Abstract
BACKGROUND:
Sustained hyperglycemia leads to multiple health complications associated with oxidative balance and metabolic pathways alterations. Current treatments for hyperglycemia are not entirely effective thus, the identification of natural products from food sources, such as Ugni molinae berries, to reduce hyperglycemia and prevent the deleterious effect of oxidative stress is attractive to develop new therapeutics.
OBJECTIVE:
Our aim was to evaluate the antioxidant capacity and the inhibitory activity on α-glucosidase and glycogen phosphorylase A of polyphenolic extracts from different genotypes of U. molinae berries and to comparatively analyze their polyphenolic profile.
METHODS:
Berry extraction was performed by exhaustive maceration with increasing-polarity solvents. The antioxidant capacity, and inhibitory activity on enzymes were analyzed by different spectrophotometric methods. Moreover, the chemical profile of bioactive extracts was comparatively evaluated through LC-MS.
RESULTS:
16 semi-purified extracts were obtained and showed antioxidant capacity and inhibitory activity on the evaluated enzymes. Moreover, the chemical analysis showed differences in phenolic profile among the extracts and, particularly, the acetonic extracts were more concentrated in phenolic compounds, which is associated with the more potent activities.
CONCLUSIONS:
Our results indicate that murtilla berries’ acetonic extracts include a mixture of phenolic compounds that inhibit the activity of two enzymes related to carbohydrate metabolism and have a promising antioxidant capacity.
Introduction
Sustained hyperglycemia is a common clinical feature in people suffering from pathological conditions such as insulin resistance (IR), diabetes mellitus (DM), or metabolic syndrome (MS). Different treatments for these diseases are available, but they are not entirely effective. IR, DM, and MS are characterized by damage at the cellular level and over different signaling pathways due to the formation of advanced glycation end products (AGEs), oxidative stress, and activation of protein kinase C isoforms. Taken together, these events could be responsible for the complications observed in DM [1–3].
Different strategies have been proposed to counteract the deleterious effects of sustained hyperglycemia. Among them, the antioxidant activity, regulation of the inflammatory response, and modulation of enzymes that participate in carbohydrate metabolism have been described [4, 5]. One enzyme that participates in carbohydrate metabolism is α-glucosidase. This enzyme is localized in the surface membrane of the microvilli of enterocytes, and catalyzes the hydrolysis of complex carbohydrates and disaccharides to absorbable monosaccharides, thus causing an increase in postprandial glycemic levels. Another example is glycogen phosphorylase A (GPa) which is localized in the liver, muscle, and brain. This enzyme is responsible for the release of glucose from glycogen, depending on energy requirements. Consequently, the inhibition of the catalytic activity of α-glucosidase decreases the influx of glucose from the intestinal tract, resulting in a decrease in postprandial glycemia [6, 7]. On the other hand, inhibiting the activity of the hepatic isoform of GPa would reduce the production and release of glucose into the blood [8, 9].
Therefore, there is an opportunity for new multitarget therapeutic alternatives that complement conventional therapies, particularly for those that manifest both hypoglycemic and antioxidant properties. Thus, the search for bioactive compounds in extracts obtained from medicinal plants is a promising alternative [10, 11].
Multiple studies for hypoglycemic or antihyperglycemic properties in traditional medicine, among which leaves and berries of the native Chilean species Ugni molinae Turcz, Myrtaceae, popularly known as murtilla, has been described. U. molinae is a perennial shrub that produces globose, fleshy, sweet, and aromatic berries with many seeds. Murtilla berries have a great nutritional value and are consumed fresh or in preparations such as jams, syrups, desserts, and liqueurs. Moreover, murtilla leaves have been used by traditional medicine for the treatment of diarrhea and dysentery, as well as for treating different types of pain [12–14].
Since 1996, the Instituto de Investigaciones Agropecuarias (INIA) has domesticated this species, genotyping different ecotypes and developing a germplasm bank and controlled crops to enable its commercial exploitation. Furthermore, at INIA’s Carillanca experimental station, the different murtilla genotypes are cultivated under the same edaphoclimatic conditions. Overall, this work by INIA led to the patenting of 2 commercial varieties: the genotypes 19-1 and 27-1, identified as the Red Pearl INIA and the South Pearl INIA, respectively [14, 15].
