tocopherol
α-Tocopherol was determined as described by Fiorentino et al. (2009). Leave flours (100 mg) were extracted with 5 mL of hexane/ethyl acetate (17:3 v/v) added with 4 μg/mL of BHT (butylated hydroxytoluene) as antioxidant. The mixture was sonicated for 1 h and filtered through cheesecloth; the residue was re-extracted one more time and the extracts mixed. The solvent was eliminated under vacuum (40 °C) with a rotary evaporator, and the residue was re-suspended in 1 mL of hexane; the sample was passed through a syringe filter (17 mm × 0.45 μm, PVDF, Titan) and analyzed with a gas chromatograph HP6890 coupled with a mass spectrometer 5973N (Agilent Technologies, USA). Separation was done in a fused silica capillary column QUADREX 007 CARBOWAX 20 M (30 m × 0.25 mm i.d., film thickness 0.25 μm) using helium as carrier gas; the flow was 0.9 mL/min for 60.5 min. Temperature conditions were: injector port at 250 °C; initial oven temperature 60 °C for 1 min, gradient at 5 °C/min to 200 °C and then at 10 °C/min to 275 °C, and held at 275 °C for the rest of analysis. A 5 μL volume sample was injected in splitless mode. Full-scan mass spectra were collected between 15 and 800 u at 2 scan/s. The MS was operated in the electron impact (EI) ionization mode with electron energy of 70 eV. The ion source and quadrupole temperatures were maintained at 245 and 150 °C, respectively. α-Tocopherol was quantitated by using a calibration curve prepared with an authentic standard.
2.6.4 Total phenolics
Total phenolics were quantitated by the Folin–Ciocalteu method as described by Singleton and Rossi (1965) with slight modifications. Echeveria ME solutions were prepared in methanol (E. craigiana and E. kimnachii, 1.5 mg/mL; E. subrigida, 1 mg/mL). Echeveria solution or standard (0.02 mL) was mixed with distilled water (1.58 mL) and 0.1 mL of the Folin–Ciocalteu reagent. The mixture was stirred for 2 min (40 °C/darkness) and then added with 0.3 mL of a saturated solution of Na2CO3; the mixture was allowed to stand at 40 °C for 30 min and then the absorbance was measured at 765 nm (Spectronic Genesis 20, Spectronic Instruments, USA). Water was used instead of sample as negative control. Quantitation was carried out by using a calibration curve of gallic acid (0–500 μg/mL) and the results expressed as Gallic Acid Equivalents (GAE) (mg GAE/g f.w.).
2.6.5 Total flavonoids
Total flavonoids were determined for the ME of the three Echeveria species as reported by Quettier-Deleu et al. (2000). ME were dissolved in methanol (2 mg/mL) and an aliquot (1 mL) of each extract was mixed with 2% methanol solution of AlCl3⋅6H2O (1:1 v/v), allowed to stand for 1 h (25 °C/darkness) and the absorbance was measured at 415 nm. Quantitation was carried out by using a calibration curve of quercetin (0–50 μg/mL) and the results were expressed as Quercetin Equivalents (QE) (mg QE/g f.w.).
2.7 Determination of the antioxidant activity
2.7.1 DPPH method
The DPPH method measures the ability of substances to donate electrons or hydrogen atoms, which is associated with the bleaching of the purple colored solution of the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical (Brand-Williams et al., 1995). For the evaluation, Echeveria ME were dissolved in methanol (100–400 μg/mL). Trolox solutions were used as standards. A 150 mM DPPH solution in methanol (1.8 mL) was mixed with 0.2 mL of the sample, allowed to stand for 20 min (37 °C/darkness) and the absorbance was measured at 515 nm. Before sample readings, the spectrophotometer was adjusted to zero with a mixture of 0.2 mL of the sample and 1.8 mL of methanol. A calibration curve was prepared with Trolox solutions (0–100 μg/mL) processed as described for the sample and used for quantitation as Trolox Equivalents (TE) (μmol TE/g f.w.).
