West Mexico Berries Modulate α-Amylase, α-Glucosidase and Pancreatic Lipase Using In Vitro and In Silico Approaches

The objective was to evaluate the antioxidant and biological potential of eight freeze-dried berry varieties of southern Jalisco using in silico and in vitro approaches. Fourteen tentative phenolic compounds were identified in berries by ESI-QToF, including anthocyanins, phenolic acids, flavanols and flavonols. In silico assays of phytochemicals in the berry inhibiting enzymes related to obesity and diabetes showed predicted binding energy interactions (ranging from −5.4 to −9.3 kcal/mol). Among the cultivars, antioxidant potential for DPPH IC50 ranged from 1.27 to 3.40 mg/mL, ABTS IC50 from 2.26 to 7.32 mg/mL and nitric oxide (NO) inhibition IC50 from 4.26 to 11.07 mg/mL. The potential to inhibit α-amylase IC50 ranged from 4.02 to 7.66 mg/mL, α-glucosidase IC50 from 0.27 to 4.09 mg/mL, lipase IC50 from 1.30 to 4.82 mg/mL and DPP-IV IC50 from 1.36 to 3.31 mg/mL. Blackberry cultivars from the southern Jalisco region showed outstanding biological potential compared to other evaluated berries and could be used in the formulation of functional foods in the prevention of noncommunicable diseases.


Introduction
Mexican berries represent an important product in the national agricultural sector. Mexico ranks first place in blackberry production, second in raspberry and sixth in blueberry, worldwide [1]. Mexico has been ranked as the second exporter of these fruits worldwide [2]. Jalisco state (located in west Mexico) is the principal producer of blueberry and raspberry, and the second producer of blackberry in the country [1]. The global demand for berries is increasing and it is estimated that this demand will be duplicated by 2030. The growing demand for berries is mainly due to their health benefits. Extensive evidence has linked berries' phytochemicals with the attenuation of chronic metabolic diseases [3].
Although multiple efforts have been applied by government agencies worldwide, cost-effective alternatives for the prevention and treatment of noncommunicable diseases (NCDs) such as diabetes and obesity are needed. Multiple studies have demonstrated that functional foods and nutraceuticals are excellent options in the development of new alternative treatments for these diseases. Bioactive compounds derived from food may influence health beyond nutrition [4]. Berry polyphenols exert anti-hyperglycemic, lipid-lowering and anti-inflammatory effects [5]. In the treatment of hyperglycemia, polyphenols could act through insulin-dependent and insulin-independent mechanisms. The insulin-dependent mechanism involves the enhancement of β-cell function and insulin sensitivity of peripheral tissue. Otherwise, the insulin-independent mechanism is related to the inhibition Table 1 shows the concentration of phenolic compounds identified in the extracts. Among cultivars, the highest concentration of total polyphenols was observed in the two varieties of blackberry with a mean of 10.46 mg GAE/g DW. Blueberry varieties had the lowest concentration (4.57-5.62 mg GAE/g DW), with no differences among them (p > 0.05). The variety Ras3 had a similar concentration (5.89 mg GAE/g DW) to blueberry varieties, but the lowest concentration compared to the other raspberry varieties. The phenolic composition in several berries has shown similarities with our findings; blackberries have the highest amount of phenols, followed by raspberries and blueberries [10][11][12][13]. Sariburun et al. [13] reported a total phenol content of four blackberry cultivars from 2279.8 ± 12.5 mg GAE/100 g FW (fresh weight) to 2786.8 ± 21.9 mg GAE/100 g FW; and the content of five raspberry cultivars from 1040.95 ± 15.91 mg GAE/100 g FW to 2062.27 ± 4.13 mg GAE/100 g FW. In Korean berries, Kim [11] reported a total phenolic composition of 1307.33 ± 9.24 mg GAE/100 g for blackberry, 936.67 ± 2.08 mg GAE/100 g for blueberry and 489.67 ± 2.52 mg GAE/100 g for raspberry. In Romanian berries, blueberries have obtained a higher value of polyphenols with 678 mg GAE/100 g FW, followed by blackberries (about 440 mg GAE/100 g FW) and raspberries with approximately 410 mg GAE/100 g FW [14].
For anthocyanin concentration, raspberry varieties showed the lowest concentration among cultivars (0.41-1.47 mg C3GE/g DW). Blueberry varieties showed the highest concentration (p < 0.05) among cultivars and similar concentration with blackberry varieties. The highest concentration was observed in Blu1 variety (3.61 mg C3GE/g DW). The highest concentration of anthocyanins has been reported in blackberries, followed by blueberries and raspberries [10,12,13]; however, in this work, one of the blueberry varieties showed the highest concentration among cultivars (4.98 ± 0.25 mg C3GE/g DW), while the other blueberry and blackberry varieties showed similarities, and raspberries had a significantly lower concentration (0.65 to 2.24 mg C3GE/g DW). Between four blackberry cultivars, total anthocyanins have been reported from 41.3 ± 0.3 mg C3GE/100 g FW to 87.1 ± 1 mg C3GE/100 g FW; and from five raspberry cultivars, from 12.4 ± 0.3 mg C3GE/ 100 g FW to 69.5 ± 0.5 mg C3GE/100 g FW [13]. For flavonoid concentration, the Blu3 variety presented the highest value (5.74 mg RUE/g DW), and no differences were observed between Blu2 and Blu1 (p > 0.05). Similar concentrations were obtained in Bla1 and Bla2 varieties with a mean of 4.28 mg RUE/g DW. The lowest concentration was observed in the Ras1 variety. This value was almost half of the maximum concentration obtained in the Blu3 variety. Similar to our findings, Sariburun et al. [13] found a higher amount of total flavonoids in blackberry varieties (29.07 ± 0.49 mg/100 g of (+)-catechin equivalents (CTE) to 82.21 ± 1.34 mg CTE/100 g FW), than in raspberries (15.41 ± 0.12 mg CTE/100 g FW to 41.08 ± 0.92 mg CTE/100 g FW).