Previous studies with extracts obtained from murtilla leaves have shown anti-inflammatory, antimicrobial, wound healing, antioxidant and inhibitory activities on enzymes such as α-amylase and α-glucosidase, mainly due to the presence of polyphenols and terpenoids derivatives [12, 16–25].
Moreover, studies with murtilla berries have reported the presence of pectic substances, phenolic acids, flavonoids, and anthocyanins, as well as antimicrobial, antioxidant, and inhibitory effects on α-amylase and α-glucosidase enzymes. However, except for the work by Alfaro et al. (2013) [26], who used three INIA genotypes (14-4, 19-1, 27-1) in antioxidant capacity studies, all other investigations have been carried out with wild specimens [7, 25–34]. Furthermore, our group recently published a study about the neuroprotective properties of semi-purified acetonic and ethanolic acid extracts from murtilla berries in a cellular model of Huntington’s disease. Additionally, in this work, the total phenolic content (TPC) for acetonic and ethanolic acid extracts were determined, including the chemical profile of selected ethanolic acid extracts [35].
Nevertheless, the antioxidant properties and the modulation of the activity of enzymes involved in carbohydrate metabolism of the murtilla berries have not yet been explored.
This work aimed to determine the in vitro antioxidant capacity through the DPPH and ORAC assays, as well as the inhibitory potency on the activity of the α-glucosidase and glycogen phosphorylase A enzymes of acetone and ethanolic acid extracts obtained from berries of 8 INIA genotypes of U. molinae. These genotypes were grown under the same edaphoclimatic conditions and agronomical management, which allows to correlate the phenolic composition of the extracts with the aforementioned pharmacological activities and to identify the most active genotypes. Thus, our study will contribute to define the potential of murtilla extracts for the management of diseases as DM and to promote the cultivation of murtilla not only by the agronomic and commercial value of its fruits but also by its pharmacological properties.
Methodology
Materials
Chemicals
Dichloromethane, acetone, ethanol, acetic acid, methanol, acetonitrile, dimethyl sulfoxide, sodium hydroxide, potassium chloride, magnesium chloride, and quercetin were purchased from Merck S.A (Darmstadt, Germany). Sodium phosphate, sodium bisphosphate, sodium phosphate monobasic, ammonium molybdate, malachite green, AAPH (2,2-azobis (2-methylpropionamidine) dihydrochloride), fluorescein (as disodium salt), pyrogallol red, 6-hydroxy-2,5,7,8-tetramethyl-chroman-2-carboxylic acid (Trolox), 2,2-diphenyl-1-picrylhydrazyl (DPPH), acarbose, p-nitrophenyl-D-A-glucopyranoside, α-glucosidase, N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES), Ethylene-bis (oxyethylenenitrilo) tetraacetic acid tetrasodium (EGTA), α-D-Glucose 1-phosphate, caffeine, glycogen, and glycogen phosphorylase A (GPa) were purchased from Sigma-Aldrich (Chile). Milli-Q water was used for dilutions in all measurements.
Plant material
In April 2016, ripe fruits of 8 different genotypes of murtilla (U. molinae Turcz, Myrtaceae), grown under the same edaphoclimatic conditions, were collected at the Carillanca experimental station of the Instituto de Investigaciones Agropecuarias (INIA Carillanca) located in Tranapuente, Temuco, Chile (38°41’29.6’S; 73°21’13.3’W). The genotypes were selected based on the agro-commercial quality of their fruits informed by INIA. The berries were ripe at the time of harvest, according to the information reported by INIA Carillanca and what is described in the literature for the geographical area where the crops are located [36]. The collected fruits were lyophilized, milled, and stored at –20°C until extraction was performed.
A sample of each genotype was stored in the Herbarium of the Facultad de Ciencias Químicas y Farmacéuticas of the Universidad de Chile. The number of accessions corresponds to 14-4 (SQF-22549), 19-1 (SQF-22554), 19-1ha (SQF-22553), 19-2 (SQF-22557), 22-1 (SQF-22552), 23-2 (SQF-22556), 27-1 (SQF-22555) and 31-1 (SQF-22551). It should be noted that among the accessions are the varieties registered by INIA, Red Pearl (19-1), and South Pearl (27-1).