2.7.2 ABTS method
In the ABTS (2,2′-azinobis-3-ethylbenzotiazoline-6-sulfonic acid) method, the reagent is oxidized to its corresponding radical ABTS•+, which shows an intense coloration in solution. The antioxidant substances transfer electrons or hydrogen atoms to the radical ABTS•+ species being inactivated and measured as the loss of color (Re et al., 1999). The ABTS•+ was prepared by mixing 5 mL of 7 mM ABTS reagent and 88 μL of potassium persulfate (140 mM) and the mixture was allowed to stand for 12–16 h at 25 °C in darkness. The ABTS•+ radical was diluted with phosphate buffer solution (PBS) (7 mM, pH 7.4) to get an absorbance of 0.700 ± 0.020 at 734 nm. In assay tubes, 0.5 mL of samples diluted in methanol (E. craigiana and E. subrigida, 100 μg/mL; E. kimnachii, 300 μg/mL) was mixed with 1.95 mL of the radical solution; the mixtures were allowed to stand for 10 min at 37 °C in darkness and their absorbances were measured at 734 nm. A Trolox calibration curve was prepared in the range of 0–200 μg/mL and the antioxidant activity expressed as Trolox Equivalents (μmol TE/g f.w.).
2.7.3 ORAC (oxygen radicals absorbance capacity) method
The ORAC assay measures the ability of an antioxidant, acting by hydrogen transfer mechanism, to stabilize fluorescein attacked by peroxyl radicals generated by AAPH; this activity is associated with a decreased rate in the loss of the fluorescence over time (Prior et al., 2005). Briefly, the Echeveria ME were dissolved in methanol (10 mg/mL) and diluted 1:500 v/v with PBS (75 mM, pH 7.4). A 96-well black microplate with flat bottom was filled with 25 μL of sample or 25 μL of PBS (blank) or 25 μL of Trolox (25–125 μM) (standard curve). The microplate was placed in the fluorescence reader (Synergy 2 SL, BioTek Instruments, USA), where 200 μL of fluorescein (0.96 μM) and 75 μL of APPH (95.8 mM) were automatically added. The fluorescence intensity (485 nm (ex)/525 nm (em)) was measured for 75 min (37 °C) with 1 min intervals (Huang et al., 2002). The antioxidant capacity was determined by contrasting the area under the curve obtained with Trolox and that obtained with our sample. Results were expressed as Trolox Equivalents (μmol TE/g f.w.).
2.7.4 β-Carotene bleaching method (β-CBM)
The β-carotene bleaching method measures the ability of an antioxidant to avoid the loss of the yellow color of a β-carotene solution due to its reaction with radicals formed by linoleic acid oxidation in an emulsion at elevated temperature (Wang et al., 2006). Briefly, the reaction mixture was prepared with 50 mg of Tween 40, 6.25 μL of linoleic acid and 500 μL of β-carotene (2 mg/mL in CH2Cl2); solvent was eliminated under N2(g) and 25 mL of H2O2 was added and stirred vigorously to form an emulsion. A blank mixture was prepared with the same procedure but without β-carotene. Using 96 microwell plates, a blank was prepared by mixing 50 μL of DMSO and 250 μL of the blank mixture; 50 μL of the sample and 250 μL of the reaction mixture were used for sample evaluation; negative and positive controls contained 50 μL of DMSO or BHT (at the same concentrations used for the samples), respectively. Plates were incubated at 50 °C and the absorbance was measured at 492 nm immediately and at 120 min using a UV/Vis Microplate reader (Multiskan Bichromatic, Fisher Scientific, USA). Degradation rate (DR) was calculated using the equation DR = [ln(a/b)]/t (Uguzlar et al., 2012); where “a” is the absorbance at initial time, “b” is the absorbance at 120 min and “t” is the time in minutes. Antioxidant activity (AA) was expressed as percent of inhibition relative to the control, using the equation %AA = [(DRcontrol − DRsample)/DRcontrol] × 100; where DRcontrol is the degradation rate of the control, DRsample is the degradation rate of the extract or of the antioxidant used as standard.
