Inhibition of α-amylase
R. echinocarpa methanolic extracts inhibited α-amylase enzymatic activity, corresponding to the first step in for the hydrolysis of starch and other carbohydrate polymers into shorter oligosaccharides. The α-amylase inhibitory effect of the extracts tested at 1 mg/mL was significantly similar to, or greater than the observed for acarbose at 25 mM (Table 1). Extracts from Tyr_50 and 100 had better (p < 0.05) inhibitory activity than the control (Ctrl); while the obtained for Tyr_200 (34.20 ± 0.38%) was similar (p > 0.05) to acarbose 50 mM (32.36 ± 1.85%). Extracts from other plants and algae have also shown similar activities (Picos-Salas et al., 2021; Landa-Cansigno et al., 2020). When tested at 400 µg/mL, extracts from Lippia graveolens inhibited α-amylase by 43.41% while those from Lippia palmieri did it only by 12.19% (Gutiérrez-Grijalva et al., 2019). Methanolic extracts of Vernonia antihelmitica callus and leaf showed an IC50 of 0.499 mg/L and 0.372 mg/L, respectively (Rajan et al., 2022). Moderate inhibition (30–50%) of α-amylase is desired since excessive bacterial fermentation of non-digested carbohydrates in colon could cause diarrhea and flatulence (Kashtoh & Baek, 2023).
Inhibition of α-glucosidase
Contrasting to the α-amylase activities described above, no enzymatic inhibition was observed for α-glucosidase (data not shown). A previous study carried out by Cuevas-Juárez et al. (2014), reported that partially purified melanins from the pulp fruit of R. echinocarpa presented higher α-glucosidase inhibitory (IC50 1.00–1.17 mg/mL) activities when compared with acarbose (8.38 mg/mL). On the other hand, different plant extracts showed better inhibitory activity on α-amylase than the observed for α-glucosidase. Hua et al. (2018) reported flavone glycosides isolated from Lu’an GuaPian tea (Camelia sinensis L.O. Kuntze) with better inhibition effects on α-amylase (IC50 0.50–12.05 µM) than for α-glucosidase (IC50 28.11–106.65 µM). Similarly, different fractions from Persicaria hydropiper presented higher inhibition activities on α-amylase than α-glucosidase (Mahnashi et al., 2022). The IC50 of a methanolic extract of V. antihelmitica callus was also higher for α-glucosidase inhibition than the observed for α-amylase (Rajan et al., 2022). Methanolic extracts of Carthamus tinctorius callus showed a IC50 for α-glucosidase of 0.63 g/L that was mainly attributed to the presence of different chlorogenic acids (Liu et al., 2023). The fact that only α-amylase activity was inhibited by the methanolic extracts of R. echinocarpa can be attributed to the complex mixture, and to synergic or antagonist effects of the compounds (Landa-Cansigno et al., 2020).
Table 1
Inhibition of α-amylase by crude extracts (1 mg/mL) of R. echinocarpa cell suspensions treated with 50, 100 or 200 mg/L of L-tyrosine compared to acarbose 25–150 µM.
Treatment | Inhibition of α-amylase (%) |
Ctrl | 13.90 ± 1.43 e |
Tyr_50 | 20.84 ± 0.83 d |
Tyr_100 | 24.79 ± 1.27 d |
Tyr_200 | 34.20 + 0.38 c |
25 µM Acarbose | 9.46 ± 3.06 e |
50 µM Acarbose | 32.36 ± 1.85 c |
100 µM Acarbose | 61.87 ± 1.14 b |
150 µM Acarbose | 69.20 ± 1.25 a |
Ctrl, control; Tyr_50, L-tyrosine at 50 mg/L; Tyr_100, L-tyrosine at 100 mg/mL; Tyr_200, L-tyrosine at 200 mg/L.
Hierarchical Clustering Analysis
Mass spectrometric analysis detected 494 Rt-m/z signals in positive mode. The addition of Tyr at different concentrations induced significant changes in the chemical profile of the R. echinocarpa cell suspensions (Fig. 1a). In general, control sample (Ctrl) showed decreased (blue) intensities of the Rt-m/z signals compared to the Tyr treatments. Tyr_100 and Tyr_200 treatments showed similar signal intensities. This was further supported by the 3D-PCA diagrams, whose components explained 95.2% of the total variance (Fig. 1b). To represent the number of statistically significant Rt-m/z signals shared between Tyr treatments, a Venn diagram was created (Fig. 1c). The core metabolism consisted of 221 Rt-m/z signals. Tyr_100 treatment presented the highest number of unique Rt-m/z signals (59), while Ctrl presented the lowest number of Rt-m/z signals (44). In a similar way, Ctrl and Tyr_50 treatments showed similar number of unique Rt-m/z signals with 44 and 45, respectively. These changes in the intensities detected represent the changing accumulation of the compounds due to the activation, or inactivation, of different metabolic pathways.