Preparation of extracts
In October 2016, 200 g of lyophilized and milled ripe fruit of each genotype were subjected to a seriated extraction process by simple maceration until complete exhaustion with 1000 mL of the following solvents: dichloromethane, acetone, and a mixture of ethanol: acetic acid 1 %, representing a plant material: solvent ratio of 1:5. The extraction process was performed at room temperature and protected from light.
The obtained extracts were concentrated under reduced pressure and dried in a vacuum oven, obtaining the dry extracts of dichloromethane (DCEs), acetone (ACEs), and acid ethanol (AEEs). The dry extracts were stored at –20°C until their use. Considering the polarity of the used solvents, ACEs and AEEs were selected for this study as these extracts concentrate the polyphenolic compounds found in the murtilla berries.
Determination of antioxidant capacity
Reduction of the 2,2-diphenyl-1-Picrylhydrazyl radical
The antioxidant capacity against the 2,2 diphenyl-1-picrylhydrazyl radical (DPPH) assay was adapted with slight modifications from Castro et al. (2014) [37]. The assay was performed in 96-well plates, in a room protected from direct light, using the Thermo Scientific® Multiskan® GO 3.2 spectrophotometer. Briefly, extracts were dissolved in methanol at 200 ppm for the ACEs and 500 ppm for the AEEs. Different dilutions were prepared in a range of 10 to 100 ppm for the ACEs and a range of 25 to 500 ppm for the AEEs. Then, 25μL of each extract dilution was mixed with 235μL of methanolic DPPH solution at 40μg/mL. The mixture was subjected to successive readings every 10 seconds for 90 minutes, at 20°C and a wavelength of 517 nm. The absorbance decay curves were plotted for each extract concentration and compared against methanol as a blank. The areas under the curve (AUC) were obtained and the percentages (%) of remaining DPPH were calculated as follows = (AUCSample / AUCBlank) x 100. The concentrations of each extract were plotted against the remaining percentage of DPPH to calculate the effective concentration 50 (EC50). Quercetin (reference standard) was tested under the same conditions previously described [37].
ORAC assay
The antioxidant capacity against the peroxyl radical (ROO·) was carried out in the Synergy HT equipment from Bio-Tek Instruments, Inc, controlled by Gen 5 software. This assay was used to determine the ORAC values for total antioxidant capacity (TAC) from extracts of dry and milled fruit (as described in 2.3) obtained with a mixture of acetone: water: acetic acid (70:29.5:0.5 v/v/v, AWA) at 40 mg/mL, and to determine the ORAC values for methanolic solutions of ACEs and AEEs at 10 mg/mL, diluted with phosphate buffer pH 7.4 [22, 37–39].
2.3.2.1. ORAC-FL assay. 96-well white polystyrene microplates were used to evaluate the fluorescence from the top with an excitation wavelength of 485/20 nm and with an emission filter of 528/20 nm. The reaction was carried out in 75 mM sodium phosphate buffered medium (pH 7.4) at a final volume of 200μL. 150μL of fluorescein (FL) at 48 nM and 25μL of extract or standard were added to each well. The mixture was pre-incubated for 7 minutes at 40° C, before adding 25μL a solution of 2,2-azobis (2-methylpropionamidine) dihydrochloride (AAPH) as a source of peroxyl radical, at a final concentration of 18 mM. Fluorescence decay was recorded every 1 minute for 120 minutes. As a reference standard, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) was used to elaborate a calibration curve. The blank corresponded to a mixture of FL and AAPH, replacing the antioxidant solution (extract or standard) with phosphate buffer.
The fluorescence decay curves were plotted against time to calculate the area under the curve (AUC) of the extract, standard and blank, with F0 being fluorescence at 0 minutes and F being fluorescence at different times. The corrected AUC was obtained as follows = AUCSample - AUCBlank. The corrected AUC values of each extract were interpolated in the Trolox calibration curve (Y = 0.124X+103.34, R2 = 0.997), expressing the antioxidant capacity as ORAC values of Trolox equivalents (TE in μmol/L) per mg of dry fruits (DF) or dry extract (DE).