2.8 α-Glucosidase inhibition
The α-glucosidase inhibition was measured as reported by da Silva Pinto et al. (2008) with slight modifications. In a 96-well microplate, sample aliquots (50 μL) at different concentrations were mixed with 100 μL of α-glucosidase (0.5 U/mL) in phosphate buffer (0.1 M, pH 6.9); the microplate was incubated for 10 min at 37 °C (Stat Fax-2200, Awareness Technology, USA) and 50 μL of p-nitrophenyl-α-d-glucopyranoside in phosphate buffer (5 mM) was added to each well. Microplate was incubated (37 °C/10 min) and the absorbance was measured with a Multiskan Bichromatic Microplate reader (Fisher Scientific, USA). A solution without sample and a solution without substrate were used as blanks. Acarbose was used as positive control. The assay was carried out by triplicate and the percentage of α-glucosidase inhibition was calculated using the equation (%) = [(A − B)/A] 100; where A is the control absorbance (reaction mixture without sample) and B is the absorbance of the reaction mixture with the sample/standard.
2.9 Antibacterial activity
Minimal inhibitory concentration (MIC) and minimal bactericide concentration (MBC) were determined by the microdilution assay in 96-well plates and plate assay, respectively (Cos et al., 2006). Strains were cultured in Petri dishes (TSA, trypticase soy agar) at 37 °C for 18–20 h; 2–5 colonies were suspended in 1 mL of 0.85% NaCl (w/v) and the density adjusted to 108 CFU/mL (0.5 McFarland value). Bacterial culture was diluted to 106 CFU/mL and 50 μL was deposited per well plus 50 μL of extract at each concentration. Gentamicin (0.125–8 μg/mL) was used as positive control, microorganisms without additives as negative control and the bacterial toxicity of solvents was also evaluated. The 96-well microplates were incubated at 37 °C for 18–20 h. After incubation, MIC corresponded to the concentration of the first well without turbidity or button formation; whereas for MBC determination, a sample of every well without growth was inoculated in a Petri dish with MacConkey or blood agar for Gram negative or Gram positive bacteria, respectively. Plates were incubated (37 °C/18–20 h) and the plate without growth obtained from the lowest extract concentration was selected as the MBC value.
2.10 Statistical analysis
One way analysis of variance and multiple contrast of means using the Fisher test (LSD, α = 0.05) were carried out by the STATGRAPHICS Centurion XVI software (Statpoint Inc., Warrenton, VA, USA). Data were analyzed for Pearson’s correlation using the PASW Statistics v. 18 software (SSPS Inc., USA). The IC50 values of α-glucosidase were calculated with the GraphPad Prism v. 5.03 software (GraphPad Software Inc. USA). For every experiment, evaluations were done at least by triplicate.
3 Results and discussion
3.1 Echeveria phytochemicals
The three Echeveria species showed six out of the eight analyzed family compounds (Table 1); alkaloids and cardiotonics were absent. The highest diversity of compounds was observed in E. craigiana, followed by E. kimnachii and E. subrigida. Terpenes/sterols, saponins, free anthracenics and tannins were more abundant in E. kimnachii, while the presence of coumarins and tannins was stronger in E. subrigida (Table 1). The chloroform extract of E. leucotricha was reported previously to contain triterpenes, flavonoids, cardiotonics, lactones, alkaloids, saponins, anthraquinones and tannins (Martínez-Ruiz et al., 2012); the composition of this extract was similar to those of our extracts and the differences found may be associated with the use of different extraction solvents. Alkaloids were absent in the three Echeveria species analyzed (Table 1), contrasting with its presence in Echeveria venezuelensis (Stevens et al., 1995).