Antioxidant Potential (AP) from Different Berry Cultivars
All the berry varieties studied showed an important AP with the three different methods (ABTS, DPPH, NO). The results were expressed as IC 50 values that, at the lowest levels, indicate the highest free radical-scavenging activity of the samples.
In general, AP results ( Figure 1) show that Bla1 and Bla2 varieties have the best radicalscavenging activity. Moreover, the lowest activity for DPPH and NO was for raspberry varieties, while for ABTS, blueberry varieties showed the lowest inhibition capacity. For the DPPH assay, Figure 1A shows that the highest inhibition was in the Blu1 variety (IC 50 1.27 mg/mL), with no differences with Bla1 and Bla2 varieties (IC 50 1.37 and 1.41 mg/mL, respectively). For ABTS inhibition ( Figure 1B), the highest activity observed was in the Bla1 (IC 50 2.69 mg/mL) and Bla2 (IC 50 2.26 mg/mL) blackberry varieties. As shown in Figure 1B, the lowest activity was obtained in the Blu2 variety (IC 50 7.32 mg/mL). Figure 1C shows that the Blu1 variety had the highest inhibition activity at 4.26 mg/mL concentration for the NO inhibition assay. Moreover, the lowest inhibition was observed in the Ras1 raspberry variety (IC 50 11.07 mg/mL).
The antioxidant potential of berries has been widely reported. Diverse methods such as ORAC, FRAP, DPPH and ABTS have been used to evaluate the antioxidant capacity of blackberry, blueberry, and raspberry [11,12,15]. Similar findings were obtained by Sariburun et al. [13] using the DPPH and ABTS methods; while, for NO, the highest potential was observed in the Blu1 blueberry variety and blackberries varieties. Free radical scavenging promotes the reduction of diabetes mellitus (DM) complications associated with oxidative stress, such as β cell apoptosis and insulin resistance.

Biological Potential of Berries Cultivars
Modulation of glucose metabolism markers by phenolic compounds present in berries has been related to the inhibition of α-glucosidase and DDP-IV activity by anthocyanins and α-amylase activity by ellagitannins [8,10]. In this experiment, all berry varieties inhibited the in vitro activity of these enzymes. Figure 2 shows that the lowest concentration required to inhibit α-glucosidase, α-amylase and DPP-IV activity was observed in blackberries (IC 50 of 4.02 ± 0.12, 0.27 ± 0.02 and 1.36 ± 0.07 mg/mL, respectively), followed by some blueberry varieties (Figure 2A-D). Among the blackberry varieties, Bla2 showed the highest bioactivity presenting the lowest IC 50 values for all the assays. The highest potential activity shown in blackberries has also been reported by Fan et al. [7] (from 0.07 to 0.22 IC 50 µM) and Johnson et al. [10] (from 5.5 to 18.2 IC 50 µM).
burun et al. [13] using the DPPH and ABTS methods; while, for NO, the highest potential was observed in the Blu1 blueberry variety and blackberries varieties. Free radical scavenging promotes the reduction of diabetes mellitus (DM) complications associated with oxidative stress, such as β cell apoptosis and insulin resistance.