2.3.2.2. ORAC-PGR assay. 96-well polystyrene microplates were used to evaluate the decay of pyrogallol red (PGR) absorbance at 540 nm. The reaction was carried out in 75 mM sodium phosphate buffered medium (pH 7.4) at a final volume of 200μL. 150μL of pyrogallol red (PGR) at 5.0μM and 25μL of extract or standard were added to each well. The mixture was pre-incubated for 7 minutes at 40° C, before adding 25μL of AAPH at 10 mM. Subsequently, the consumption of the probe molecule was recorded by the progressive decay of the absorbance every 1 minute for 120 minutes.
The ORAC-PGR values were calculated as described for ORAC-FL, interpolating the corrected AUC values of each extract in the Trolox calibration curve (Y = 3.1271X –22.064, R2 = 0.997), expressing the antioxidant capacity as ORAC values of Trolox equivalents (TE in μmol/L) per mg of dry fruits (DF) or dry extract (DE).
Enzyme activity inhibition assay
The in vitro inhibitory potency of the murtilla extracts on the enzymatic activity of α-glucosidase and glycogen phosphorylase A (GPa) was analyzed through spectrophotometric assays in 96-well plates, using the Thermo Scientific® Multiskan® GO 3.2 equipment.
α-glucosidase assay
The ACEs, AEEs, and acarbose (positive control) were dissolved in 11.5 % dimethyl sulfoxide (DMSO) at 1.15 mg/mL. Different dilutions were obtained from each extract, or acarbose solution to carry out the assay. The reaction was carried out at a final volume of 230μL. 120μL of phosphate buffer (pH 6.8) at 100 mM, 20μL of extract, positive control or blank (11.5 % DMSO), and 60μL of p-nitrophenyl-α-D-glucopyranoside (pNPG) at 5 mM were added to each well. The mixture was incubated for 15 minutes at 37°C before adding 30μL of an enzymatic solution of α-glucosidase, from Saccharomyces cerevisiae at 0.1 IU/mL.
Immediately after starting the reaction, the absorbances of each sample were recorded at 400 nm (Time 0), and then, the samples were incubated for 30 minutes at 37° C, the period in which the pNPG released a yellowish p-nitrophenol group. The absorbances of each sample were recorded at 400 nm for a second time (Time 30). Along with the extracts, a non-enzymatic control (without enzyme), a negative control (without extract), and a blank (without extract and enzyme) were evaluated. For the determination of the enzyme inhibition (%), the absorbance was considered between the difference in readings time 30 and 0. The % inhibition of the samples were calculated as follows = [(NCA - BA) - (SA - NECA)] x 100 / (NCA - SA), where: NCA = Negative control absorbance; BA = Blank absorbance; SA = sample absorbance (extracts or standard); NECA = Non-enzymatic control absorbance. Furthermore, the obtained % inhibition were plotted against the logarithm of the concentration of each extract tested to obtain the inhibitory concentration 50 (IC50). The IC50 values of the extracts were compared to each other and to the IC50 value of the acarbose [6].
Glycogen phosphorylase A (GPa) assay
The inhibitory activity on the GPa enzyme (from rabbit muscle) was determined in the direction of glycogen synthesis through the release of phosphate from glucose-1-phosphate at 22° C. The reaction was carried out at a final volume of 300μL with a solution of DMSO at 14 % in the saline buffer that included 50 mM HEPES, 100 mM KCl, 2.5 mM EGTA, and 2.5 mM MgCl2, a solution of glucose 1-phosphate (G-1-P) at 0.25 mM in 50 mM Hepes buffer, a solution of glycogen at 1 mg/mL, and a solution of glycogen phosphorylase A at 60μg/mL. The ACEs, AEEs, and caffeine (positive control) were dissolved in saline buffer at 1.5 mg/mL. Different dilutions of the extracts or caffeine were used to carry out the assay. 60μL of extract dilution, positive control or blank (DMSO in saline buffer), 35μL of glycogen, 35μL of G-1-P, and 20μL of glycogen phosphorylase A were added to each well. The mixture was incubated for 20 minutes at 22°C, before adding 150μL of stopping solution composed of ammonium molybdate at 10 mg/mL and malachite green at 0.38 mg/mL in acid medium. The mixture was incubated for 5 minutes at 22°C, the period in which the G-1-P released phosphate, generating a green-colored adduct. Then, the absorbances of each well were recorded at 621 nm. IC50 calculations and comparison of the IC50 values for each extract were performed as described for α-glucosidase, but the absorbances were only recorded at the end of the assay [40].