Enrichment of Metabolic Pathways
In Table S1, the total number of metabolites in the pathway and the number of metabolites identified in the data (hits) were summarized along with the raw p-value calculated from the enrichment analysis and the impact value. The functional analysis considering all signals detected by the metabolomic approach serves to identify the main metabolic pathways (pathway impact values ≥ 0.15 and p-values ≤ 0.1) present in the cell suspensions of R. echinocarpa from all treatments (Fig. 2).
Galactose and starch/sucrose metabolism were among the main metabolic pathways identified by the functional analysis (Fig. 2); both are involved in the over-accumulation of compounds related with carbohydrate utilization. Other metabolic pathways affected by the Tyr treatments included steroid biosynthesis, ubiquinone and other terpenoid-quinone biosynthesis, sphingolipid metabolism and Tyr metabolism. It was expected an increase in the metabolism of Tyr and other metabolic pathways involved in its utilization, like the biosynthesis of ubiquinone and other terpenoid-quinones, since Tyr serves as a precursor for the biosynthesis of specialized metabolites involved in those metabolic pathways (Xu et al., 2020).
The brassinosteroid and steroid biosynthesis, and the sphingolipid metabolism pathways were also identified in the R. echinocarpa cell suspensions treated with Tyr. Brassinosteroids are considered the sixth class of plant regulators, being present in all plant organs, and have a fundamental role in the development and growth of plants, inducing a wide range of morphological and physiological responses, including tolerance against abiotic and biotic stress (Bajguz et al., 2020; Wei & Li, 2020). On the other hand, in plants, cell membranes are primarily composed of glycerolipids, sterols and sphingolipids (Valitova et al., 2016), thus the high impact on the steroid biosynthesis and the sphingolipid metabolism in the cell suspensions of R. echinocarpa was also expected to occur. Plant sterols are used for the biosynthesis of brassinosteroids (Sonawane et al., 2016). On the other hand, sphingolipids are also crucial for maintaining the morphology of membrane systems and modulating membrane functions like signaling, cell polarity, and cellular responses to abiotic and biotic conditions (Haslam & Feussner, 2022).
The metabolites over-accumulated in the Tyr_100 and 200 methanolic extracts, in comparison to Ctrl, were tentatively identified and enlisted in Table 2. In Tyr_100, the compound with the highest FC value (3343) was homovanillic acid 4-glucuronide, while in Tyr_200 the highest FC value (622.36) corresponds to 5'-phosphoribosyl-N-formylglycinamide, a compound also identified in Tyr_100. Wang et al. (2019) reported an increase of homovanillic along with salicylic, caffeic, and ferulic acids after short-term heat shock treatment in Festuca trachyphylla. On the other hand, application of LED light stimulated the production of several phenolics, including homovanillic acid, in seedlings of Fagopyrum esculentum Moench (Hornyák et al., 2022).
In plants, Tyr can be consumed to synthetize L-DOPA, a precursor of homovanillic acid, and it has been reported as an important regulator of the synthesis of specialized metabolites like those derived from the phenylpropanoid pathway (Breitel et al., 2021). Although the presence of L-DOPA was not observed as an accumulated compound in the methanolic extracts, this can be attributed to its utilization for the synthesis of homovanillic acid. Moreover, other compounds can be synthesized from L-DOPA, such as melanins (Soares et al., 2014). In this metabolic branch, Tyr is oxidized to L-DOPA by the enzyme tyrosinase oxidase (TYROX), which is also involved in the synthesis of melanins (Oviedo-Silva et al., 2018). Melanins can also be produced by enzymatic browning reactions catalyzed by polyphenol oxidases (PPOs) from phenolic substrates (Glagoleva et al., 2020).
The tentative identification of flavonoid-o-glycosides, oligosaccharides, methyl 3,4-dicaffeoylquinate, and diferuloylquinic acid, was also observed in Tyr_100. Further analyses with standards are required to corroborate the presence of these compounds in the methanolic extracts of R. echinocarpa cell suspensions. Flavonoids have been reported to inhibit α-amylase (Martínez-González et al., 2019). This inhibition is attributed to the ability of flavonoids to interact with proteins, either with covalent or non-covalent bonds (Takahama & Hirota, 2018).