Biological Potential of Berries Cultivars
Modulation of glucose metabolism markers by phenolic compounds present in berries has been related to the inhibition of α-glucosidase and DDP-IV activity by anthocyanins and α-amylase activity by ellagitannins [8,10]. In this experiment, all berry varieties inhibited the in vitro activity of these enzymes. Figure 2 shows that the lowest concentration required to inhibit α-glucosidase, α-amylase and DPP-IV activity was observed in blackberries (IC50 of 4.02 ± 0.12, 0.27 ± 0.02 and 1.36 ± 0.07 mg/mL, respectively), followed by some blueberry varieties (Figure 2A-D). Among the blackberry varieties, Bla2 showed the highest bioactivity presenting the lowest IC50 values for all the assays. The highest potential activity shown in blackberries has also been reported by Fan et al. [7] (from 0.07 to 0.22 IC50 μM) and Johnson et al. [10] (from 5.5 to 18.2 IC50 μM).  The IC 50 for pancreatic lipase inhibition ( Figure 2C) indicated the highest activity by blackberries (1.36 to 1.42 mg/mL), followed by the Ras3 raspberry variety (IC 50 1.30 ± 0.14 mg/mL) and blueberry varieties (IC 50 1.80 to 1.87 mg/mL), while the lowest activity was shown by Ras1 and Ras2 (IC 50 4.35 and 4.82 mg/mL). Similarly, McDougall et al. [16] reported that a tannin-rich raspberry extract effectively inhibited pancreatic lipase activity in vitro, while blueberry caused a slight inhibition.