Phytochemical
Methanol extract of Echeveriaa
craigiana
kimnachii
subrigida
Yield (% d.w.)
32.3 ± 0.8
40.3 ± 1.2
20.7 ± 0.9
Terpenes/sterols
+
++
−
Flavonoids
+
−
+
Tannins
+
+++
+++
Saponins
+
++
−
Coumarins
+
+
++
Free anthracenics
+
++
−
Alkaloids
−
−
−
Cardiotonics
−
−
−
3.2 Antioxidant compounds in species of Echeveria
E. subrigida showed the highest content of three out of the five analyzed components (i.e. ascorbic acid, total phenolics and α-tocopherol), while E. kimnachii and E. craigiana showed the highest content of β-carotene and total flavonoids, respectively (Table 2). These components have not been previously reported in Echeveria spp. However, Kalanchoe diagremontiana (another Crassulaceae) showed up to six times higher content of β-carotene (2.6 mg/100 g f.w.) (Anisimov et al., 2009) and up to 3.6 times lower values of α-tocopherol (1.7–2.5 mg/100 g f.w.) (Kruk et al., 2011) than the three studied Echeveria species (Table 2). E. subrigida showed two times more total phenolics than Kalanchoe pinnata (149.3 mg GAE/g ME) (Sharker et al., 2012).
Echeveria species
β-Carotene
Ascorbic acid
α-Tocopherol
Total phenolicsb
Total flavonoidsc
mg/100 g f.w.
mg GAE/100 g f.w. (mg GAE/g ME)
mg QE/100 g f.w. (mg QE/g ME)
E. craigiana
0.31 ± 0.02x
10.4 ± 1.4x
2.9 ± 0.5x
152.2 ± 7.0x
36.4 ± 1.3x
(102.1 ± 4.7)
(24.4 ± 0.9)
E. kimnachii
0.43 ± 0.02y
10.3 ± 1.2x
7.6 ± 0.8y
153.7 ± 12.3x
17.9 ± 1.8y
(108.8 ± 8.7)
(12.7 ± 1.3)
E. subrigida
0.043 ± 0.002z
16.7 ± 1.6y
9.0 ± 1.3y
400.5 ± 9.6y
31.6 ± 3.4z
(302.8 ± 7.3)
(23.9 ± 2.6)
Contrasting our results with those obtained for the aqueous extract of Aloe vera, a Crassulacean Acid Metabolism Plant (CAM) as the Echeveria spp., the A. vera extract showed higher content of ascorbic acid (42.2 mg/100 g f.w.) and β-carotene (1.5 mg/100 g f.w.) and lower content of α-tocopherol (0.147 mg/100 g f.w.), total phenolics (24.1 mg GAE/100 g f.w.) and flavonoids as Catechin Equivalents (CE) (24.5 mg CE/100 g f.w.) (Ozsoy et al., 2009).
Contrasting our results with those of other vegetables, the β-carotene contents of the three Echeveria species were similar to those reported for broccoli (Brassica oleracea) (0.414 mg/100 g f.w.), green asparagus (Asparagus officinalis) (0.320 mg/100 g f.w.) and artichokes (Cynara scolymus L.) (0.047 mg/100 g f.w.) (Beltrán et al., 2012), whereas the ascorbic acid contents were similar to those reported for some fruits such as Andean blackberry (Rubus glaucus Berth) (10–11 mg/100 g f.w.), grenadia (Passiflora ligularis L.) (16–25 mg/100 g f.w.), and naranjilla (Solanum quitoense Lam.) (11–13 mg/100 g f.w.) (Vasco et al., 2008). On the other hand, except for E. craigiana, the α-tocopherol contents of the Echeveria species were similar to foods rich in this nutrient: e.g. maize (8.1 mg/100 g f.w.) and peanuts (8.2 mg/100 g f.w.) (Wyatt et al., 1998). Total flavonoid contents of the studied Echeveria species were similar to those found in broccoli (B. oleracea L var botrytis L subvar cymosa) (31.6 mg QE/100 g f.w.) and cabbage (B. oleracea L var botrytis L) (17.2 mg QE/100 g f.w.) (Bahorun et al., 2004). The total phenolics of the three Echeveria species were high and similar to those reported for bitter melon (Momordica charantia L.) (143.6 mg GAE/100 g f.w.), and highbush blueberry (Vaccinium corymbosum L. Hybrids) (399.3 mg GAE/100 g f.w.) (Lin and Tang, 2007; Sellappan et al., 2002).
