Identification of Bioactive Compounds and Analysis of Inhibitory Potential of the Digestive Enzymes from Syzygium sp . Extracts

Laboratório de Bioquı́mica, Instituto de Ciências Biológicas e Naturais, Universidade Federal do Triângulo Mineiro–UFTM, Uberaba, MG, Brazil Laboratório de Biof́ısica Teórica, Departamento de Fı́sica, Instituto de Ciências Exatas, Naturais e Educação, Universidade Federal do Triângulo Mineiro–UFTM, Uberaba, MG, Brazil Laboratório de Bioquı́mica, Departamento de Quı́mica, Universidade Federal de Lavras–UFLA, Lavras, MG, Brazil Laboratório de Farmacologia, Instituto de Ciências Biológicas e Naturais, Universidade Federal do Triângulo Mineiro–UFTM, Uberaba, MG, Brazil Instituto de Ciências Tecnológicas e Exatas, Universidade Federal do Triângulo Mineiro–UFTM, Uberaba, MG, Brazil Departamento de Ciências da Saúde, Universidade Federal de Lavras–UFLA, Lavras, MG, Brazil


Introduction
Diabetes mellitus and obesity represent some of the greatest public health problems today.Around 90% of individuals who develop type 2 diabetes are obese, and most of these patients have high blood pressure and/or dyslipidemia, in addition to being overweight.Type 2 diabetes is a metabolic disorder that has insulin resistance as its main factor.Type 2 diabetes is the most common type of diabetes, accounting for around 90% of all cases of diabetes.In type 2 diabetes, hyperglycemia is the result of an inadequate production of insulin and inability of the body to respond fully to insulin, defined as insulin resistance.e obese individual has increased adipose tissue producing a number of substances that decrease the body's ability to convert glucose into energy.Type 2 diabetes already affects children and adolescents [1][2][3][4].
Medicinal plants are rich sources for the development of new pharmaceutical products, such as diabetes and obesity, due to the high presence of secondary metabolites [4][5][6].An example of a medicinal plant is Syzygium sp., which is referenced as a potential hypoglycemic and slimming agent [7,8].Known in Brazil as "jambolão," Syzygium sp. is a tree belonging to the Myrtaceae family.Chanudom and Tangpong [9] worked with aqueous extracts of Syzygium cumini seeds and identified betulinic acid and luteolin as the main inhibitors of pancreatic α-amylase enzyme.
e growing search for safe and effective treatment methods to combat diabetes and obesity has pointed to the use of medicinal plants as a valuable source of inhibitors of digestive enzymes, which inhibit the breakdown of complex carbohydrates and fats in the intestine.
ese inhibitors exert their antidiabetic effect by hindering the activity of starch hydrolysis, such as α-amylase and α-glucosidase (enzymes present on the small intestinal brush border).α-amylase catalyzes the initial hydrolysis of starch into shorter oligosaccharides and disaccharides by cleavage of glycosidic linkages between the glucose residues.α-amylase inhibitors can effectively delay the digestion of the starch, i.e., delay the formation and absorption of glucose by inhibiting the glycolytic enzymes.
is may reduce the rate of glucose uptake into the bloodstream and, thus, alleviate the postprandial increase in plasma glucose in diabetic patients possibly inducing weight loss [10][11][12].In addition, trypsin inhibitors present in plant extracts may lead to a decrease in protein digestibility, which may also lead to a reduction in weight [13], although it is nutritionally undesirable.
ere is a lack of information on the profile of inhibition of the digestive enzymes contained in Syzygium sp.extracts, considering its possible potential to be used in therapy.Also, there is a need for further studies related to its efficacy and mechanism of hypoglycemic and antioxidant action.In order to do that, the procedures performed in this study were to obtain samples of Syzygium sp.commercial teas and prepare crude extracts by using a 1 : 200 infusion method simulating the domestic use; perform enzymatic inhibition trials before and after exposure to gastric fluid; determine the presence of total phenolics, alkaloids, tannins, flavonols, and flavones; evaluate antioxidant capacity; evaluate enzymatic kinetics against inhibitors; investigate the presence of α-amylase inhibitors and perform theoretical studies to elucidate the mode of interaction of amylase by the recognized inhibitors; and investigate the presence of trypsin inhibitors that are nutritionally undesirable.
erefore, using the extracts of Syzygium sp., the objectives of the study were to (a) investigate the presence of bioactive compounds inhibiting digestive enzymes alpha-amylase and trypsin, (b) elucidate the mode of interaction between alpha-amylase and the potential inhibitor luteolin, and (c) clarify the mechanism of the proposed hypoglycemic action of Syzygium sp.