Molecular Docking of Berry Phytochemicals on Enzymes Related to Obesity and Diabetes
According to UPLC-ESI-QToF-MS/MS analysis, the extracts may contain diverse phenolic compounds that are present in other berry varieties. However, it is important to use pure standards to confirm the presence of phenolic acids and specific flavonoids.
In computational docking analysis, the theoretical free energy values showed the potential of the berries' bioactive compounds to interact with the target enzyme. Table 3 shows the minimum estimated free energy of polyphenols identified in berry extracts with α-glucosidase, α-amylase, DPP-IV and lipase. Figure 3 shows an example of ellagic acid in the catalytic site of pancreatic lipase. Interaction with lipase presented negative affinity values, from −6.1 kcal/mol (gallic acid) to −9.3 kcal/mol (ellagic acid). For DPP-IV, the binding energies were −5.4 kcal/mol (salicylic acid) to −7.4 kcal/mol (ellagic acid). The theoretical binding energy for α-glucosidase ranged from −5.4 kcal/mol (malvidin 3-O-glucoside and salicylic acid) to −7.7 kcal/mol (ellagic acid), while for α-amylase, the energy ranged from −5.9 kcal/mol (salicylic acid) to −8.9 kcal/mol (catechin). For positive controls, orlistat presented a free energy value of −6.2 kcal/mol for lipase; sitagliptin had a free energy value of −6.7 kcal/mol for DPP-IV; and acarbose presented a free energy value of −6.6 and −7.2 kcal/mol for α-glucosidase and α-amylase, respectively. Among the interaction of polyphenols with pancreatic lipase, DPP-IV, α-glucosidase and αamylase showed similar binding energies compared to positive controls (orlistat, sitagliptin, acarbose). However, most of the berry polyphenols showed a higher inhibition potential compared to drugs.
For lipase, p-coumaric acid, gallic acid, salicylic acid and caffeic acid presented a similar affinity to inhibit the enzyme, compared to the positive control; conversely, the rest of the identified compounds showed greater inhibition potential compared to Orlistat. The strongest theoretical activity was shown by ellagic acid (−9.3 kcal/mol). The main Pharmaceuticals 2022, 15, 1081 9 of 16 theoretical interactions between ellagic acid and the catalytic site of lipase were through hydrogen and Van Der Waals bonds, as well as cation-π interactions. In another case, molecular docking analysis showed that caffeic acid, p-coumaric acid and quercetin were able to inhibit the activity of lipase [19]. In our biochemical study results, the highest inhibition potential was shown by phenolic compounds, ellagic acid, (-) epicatechin and catechin, which were exclusively identified in raspberry and blackberry varieties.
Theoretical binding affinity (kcal/mol) of polyphenols identified in berry extracts. "-" indicates that the compound was not tested as an inhibitor.
Pharmaceuticals 2022, 15, x FOR PEER REVIEW 9 of 16 oretical binding energy for α-glucosidase ranged from −5.4 kcal/mol (malvidin 3-O-glucoside and salicylic acid) to −7.7 kcal/mol (ellagic acid), while for α-amylase, the energy ranged from −5.9 kcal/mol (salicylic acid) to −8.9 kcal/mol (catechin). For positive controls, orlistat presented a free energy value of −6.2 kcal/mol for lipase; sitagliptin had a free energy value of −6.7 kcal/mol for DPP-IV; and acarbose presented a free energy value of −6.6 and −7.2 kcal/mol for α-glucosidase and α-amylase, respectively. Among the interaction of polyphenols with pancreatic lipase, DPP-IV, α-glucosidase and α-amylase showed similar binding energies compared to positive controls (orlistat, sitagliptin, acarbose). However, most of the berry polyphenols showed a higher inhibition potential compared to drugs. For lipase, p-coumaric acid, gallic acid, salicylic acid and caffeic acid presented a similar affinity to inhibit the enzyme, compared to the positive control; conversely, the rest of the identified compounds showed greater inhibition potential compared to Orlistat. The strongest theoretical activity was shown by ellagic acid (−9.3 kcal/mol). The main theoretical interactions between ellagic acid and the catalytic site of lipase were through hydrogen and Van Der Waals bonds, as well as cation-π interactions. In another case, molecular docking analysis showed that caffeic acid, p-coumaric acid and quercetin were able to inhibit the activity of lipase [19]. In our biochemical study results, the highest inhibition potential was shown by phenolic compounds, ellagic acid, (-) epicatechin and catechin, which were exclusively identified in raspberry and blackberry varieties.
Johnson et al. [10] reported that anthocyanins identified from blueberries and black- Johnson et al. [10] reported that anthocyanins identified from blueberries and blackberries had a strong potential to inhibit DPP-IV enzyme, similarly to the control diprotin A; conversely, Fan et al. [7] demonstrated that flavonoids from blueberry-blackberry wine could inhibit DPP-IV enzyme. In our case, most of the compounds inhibit the enzyme with similar values compared to sitagliptin; however, cyanidin 3-glucoside, quercetin 3-D-glucoside and ellagic acid showed a stronger potential.
Blackberry varieties showed the greatest potential to inhibit α-glucosidase and αamylase by biochemical analysis. This result was similar to the in silico analysis in which the catechin, only identified in blackberries, demonstrated a potential inhibition greater than acarbose. As can be seen from these results, some compounds showed higher potential than the positive controls to inhibit the enzymes.

Sample Collection
Three varieties (Ras1, Ras2, Ras3) of raspberry (Rubus idaeus), three (Blu1, Blu2, Blu3) of blueberry (Vaccinium spp.) and two (Bla1, Bla2) of blackberry (Rubus ulmifolius) were obtained by cultivar zones in southern Jalisco during one production cycle (December 2018-January 2019). Fresh fruits were stored at −20 • C until the lyophilization process. After, frozen fruits were separated individually and lyophilized using a Harvest Right™ freeze dryer. The freeze-dried samples were stored in a vacuum package until required for analysis.

Polyphenol-Rich Extracts Preparation
Lyophilized berries samples were grounded using an IKA ® A11 analytical mill, then were sieved in 40 US-mech (standard testing sieve, Advantech a W.S. Tyler®Company, Mentor, OH, USA) and stored in a plastic bag. For every independent replication, an acidified ethanol extract (85:15 ethanol:HCl) was obtained in a 1:10 (solid:solvent) ratio of the powdered sample of each fruit. Sonication at room temperature (3 times × 15 min each) in an Ultrasonic Cleaner SB-5200DTN (Ningbo Scientz ® Biotechnology Co., Ltd., Tech Park, Ningbo, Zhejiang, China) was used to assist the extraction, followed by centrifugation for 10 min at 10,000 rpm. The supernatant was collected in an Eppendorf ® microtube and stored at −20 • C until analysis.