3.3 Antioxidant activity
The highest antioxidant activities of the ME were for E. craigiana (DPPH and ABTS) and E. subrigida (ORAC and β-CBM). The activity of E. kimnachii by the β-CBM was similar to that of E. subrigida (Table 3). ABTS values were about two times higher than DPPH values, perhaps by the additive effect of hydrophilic and lipophilic components detected by the ABTS method (Floegel et al., 2011). The pertinence of using several antioxidant methods is supported by the differences found in the activities evaluated by ABTS, DPPH, ORAC and β-CBM, which differ in some chemical principles and capability to detect antioxidants of different polarity.
Echeveria species
Antioxidant activity method
DPPH
ABTS
ORAC
β-Carotene bleaching
μmol TE/g f.w. b
% AAc
E. craigiana
26.19 ± 0.24x
65.91 ± 2.58x
32.52 ± 1.48x
68.5 ± 1.2x
E. kimnachii
12.01 ± 0.63y
23.36 ± 0.14y
32.25 ± 1.91x
76.7 ± 1.5y
E. subrigida
24.83 ± 0.88z
51.58 ± 1.16z
37.58 ± 2.63y
79.3 ± 2.3y
The antioxidant activities of the Echeveria ME were similar to those of fruits recognized by their antioxidant capacity such as berries (Paredes-López et al., 2010). The ABTS values for the three Echeveria species were higher than those of highbush blueberry (V. corymbosum) (14.0 μmol TE/g f.w.) and strawberry (Fragaria x. ananassa) (11.9 μmol TE/g f.w.); whereas DPPH and ORAC results were similar to those of raspberry (Rubus idaeus) (25.3 μmol TE/g f.w.) and lingonberry (Vitis vitis-idea) (31.8 μmol TE/g f.w.), respectively (Paredes-López et al., 2010).
The ME of the three Echeveria species showed high activity by the β-CBM, inactivating the free radicals derived from linoleic acid oxidation. The antioxidant values were slightly lower than those obtained for the commercial antioxidant BHT (86.6%) and raspberry (R. idaeus) (86.0%, BHA 88.1%), and higher than that of highbush blueberry (V. corymbosum) (19.5%, BHT 83%) (Rodrigues et al., 2011; Tosun et al., 2009). Thus, E. subrigida and E. kimnachii are species with high antioxidant capacities (>70%) (Hassimotto et al., 2005).
Total antioxidant activities of the analyzed samples are associated with the combined effect of phytochemicals acting by additive or synergic effects. Some phytochemicals, such as phenolics, show a higher contribution and their content correlate positively with the antioxidant activity (Teow et al., 2007). For the studied Echeveria species, antioxidant activities were correlated with hydrophilic components for the DPPH and ABTS (flavonoids) and ORAC (phenolics and ascorbic acid), as well as with lipophilics for the β-CBM (α-tocopherol) (Table 4). However, the antioxidant activities of the different methods did not correlate (P ⩾ 0.16); the highest correlation value was between DPPH and ABTS methods (r = 0.97, P = 0.16).