Determination of the phenolic compounds profile in extracts with the highest antioxidant capacity and inhibitory activity on both enzymes
The ACEs of the 8 genotypes were analyzed by HPLC-UV-ESI-MSn in Esquire 4000 ESI-IT equipment (Bruker Daltonics GmBH, Germany), using a Hibar Purospher Star RP-18 column (Waters) with an end-capped of 5μm and 250 mm×4 mm. The analyzes were performed at room temperature with an injection of 20μL of each extract dissolved in methanol (used as a blank) at 100 mg/mL. A gradient system composed of 2 phases was used, (A) 4.5 % formic acid in water and (B) Acetonitrile, according to the methodology described by Pedro et al. (2016) [41]. The elution gradient was: 0–22 min 3 % B, 22–31 min 22 % B, 31–40 85 % B, 40–46 min 85 % B, 46–56 min 100 % B, and 56–65 min 3 % B at a flow rate of 1.0 mL/min. UV detection was carried out at 280 nm. The ionization process (nebulization) was developed at 3,000 V using nitrogen as the nebulizer gas at 365°C, a pressure of 60 psi, and a flow of 10 L/min. Mass spectra were obtained in negative polarity. For the analysis of the chromatograms and spectrograms, the Bruker DataAnalysis 3.2 software (Bruker Daltonik GmbH, DE) was used. The tentative identification of the compounds was carried out through the literature review, using databases such as Respect for Phytochemicals, Massbank, and a compound library obtained at the Centro de Estudios Químicos (CEPEDEQ) of the Universidad de Chile [22, 42].
To compare the phenolic profile among the analyzed extracts, the relative amount of each tentatively identified compound was determined. The molecular ion of each compound was selected in the ion selection mode and its areas were integrated. The peak area was normalized by the sum of the areas of all the peaks analyzed. Subsequently, the largest normalized area among the extracts was assigned an abundance of 100%. Using a semi-quantitative analysis, it was possible to compare the phenolic profile among the extracts, as well as to determine if the identified compounds could be related to the pharmacological activities.
Statistical analysis
The results were expressed as means±standard deviation (SD) of 3 independent measurements. The analyzes and the comparison of the extracts were carried out in GraphPad Prism 6 software, applying one-way ANOVA and Tukey’s test of multiple comparisons, considering significant differences with p-values≤0.05.
Results and discussion
Antioxidant capacity
The antioxidant capacity was evaluated through two in vitro methodologies: the DPPH radical assay and the ORAC assay. Table 1 shows the results for the antioxidant assays of both series of extracts and the reference compounds, showing the statistical differences with letters.
Antioxidant capacity of U. molinae fruits extracts from different genotypes
Antioxidant capacity of U. molinae fruits extracts from different genotypes
Different letters on each column represent significant differences (p < 0.05) analyzed by one-way ANOVA and Tukey’s multiple comparisons test. ACEs = Acetone extracts; AEEs = Acid ethanolic extracts. The more potent extract was marked in bold. DPPH expressed at effective concentration 50 (EC50) in μg/mL. *All the extracts were statistically different from quercetin. ORAC FL (Fluorescein) y PGR (Pyrogallol Red) expressed in μmol of Trolox equivalent (TE) / mg DE. 1TAC = Total antioxidant capacity expressed in μmol TE / mg Dry fruits (DF). SD = Standard deviation. n.d. = Not determined.
ACEs series showed about 6 to 11-fold more potent scavenging effect over the DPPH radical (EC50 = 34.15 to 58.30μg/mL; p≤0.05) compared to the AEEs series (EC50 = 197.48 to 365.74μg/mL; p≤0.05). However, compared to quercetin, murtilla berry extracts showed a lower DPPH scavenging potency.