Total Phenolic Compounds
Total phenolic compounds were measured using the Folin-Ciocalteu method adapted to a microassay using gallic acid (GA) as standard [22]. Samples were diluted to a factor of 1:10 and 1:20 with deionized water. Then, 50 µL microliters of the samples, GA standard (40-100 µg/mL) or blank (deionized water) were placed in a 96-well flat-bottom plate and then 50 µL of 1 N Folin-Ciocalteu's phenol reagent were added. After 5 min, 100 µL of 20% Na 2 CO 3 were added, and the mixture was incubated for 10 min. The absorbance was read at 690 nm in a Tecan Infinite ® M200 PRO (Tecan Group, Ltd., Männedorf, Switzerland). Final concentrations were expressed as mg of gallic acid equivalents (GAE) per g of DW (dry weight) (y = 0.0094x − 0.0521, R 2 = 0.9975).

Tannins
For total tannins concentration, catechin (CAE) was used as standard. The method was done as reported by Mojica et al. [22]. Samples of extract were diluted to a factor of 1:20 and 1:40 with deionized water. In a 96-well flat-bottom plate, 50 µL of these diluted samples, CAE standard (0.1-0.8 mg/mL) or blank (50 µL of methanol 99.8% and 200 µL of 4% acidified methanol) were added, followed by the addition of 200 µL of 8% acidified methanol and 1% vanillin methanol solution to a factor of 1:1. The absorbance was read at 500 nm using a Tecan Infinite ® M200 PRO (Tecan Group, Ltd., Männedorf, Switzerland). Final concentrations were expressed as mg catechin equivalents (CAE) per g of DW (y = 0.2279x + 0.0167, R 2 = 0.997).

Nitric Oxide (NO) Radical Scavenging Assay
Scavenging of NO was determined as previously reported by Oseguera-Toledo et al. [25], with some modifications. Sodium nitroprusside (SNP) was used as the NO donor. Samples were diluted to a factor of 1:5 to 1:40 with phosphate-buffered saline 0.01 M, pH 7.4. In a 96-well flat-bottom plate, either 50 µL of the diluted sample or blank (PBS) were incubated with SNP solution at 27 • C. After 120 min, 50 µL of the incubated solution was mixed with 50 µL of Griess reagent and incubated again for 10 min. After a time, absorbance was immediately read at 550 nm using a Tecan Infinite ® M200 PRO (Tecan Group, Ltd., Männedorf, Switzerland). Results were presented as IC 50 values mg/mL and were determined from the free radical scavenging activity (%).
3.6. Biological Potential 3.6.1. α-Amylase Inhibition Biochemical Assay For the α-amylase assay, the method was used as reported by Mojica et al. [23], with some modifications. Samples were diluted to a factor of 1:10 to 1:40 with PBS 0.01 M, pH 7.4. In microtubes, 50 µL of each diluted sample, 1 mM acarbose (positive control), or PBS, 0.02 M, pH 6.9 (negative control) were added to 50 µL of 13 U/mL α-amylase solution (PBS 0.02 M, pH 6.9), and incubated at 37 • C for 10 min. Afterward, 50 µL of 1% soluble starch solution (previously dissolved in PBS 0.02 M, pH 6.9, and boiled for 10 min) was added to each tube and incubated at 37 • C for another 10 min. Finally, 1 mL of dinitro salicylic acid reagent (DNS) was added, and the tubes were placed in a 100 • C water bath for 5 min. The mixture was diluted with 1 mL of distilled water. In a 96-well plate, 200 µL of each mixture was added, and absorbance was read at 540 nm. The same procedure was carried out in microtubes, where 50 µL of PBS 0.02 M, pH 6.9 solution were added instead of α-amylase solution. The results obtained by these three independent experiments were considered to calculate the inhibition percentage with the following equation, where: A = the subtraction of the absorbance obtained in the sample with α-amylase enzyme solution and the absorbance obtained in the sample with PBS solution; B = mean of the absorbance obtained in the negative control PBS with α-amylase enzyme. Results were presented as IC 50 . The assay was based on the method reported by Mojica et al. [23], with some modifications. Each sample of acidified ethanol extract was evaporated to remove ethanol with a Thermo Scientific™ Savant, SpeedVac™ Concentrator (Thermo Fisher Scientific Inc., Waltham, MA, USA). Deionized water was added to obtain the initial volume before evaporation. For the α-glucosidase assay, samples were diluted to a factor of 1:40 to 1:700 with PBS (0.01 M, pH 7.4). In a 96-well flat-bottom plate, either 50 µL of the diluted sample, 1 mM acarbose (positive control), or PBS, 0.1 M, pH 6.9 (negative control) were added to 100 µL of 1 U/mL α-glucosidase solution (PBS, 0.1 M, pH 6.9) and incubated at 37 • C for 10 min. Later, 50 µL of 5 mM p-nitrophenyl-α-D-glucopyranoside solution (PBS 0.1 M, pH 6.9) was added to each well and incubated at 37 • C for 5 min. Absorbance was read at 405 nm. Results were presented as IC 50 values (mg/mL).