Echeveria component
Evaluation method of the antioxidant activity
DPPH
ABTS
ORAC
β-CBM
β-Carotene
−0.66
−0.47
−0.97
−0.44
(0.53)
(-0.69)
(0.16)
(0.71)
Ascorbic acid
0.42
0.20
1.00
0.68
(0.72)
(0.88)
(0.02)
(0.52)
α-Tocopherol
−0.38
−0.60
0.64
1.00
(0.75)
(0.59)
(0.56)
(0.01)
Total phenolics
0.42
0.18
1.00
0.69
(0.73)
(0.89)
(0.03)
(0.51)
Total flavonoids
0.99
1.00
0.31
-0.52
(0.11)
(0.05)
(0.8)
(0.65)
The correlation between antioxidant activity and the content of total phenolics and ascorbic acid is commonly reported (Du et al., 2009; Velioglu et al., 1998). Moreover, the lack of correlation between these components and the β-CBM was also reported by Hassimotto et al. (2005). Interestingly, the correlation between the content of α-tocopherol and antioxidant activity by the β-CBM has not been previously reported; our results supported that α-tocopherol (lipophilic) must be better antioxidant in lipophilic media and consequently better detected by the β-CBM; tocopherols, BHT and BHA are better antioxidants than phenolic acids and flavonoids by this method (Ouchikh et al., 2011; Von Gadow et al., 1997).
The results indicate that the evaluated Echeveria species are excellent sources of natural antioxidants, mainly phenolics and α-tocopherol, and they could be exploited for food and pharmaceutical uses.
3.4 α-Glucosidase inhibition
The inhibition of digestive enzymes (e.g. α-amylase and α-glucosidase) is suggested as an efficient strategy to control the blood glucose levels; consequently, these inhibitors are potential antidiabetic agents (De Luca et al., 2012; Gulati et al., 2012). Remarkably, the ME of the studied Echeveria species showed 63–143 times better α-glucosidase inhibition values than the commercial drug acarbose (IC50 3.59 ± 1.03 mg/mL) with the following values and order E. subrigida (IC50 25.21 ± 3.66 μg/mL) > E. craigiana (IC50 56.67 ± 4.2 μg/mL) > E. kimnachii (IC50 50.57 ± 1.48 μg/mL). This is the first report of β-glucosidase activity of any Echeveria spp.
The Crassulaceae genera Sedum and Kalanchoe are traditionally used against diabetes. The antidiabetic activity of Sedum dendroideum (Spanish common name, e.g., siempreviva) and K. pinnata (Lam.) Pers (Spanish common name, e.g., tronador) has been demonstrated in a mouse model and associated with sedoheptulose and flavonoids, respectively (Andrade-Cetto and Heinrich, 2005). Moreover, the number of papers registering α-glucosidase inhibitory activity of plant extracts has recently increased. In general, the best extracts show IC50 values two times higher than acarbose and lower values were only registered for pure compounds derived from such extracts (Ali et al., 2013; Kang et al., 2012; Moradi-Afrapoli et al., 2012); however, hydroalcoholic extracts of two plants from Benin used to treat diabetes show better activity than acarbose (IC50 726 μg/mL) (Polygonum senegalensis, 1.5 μg/mL; Pseudocedrela kotschyi, 5 μg/mL) (Moradi-Afrapoli et al., 2012), as well as the aqueous extract of Brickellia cavanillesii (IC50, extract 0.169 mg/mL and acarbose 1.12 mg/mL) (Escandón-Rivera et al., 2012). Remarkably, the ME of the three Echeveria species used in the present study were more active than acarbose and these plants could be promising sources of hypoglycemic compounds. E. subrigida showed the highest content of total phenolics (Table 2) which could be associated with its highest inhibition of α-glucosidase, but further studies are required to elucidate whether or not phenolics are responsible for this activity.
3.5 Antibacterial assay
The order for the antibacterial activity of the studied Echeveria ME were E. subrigida > E. kimnachii > E. craigiana; all Gram positive strains were inhibited being S. aureus the most sensitive. The antibacterial activity of the ME of the three Echeveria species was only observed for two out of the four evaluated Gram negative strains (Table 5). The gentamicin MIC values for the ATCC strains corresponded with those reported previously (Lennette et al., 1987). The three Echeveria species ME were not bactericide at the evaluated concentrations (⩽1000 μg/mL). “–” Stands for no activity up to such concentration.