Among the ACEs, genotypes 23-2 and 22-1 stood out, while among the AEEs series genotype 19-2 showed the lower EC50. Previous works by Rubilar et al. (2011) [7] reported an EC50 of 10.9μg/mL for a methanol acid extract and by Brito et al. (2014) [29] reported an EC50 of 82.9μg/mL for ethanol / water (50 % v/v) extracts. In both works, wild murtilla fruits were analyzed, and the EC50 values for DPPH radical were lower compared with other berries commonly grown in Chile, like maqui (Aristotelia chilensis), calafate (Berberis microphylla), blueberries (Vaccinium corymbosum), arrayán (Luma apiculata), chequén (Luma chequen), and meli (Amomyrtus meli).
Table 1 includes ORAC values for the total antioxidant capacity (TAC) of fruits extracted with the AWA solvent, and for both series of extracts. The ORAC methodology allowed us to evaluate the protective activity of the extracts against the attack of the peroxyl radical on a probe molecule. In ORAC-FL the probe molecule is fluorescein, while in ORAC-PGR is pyrogallol red [43].
For the TAC, in ORAC-FL, we observed values between 0.24 and 0.39μmol TE /mg of DF, where the genotype 19-2 showed the highest ORAC value. These results can be compared with previous reports by the Instituto de Nutrición y Tecnología de los Alimentos (INTA) of the Universidad de Chile for wild murtilla berries with an ORAC value of 0.43μmol TE /mg of DF (http://www.portalantioxidantes.com/). The value reported by INTA was higher than the TAC values of the 8 genotypes analyzed in this work. Additionally, Rodriguez et al. (2014) [44] reported ORAC values between 0.01 to 0.16μmol TE /mg DF for wild fruits, which is below that the ORAC values reported for INIA genotypes in this study. The discrepancies among the reported values in the literature and our results could be explained in part by the extraction methodologies, differences in ecological conditions of the collection site, whether they were cultivated or wild fruits, and the harvest years of the evaluated murtilla berries as these factors have been shown to modulate the phenolic composition of the analyzed fruits [26, 46]. For the ORAC-PGR modality, we observed values between 0.51 and 0.78μmol TE /mg DF, where the genotype 19-1 stood out with the highest value. It is important to mention that this is the first time that ORAC-PGR values are reported for murtilla berries. Other works have reported ORAC-PGR values for blueberries, blackberries, and raspberries extracts related to the content of ascorbic acid, where the authors describe the blueberry as a good protector of probe molecule [47]. Moreover, these results are not comparable to our work, since the extracts preparation and the calculations for the ORAC values were different.
Comparing both series of semi-purified extracts, the ACEs showed 4 to 5-fold higher than the AEEs in the ORAC-FL values and 3 to 10-fold in the ORAC-PGR values. These results suggest that the ACEs have a higher concentration of phenolic compounds that protect fluorescein and their mixtures of compounds were more reactive to protect pyrogallol red from radical attack. These results were expected, as we have previously reported that ACEs showed 3 to 5-fold higher TPC values than the AEEs [35].
The ACEs genotype 19-1 was the most active for ORAC-FL and ORAC-PGR. Meanwhile, for the AEEs series, genotype 23-2 was the most active in ORAC-FL, and genotype 22-1 was the most active in ORAC-PGR. These results are also in accordance with the TPC reported previously by our group. In ACEs series, 22-1 (111.7±5.0 mg gallic acid equivalents (GAE) /g dry extract (DE)) and 19-1 (107.9±4.6 mg GAE /g DE) genotypes showed the highest TPC, meanwhile in AEEs series, 19-2 (32.5±1.6 mg GAE /g DE) and 23-2 (30.0±2.0 mg GAE /g DE) genotypes [35].
Furthermore, our group has previously reported ORAC-FL values for leaf ethanolic extracts of the same murtilla genotypes. The observed values were between 8.6 and 23.8μmol TE /mg DE, which is 9 to 16-fold higher than the values obtained for the berry ACEs. These differences are probably due to a higher TPC and the mixture of pentacyclic triterpenes compounds present in leaves, which were not found in murtilla fruits [18, 48–51].