Among the compounds that were upregulated by Tyr at 200 or 100 mg/L, 5'-phosphoribosyl-N-formylglycineamide had a fold change (FC) of 624.4 and 608, respectively. This compound is involved in purine pathway. Inositol derivatives were over accumulated only increased in Tyr_200 by a FC of 124.3. Martínez-Ceja et al. (2022) reported the presence of inositol in the methanolic extracts of R. aculeata L.
Hexadecanoic acid (palmitate) was previously reported in hexane and dichloromethane extracts of Randia aculeata L. cell suspensions (Martínez-Ceja et al., 2022). Hexadecanoic acid had a FC of 6.6 in Tyr_200. In Tyr_100, methylhexadecanoic acid had a FC of 16.2. Previous studies had reported the inhibitory effects of hexadecanoic acid on the activities of α-amylase (Hoang-Anh et al., 2020) and α-glucosidase (Su et al., 2013). Isoflavones and palmitic acid derivatives have been reported to inhibit α-amylase activity (Martínez-Gonzáles et al., 2019; Hoang-Anh et al., 2020). Thus, the increased inhibition of this enzyme by the Tyr_200 can be related with the over-accumulation of these compounds in the R. echinocarpa cell suspensions.
Table 2
Tentative identification of the over-accumulated compounds in Tyr_100 and Tyr_200 treatments in comparison to control (Ctrl).
Treatment | Rt | m/z | FC | p value | Tentative compound | Adduct | Error (ppm) |
Tyr_100 | 0.47 | 104.1069 | 230.0 | 0.0350 | Choline | [M + H]+ | 6 |
0.5 | 381.0783 | 3343.0 | 0.0545 | Homovanillic acid 4-glucuronide | [M + Na]+ | 2 |
0.51 | 191.039 | 591.2 | 0.0427 | L-Glutamine | [M + 2Na-H]+ | 6 |
0.51 | 279.0383 | 608.0 | 0.0429 | 5'-Phosphoribosyl-N-formylglycinamide | [M + H-2H2O]+ | 2 |
0.51 | 527.1569 | 2249.8 | 0.0519 | Flavonoid-o-glycosides, oligosaccharides, Methyl 3,4-dicaffeoylquinate, Diferuloylquinic acid, | [M + H-H2O]+, [M + Na]+ | 3 |
0.53 | 185.0419 | 527.9 | 0.0418 | 1-Rhamnono-1,4-lactone | [M + Na]+ | 1 |
0.53 | 321.0495 | 1146.1 | 0.0475 | Imidazoleacetic acid ribotide, Alanine-betaxanthin | [M + H-H2O]+, [M + K]+ | 2, 4 |
0.54 | 427.075 | 1681.9 | 0.0500 | Ferulic acid | [2M + K]+ | 9 |
10.15 | 288.2893 | 16.2 | 2.922e-06 | Methylhexadecanoic acid | [M + NH4]+ | 1 |
11.51 | 453.1667 | 5.8 | 1.712e-05 | Flavanones | [M + 2Na-H]+ | 4 |
11.81 | 302.305 | 5.5 | 4.601e-09 | Sphinganine | [M + H]+ | 1 |
14.44 | 349.1352 | 5.9 | 0.0004 | N2-(2-Carboxymethyl-2-hydroxysuccinoyl)arginine | [M + H]+ | 1 |
Tyr_200 | 0.5 | 369.0719 | 188.5 | 3.30e-02 | L-Dopachrome, Isoflavonoids | [2M + H-H2O]+, [M + K]+ | 0, 4 |
0.5 | 219.0179 | 362.2 | 3.88e-02 | Fatty acids | [M + 2K]+ | 2 |
0.51 | 279.0383 | 622.4 | 4.31e-02 | 5'-Phosphoribosyl-N-formylglycinamide | [M + H-2H2O]+ | 2 |
0.53 | 335.0938 | 154.0 | 3.21e-02 | O-glycosyl compounds, flavonoids, Imidazolelactate | [M + H-H2O]+ | 5 |
0.53 | 185.0419 | 174.0 | 3.22e-02 | 1-Rhamnono-1,4-lactone | [M + Na]+ | 1 |
0.54 | 203.0525 | 124.3 | 2.89e-02 | Monosaccharides, Inositol derivatives | [M + Na]+ | 3 |
10.09 | 274.2738 | 6.6 | 1.45e-04 | Palmitic acid, isopalmitic acid | [M + NH4]+ | 1 |
11.51 | 453.1667 | 5.5 | 3.50e-06 | Flavanones | [M + 2Na-H]+ | 4 |
14.44 | 349.1352 | 5.6 | 5.97e-06 | N2-(2-Carboxymethyl-2-hydroxysuccinoyl)arginine | [M + H]+ | N/A |
Tyr_100: L-tyrosine at 100 mg/L; Tyr_200: L-tyrosine at 200 mg/L; Rt: retention time in minutes; m/z: mass/charge in Daltons; FC: fold change.