Microorganisms
MIC (μg/mL)a
E. craigiana
E. kimnachii
E. subrigida
Gentamicin
Gram-positive
Staphylococcus aureus ATCC 29213
500
125
62.5
1
Streptococcus group A-4
1000
1000
125
0.5
Staphylococcus aureus 3
125
31.25
15.63
0.5
Gram-negative
Escherichia coli ATCC 25922
–
–
–
1
Escherichia coli AO11
250
125
62.5
0.5
Salmonella Typhi
–
–
–
1
Shigella dysenteriae
1000
1000
125
0.5
Gram positive bacteria are usually more sensitive to plant extracts than Gram negative strains as observed in this report; this phenomenon has been associated with the cell wall structural differences between these two groups of bacteria (Sharma et al., 2012).
S. aureus is a common nosocomial infectious agent and a continuous public health concern because of its ability to acquire resistance; e.g. methicillin-resistant S. aureus (MRSA) shows resistance against most of the commercial antibiotics including some developed more recently such as vancomycin, linezolid (oxazolidinone derivative) and streptogramin quinupristin/dalfopristin mixture (Gibbons, 2004), emphasizing the importance of discovering new antibacterial agents. The studied Echeveria ME were active against S. aureus as it was previously registered for the ME obtained from the aerial parts of E. leucotricha (MIC = 20 μg/mL vs. methicillin resistant strain); this MIC value was similar to those obtained in the present study for the ME of E. kimnachii and E. subrigida against S. aureus 3 (Table 5). The ME of Bryophyllum pinnatum (Crassulaceae) leaves were active against Gram positive bacteria, particularly for S. aureus (ATCC 25213 and an isolated strain) (MIC = 32 mg/mL) (Akinsulire et al., 2007), but our extracts were much better. In another study, the ethanol extract of B. pinnatum showed activity against S. aureus ATCC 25922 (MIC 256 μg/mL) and S. enterica serovar Typhi ATCC 6539 (64 μg/mL) (Tatsimo et al., 2012); the MIC values of the Echeveria ME against the S. aureus control strain (62.5–500 μg/mL) corresponded with the value reported for the B. pinnatum ME.
The antibacterial activity of the B. pinnatum was associated with kaempferol type compounds (MIC = 1–256 μg/mL) (Tatsimo et al., 2012). Our phytochemical results suggested that phenolics, tannins and coumarins were associated with the antibacterial activity, as these compounds have showed this activity (Martínez-Ruiz et al., 2012; Sharma et al., 2012).
Considering the low MIC values obtained for the three Echeveria species ME against S. aureus (15.63–500 μg/mL) and in particular of E. subrigida against every sensitive strain (15.63–125 μg/mL), values which are much lower than the reference one of 1000 μg/mL, these plants could be a source of clinical relevant antibacterial compounds (Gibbons, 2004).
4 Conclusions
We are reporting for the first time that three Echeveria species (E. kimnachii, E. craigiana and E. subrigida) are good sources of antioxidants (e.g. α-tocopherol and phenolics) and that their methanol extracts show several biological activities (i.e. antioxidant, α-glucosidase inhibition and antibacterial). Thus, preparations or compounds of these plants could be useful for the prevention/treatment of some of the most important causes of morbidity and mortality (i.e. infectious and chronic degenerative diseases, and particularly diabetes), and future studies are warranted to support such uses.
Acknowledgments
Authors acknowledge the financial support of CONACYT and PROFAPI–“Universidad Autónoma de Sinaloa” (UAS); the technical assistance of Juan Fernando Pío-León (UAS), Laura Angulo Contreras and José Basilio Heredia of the “Centro de Investigación en Alimentación y Desarrollo de Culiacán”; as well as the writing assistance of José Ángel López-Valenzuela (UAS).
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