Although our extracts demonstrated antioxidant capacity in both the DPPH and ORAC assays, they were more potent in the ORAC assay (Table 1). This difference could be explained by the capacity of the phenolic compounds in the berry extracts to scavenge the radicals by either hydrogen atom transfer (HAT) or by a single electron transfer (SET) mechanism. The DPPH assay involves a sequential mechanism of HAT first and then SET, and the ORAC assay only involves the HAT mechanism [52]. Thus, our results suggest that phenolic compounds in murtilla berry extracts could scavenge radicals predominantly by the HAT mechanism.
The inhibitory potency of murtilla extracts on enzymes activity was evaluated through spectrophotometric assays for the determination of their inhibitory concentration 50 (IC50). Table 2 shows the results for the α-glucosidase and GPa inhibition assays for both series of extracts and the reference compounds, identifying the statistical differences by letters.
Inhibitory potency on
α -glucosidase and glycogen phosphorylase A of U. molinae fruits extracts from different genotypes
Inhibitory potency on
Different letters on each column represent significant differences (p < 0.05) analyzed by one-way ANOVA and Tukey’s multiple comparisons test. ACEs = Acetone Extracts; AEEs = Acid Ethanolic Extracts. The more potent extract was marked in bold. Inhibitory potency was expressed in IC50 in μg / mL of Dry Extract (DE). SD = Standard deviation. *All the extracts were statistically different from Acarbose. **All the extracts were statistically different from Caffeine.
For α-glucosidase assay, ACEs (1.4 –2.4μg/mL; p≤0.05) were around 160-fold more potent than acarbose, where genotype 19-1 stood out as the most potent. Meanwhile, the AEEs (21.4 –44.8μg/mL; p≤0.05) were around 12-fold more potent than the positive control, and genotype 27-1 showed the highest potency. Among both series of extracts, ACEs outperformed AEEs by 20-fold. Previous work by Rubilar et al. (2011) [7] reported an IC50 of 69.2μg/mL for an ethanolic extract of wild murtilla fruits, which is less potent than the values reported in our work for ACEs and AEEs obtained from INIA genotypes.
For the GPa assay, the ACEs (27.9 to 86.1μg/mL; p≤0.05) inhibited the enzyme activity, however, their values were among 5 and 16-fold less potent than the caffeine. Moreover, AEEs (75.3 to 303.7μg/mL; p≤0.05) were among 14 and 57-fold less potent than the reference substance. The highest inhibitory potency among the ACEs was observed for the 19-2 genotype, whereas genotype 22-1 showed the best results among AEEs. Notably, there are no reports in the literature for the inhibition of GPa enzyme by fruits of the species under study.
For both enzyme assays, the observed results were in accordance with the TPC previously reported by our group [35] as ACEs were more potent than AEEs. These results and the previous observations on the antioxidant capacity of the murtilla extracts, highlight the potential of murtilla berries as candidates for further analysis to confirm their pharmacological properties, which could be applied in the treatment of hyperglycemia and its complications.
The profile of phenolic compounds in the ACEs series was evaluated through liquid chromatography coupled to mass spectrometry. Table 3 shows 143 analyzed signals, with the tentative identification of the compounds according to the literature and different databases (as described in 2.5), and the semi-quantitative analysis with the relative amount of each compound.
Tentative identification of phenolic compounds in the ACEs of U. molinae fruits from different genotypes
Tentative identification of phenolic compounds in the ACEs of U. molinae fruits from different genotypes
RT = retention time. Semi quantification was carried out in respect to the higher normalized area obtained from selective ion mode in LC-MS analysis. aCEPEDEQ library. bReSpect for Phytochemicals Database. cMassbank. dReference: Peña-Cerda et al., 2017.
It can be noticed that the profiles of compounds among the murtilla genotypes analyzed were similar, highlighting the tentative identification of flavonoid derivatives, such as quercetin, myricetin, luteolin, and kaempferol, ellagic tannins, as well as derivatives of gallic, caffeic, and quinic acid. Furthermore, our group previously reported the phenolic profiles of 5 AEEs from murtilla fruits of genotypes 14-4, 19-1, 19-1ha, 23-2, and 27-1 [35] and we observed that the tentative identification of compounds was similar to ACEs.
Our results are consistent with previously reported in the literature for murtilla berries, describing the presence phenolic acids, different flavanols, flavones, and flavan-3-ols, such as caffeic acid 3-O-glucoside, quercetin 3-O-glucoside, quercetin, and other minority compounds like gallic acid, rutin, quercitrin, luteolin, kaempferol, kaempferol 3-glucoside, p-coumaric acid, and myricetin [25, 30].