Lipase Inhibition Biochemical Assay
Lipase inhibition activity was determined by using PHIBLA (pH Indicator-Based Lipase Assay) as reported by Camacho-Ruiz et al. [26] with some modifications. Each sample of the extract was diluted to a factor of 1:10 to 1:100 with PBS (0.01 M, pH 7.4). To measure indirectly the release of free fatty acids by lipase-catalyzed hydrolysis of short-chain tributyrin substrate TG (4:0), 100 mM and 4-nitrophenol (pH indicator) were dissolved in tert-butanol. A substrate emulsion (5 mM) was obtained by vigorous mixing on a vortex of 8:1:1 proportion of buffer solution, TG (4:0) and 4-nitrophenol. Buffer solution included 2.5 mM MOPS, 0.5 mM NaTDC, 150 mM NaCl and 6 mM CaCl 2 . In a 96-well flat-bottom plate, either 10 µL of the diluted sample, or Orlistat (positive control), or buffer (negative control) were added to 10 µL of lipase enzyme solution and incubated at 37 • C for 60 min in a microplate shaker. After incubation, 100 µL of substrate emulsion (prepared no more than 3 min early) was quickly added using an eight-channel pipette. Immediately, the plate was placed in a microtiter plate scanning spectrophotometer (x-Mark TM , Bio-rad Laboratories, Inc. Hercules, CA, USA) and shaken for 5 s before each reading. The decrease in absorbance at a wavelength corresponding to the λmax of the pH indicator was recorded at 410 nm every 30 s at 37 • C. Results were presented as IC 50 values (mg/mL).
Mass spectrometry (MS) analysis was performed on a Water Xevo G2-XS QToF quadrupole time-of-flight mass spectrometer, with an electrospray ionization (ESI) in-terface (Milford, MA, USA). The MS acquisition was operated in negative ion mode, with a mass range of m/z 50 to 800. The parameters were: capillary voltage: 2.5 kV, cone voltage: 40 kV, source temperature: 100 • C, desolvation temperature: 250 • C, collision energy: 6.0 eV. MassLynx 4.1 MS software (Milford, MA, USA) was used to process data.

Statistical Analysis
Statistical analysis was evaluated considering at least three independent replications. The data were analyzed using the Statistical Package for the Social Sciences version 24 (IBM ® SPSS Statistics, Chicago, ILL, USA). One-way analysis of variance (ANOVA) with Tukey's post hoc analyses were used to determine differences among fruits and varieties. An α level of p < 0.05 was considered statistically significant. Data are expressed as means ± standard deviation. The IC 50 and correlation among parameters measured were calculated using GraphPad Prism (Version 8.0.2; GraphPad Software, Inc.; San Diego, CA, USA). The graphs were made with the SigmaPlot 10.0 software for Microsoft Windows.

Conclusions
The differences between studies results in the concentration of compounds, their antioxidant capacity and their biological activity are associated to the method of extraction, the solvent type, the variety, maturity of the berry, storage, cultivation conditions and environmental conditions, among others [13,15,18]. Despite this, it can be inferred that our results highlight the characteristics of blackberries among the reviewed studies and in the results of this work, regardless of the variety, geographical area of cultivation, extraction method and some other conditions mentioned previously.
In conclusion, this work presents important new information related to berries grown in the region of Jalisco, Mexico. These compounds could act and interact synergistically to enhance the antioxidant, hypoglycemic and lipid-lowering effects observed in in vitro tests. The evaluated blackberry varieties could be highlighted as the fruits that showed the best results both for the in vitro and in silico assays. These results could be useful in the development of nutraceutical products that can be evaluated in clinical trials and may impact the development of economically sustainable alternative treatments in the prevention and management of metabolic diseases.