Molecular docking
The molecular docking analysis revealed that compounds identified in R. echinocarpa cell cultures could interact with several amino acid residues within the α-amylase catalytic site. These compounds exhibited predicted energy binding values ranging from − 5.6 to -3.4 kcal/mol, which are considered stable interactions (Yañez-Apam et al., 2023). These results were in accordance with estimated free binding energies predicted among α-amylase and small molecules such as flavonoid or pirazole derivatives (Rydberg et al., 2002; Mai et al., 2023). The enzyme α-amylase exists in two isoforms, present in both saliva and pancreatic secretions (Freitas et al., 2023). Both isoforms are composed of 496 amino acids and could be divided into three different domains (A, B, and C), with the catalytic triad (Asp197, Glu233, and Asp300) responsible for the cleaving function of the enzymes located in the A domain. Since the center of the binding site is exposed to the center of a large cavity that is found on the side of the A domain, it is more likely for smaller structures to access the binding site (Rydberg et al., 2002). The stereochemistry appears to play a role in the biological activity of the compounds inhibiting α-amylase (Akshatha et al., 2021). The compounds identified in R. echinocarpa, specially homovanillic acid 4-glucuronide, cultures were able to interact with several binding site residues of the enzyme; therefore, they may be able to pose in the large cavity blocking access to the substrate (Fig. 3A). The interactions between α-amylase and this compound were able to generate a hydrophobicity surface (Fig. 3B) that stabilizes the ligand within the catalytic pocket of the enzyme, and the types of interactions found were mainly H-bond, hydrophobic, and π bonds as observed in Fig. 3C. Acarbose was used as a control, showing a predicted energy binding value of -8.4 kcal/mol and demonstrated the most diverse interactions within the catalytic cavity of α-amylase (Table 3). The compounds identified in R. echinocarpa cell culture treated with Tyr_100 with a FC greater than 500 (homovanillic acid 4-glucuronide, 5'-phosphoribosyl-N-formylglycinamide, diferuloylquinic acid, ferulic acid, and imidazoleacetic acid ribotide) had an estimated free binding energy of -2.4 to -5.6 kcal/mol. On the other hand, only one compound in the treatment with Tyr_200 were identified to have a greater FC of 500, with estimated free binding energies of -5.3 kcal/mol. Notably, 5'-phosphoribosyl-N-formylglycinamide were able to interact with Asp300, a key amino acid in the deformation of the α-amylase substrate and enhancement of sugar electrophilicity during nucleophilic attacks during the first steps of the enzymatic reaction degrading carbohydrates (Rydberg et al., 2002). The fact that Tyr_100 showed lower inhibitory activity in α-amylase than Tyr_200 can be attributed to the presence of other compounds with antagonist effects, as discussed previously.
Table 3. Estimated free energy binding and interacting amino acids among the compounds present in cell culture of R. echinocarpa treated with 100 or 200 mg/L of tyrosine and the catalytic site of α-amylase.
Compound
|
EBE (kcal/mol)
|
Interacting amino acids in the catalytic site
|
Homovanillic acid 4-glucuronide
|
-5.6
|
Tyr62, Gln63, Asp96
|
5'-Phosphoribosyl-N-formylglycinamide
|
-5.3
|
Trp59, Tyr62, Gln63, Leu162, Asp300
|
Diferuloylquinic acid
|
-3.5
|
Asp197, Ala198, Val234
|
Ferulic acid
|
-3.4
|
Tyr62, Gln63, Asp300, Gly306
|
1-Rhamnono-1,4-lactone
|
1.3
|
-
|
Acarbose
|
-8.4
|
Hist101, Gln63, Leu165, Tyr62, Ala198, Asp300, Glu233, His299, Leu162, His305, Trp59, Val163, Trp357, Asp356, His201, Gly306, Ile235, Trp58, Lys200, Tyr151
|
EBE: estimated free binding energy, Trp: tryptophane, Tyr: tyrosine, Gln: glutamine, Val: valine, Asp: asparagine, Ala: alanine, Leu: leucine, His: histidine.