Interestingly, we have tentatively identified compounds that have not been previously reported in murtilla berries, such as isorhamnetin, taxifolin, and epicatechin derivatives. Thus, our study contributes new knowledge about the complex chemical composition of these berries.
As previously stated, we observed that both series of extracts have significant differences in their TPC [35]. Nevertheless, there are similar compounds among the genotypes (Table 3), which suggests that the specific differences in the mixture and quantity of phenolic compounds are responsible for the differences observed in the antioxidant capacity (Table 1) and inhibitory activity over enzymes (Table 2) reported in this work.
In particular, the ACEs obtained from genotypes 19-1 and 22-1, which presented the highest inhibitory potency on both enzymes related to carbohydrate metabolism [53–56] and higher antioxidant capacities [39, 57], showed high intensities in peaks tentatively identified as gallic and ellagic acid derivatives, as well as peaks tentatively identified as derivatives of quercetin, myricetin, luteolin, and isorhamnetin. Interestingly, the 19-1 genotype tentatively shows citric acid, quinic acid, galloyl quinic acid, methoxyquercetin-O-hexoside, kaempferol-O-coumaroyl glucoside, myricetin-O-pentoside, and myricetin-O-rhamnoside in its chemical composition, compounds that were not identified in the other analyzed genotypes. This could explain, in part, the outstanding activities of ACE from 19-1 genotype in the in vitro pharmacological assays.
Isolated compounds, like phenolic acid, quercetin, rutin, or catechin have demonstrated antioxidant capacity in ORAC assay [39]. Thus, the observed differences among murtilla berry genotypes in the ORAC values could be explained by the variation in the presence of these specific phenolic derivatives and the variations in the TPC in each extract.
For its part, isolated phenolic compounds such as gallic acid, myricetin, and quercetin, compounds that have been tentatively identified in our extracts, have shown inhibitory activity on α-glucosidase, which could explain the inhibitory activity of murtilla berries extracts. Moreover, a higher presence of these kinds of compounds in ACEs could also explain the differences in potency between both series of extracts [54–56].
On the other hand, it has been reported that quercetin can inhibit GPa activity, which could explain, in part, the activities observed for murtilla berry extracts [53].
These results are promising and thus, further in vivo tests, and deepening studies on other enzymes involved in carbohydrate metabolism or redox balance could allow us to corroborate the observations made through in vitro experiments.
In this work, we have carried out a comparative study with extracts from different murtilla berry genotypes. The acetonic extracts obtained from the berries of U. molinae exhibited a promising antioxidant activity with a substantial potency in scavenging DPPH and peroxyl radicals. Moreover, acetonic extracts can inhibit the activity of two enzymes related to carbohydrate metabolism, α-glucosidase, and glycogen phosphorylase A. Noteworthy, for murtilla berry, ORAC-PGR values, and IC50 values for GPa enzyme have not been reported before in the literature. The ACEs series were more concentrated in phenolic compounds and more potent than the AEEs series in all assays. In particular, the ACEs of 22-1 and 19-1 (Red Pearl variety registered by INIA) genotypes were the best among the analyzed extracts. These genotypes are composed of a mixture of compounds with a major presence of specific flavonoids derivatives like quercetin or myricetin and phenolic acids like gallic or ellagic acid derivatives.
According to our results, murtilla fruits are a rich source of bioactive phenolic compounds and thus, their extracts are candidates for in vivo studies due to their pharmacological potential in treating hyperglycemia and the prevention of its complications. This could be achieved through the development of functional foods or phytopharmaceuticals, which could also encourage the cultivation of murtilla both for its agronomic and commercial value, as well as for its medicinal properties.
Footnotes
Acknowledgments
The authors would like to thank Instituto Nacional de Investigaciones Agropecuarias (INIA, Carillanca, Chile) for the plant material, and SouthAm freeze dry for the lyophilization of the plant material.
Funding
ANID-Chile grants for Doctoral studies N° 21150851 and N° 2115076, and FONDECYT No 1130155.
Conflict of interest
The authors have no conflict of interest to report.