Materials and Methods
e analyses were conducted in the Laboratory of Biochemistry (Institute of Biological and Natural Sciences (ICBN)), Laboratory of eoretical Biophysics (Institute of Exact, Natural and Education Sciences (ICENE)), and Laboratory of Food Engineering (Institute of Technological and Exact Sciences (ICTE))-all at the Federal University of Triângulo Mineiro (UFTM), Brazil.
2.1.Syzygium sp.Five samples (A, B, C, D, and E) of Syzygium sp.tea leaves were commercially acquired in the cities of Juiz de Fora, Lavras, and Uberaba, in the state of Minas Gerais, Brazil.For all samples, at the time of purchase, an award was provided by the supplier with information such as botanical analysis, organoleptic, physicochemical, microbiological, and general identification tests.
e tea leaves were immersed in boiling water at a ratio of 1 : 200 (gram/milliliter) for 10 minutes, simulating domestic use.
e mixture was filtered through organza tissue, and the crude extract obtained was used in all trials.
e product was read in a spectrophotometer at 540 nm.Each sample (A, B, C, D, and E) was evaluated at least three times in separate experiments.
e enzymatic inhibition of α-amylase was expressed in percentage.
e trypsin activity was determined according to the methodology proposed by Erlanger et al. [15].e absorbance of the product was read in a spectrophotometer at 410 nm.Enzymatic activity trials were performed in the presence of a simulated gastric fluid in order to simulate in vitro the digestion process in the stomach.To this end, the extracts were incubated with the simulated gastric fluid prepared according to e United States Pharmacopeia (USP) [16].e activity trials were then performed.
e enzyme inhibition was obtained by determining the slopes of the lines (absorbance vs. time) testing the activity control (without extract) and sample (with extract).e slope of the line is due to the rate of product formation per minute of reaction, and the presence of the inhibitor causes a decrease in this gradient, expressed in percentage terms.All analyses were performed in triplicate.

Enzymatic Kinetics of α-Amylase.
e enzymatic kinetics of α-amylase inhibitors were evaluated using standard experiment conditions [17] described for α-amylase activity.
e trials were performed with increasing concentrations of the substrate (10 to 100 μL starch) in the absence and presence of the samples (A, B, C, D, and E). e mode of inhibition was determined graphically from the Michaelis-Menten equation and subsequently, based on the 2 Journal of Chemistry Lineweaver-Burk plot, for determination of the affinity (K m ) and inhibition (K i ).

Quantification of Tannin Content, Flavonols, and Flavones and Qualitative Test for Alkaloids and Antioxidant
Activity.e total polyphenols were determined according to the methodology of Folin-Ciocalteu modified by Singleton and Rossi [18].e product was read in a spectrophotometer at 765 nm.Each sample (A, B, C, D, and E) was evaluated at least three times in separate experiments.Total phenol content was expressed as milligram of gallic acid equivalent per liter of infusion (mg GAE/L).
e tannin content was determined using the method described by Seigler et al. [19].Absorbance was recorded using a spectrophotometer at 725 nm, and the total tannin content was determined from a calibration curve constructed with tannic acid as standard.
e results were expressed as milligram of tannic acid equivalent (TAE) per milliliter of infusion (mg TAE/mL infusion).
e flavonoid content was determined by the colorimetric method of aluminum chloride as described by Chang et al. [20].
e flavonols and flavones content was determined by a calibration curve with different concentrations of quercetin.e results were expressed as milligram of quercetin equivalent (QE) per milliliter of infusion (mg QE/ mL infusion).
e identification of alkaloids in the extracts was performed according to the method described by Costa [21].About 50 μL of the samples were mixed with 50 μL of Mayer, Dragendorff, Wagner, and Bertrand reagents to verify the positivity of the reaction (precipitate formation).Atropine was used as positive control.
e antioxidant activity (AA) was evaluated by the ability of the samples to donate hydrogen to the DPPH, modifying the color of the solution according to the method described by Yamaguchi et al. [22].Each sample (A, B, C, D, and E) was evaluated at least three times in separate experiments.
e antioxidant activity of the infusion was obtained according to the formula:
e crystalline structure was obtained from the Protein Data Bank [25], whose PDB codes were for luteolin (4QYA) and alpha-amylase (1PPI).e docking protocol was employed based in the previous work of Rettondin et al. [26].

Statistical Analysis.
e tests were performed in triplicate, the data were entered in an Excel worksheet, and the results were analyzed using the SISVAR software.

Inhibition of α-Amylase.
e enzymatic inhibitions of α-amylase and trypsin, before and after exposure to a simulated gastric fluid, by the Syzygium sp.samples are shown in Table 1.
Considering the α-amylase activity, varied percentages of inhibition of the five samples (A, B, C, D, and E) were observed before (0% to 31.67%) and after simulated gastric fluid exposure (0% to 35.79%).It is observed that α-amylase in samples A, B, and C showed an increase in the percentage of enzymatic inhibition after exposure to the simulated gastric fluid.On the contrary, samples D and E reduced the α-amylase inhibition percentage after contact with gastric fluid.us, considering the different behaviors regarding enzymatic inhibition, it is suggested that, although all the samples are marketed as "jambolão," their phytochemicals content and biological activity may be different.It is emphasized here that the possible differences in phytochemical content and biological activity may be related to edaphoclimatic conditions, contaminations, and other factors.
Freitas et al. [27] demonstrated that infusions of Syzygium sp.leaves at different dilutions (1 : 20, 1 : 50, and 1 : 100) show high percentages of α-amylase enzyme inhibition before exposure to gastric fluid (above 90% in all dilutions).e authors suggested a possible relationship between amylase inhibition and the presence of phenolic compounds.Poongunran et al. [28], working with dried leaves of Syzygium cumini, found that extracts of methanol and water had high α-amylase inhibitory activity (98.3 ± 2.3 and 98.6 ± 1.6, respectively).e authors reported that the strong inhibition of the enzyme is due to the presence of oleanolic acid and ursolic acid, isolated from the leaves of Syzygium.
e extracts of Syzygium sp. at the 1 : 200 dilution showed lower enzymatic inhibition than those obtained in other studies, probably due to the higher dilution of the samples.It is suggested that the dilution is directly related to the possible hypoglycemic potential, considering the domestic use of leaf infusions of Syzygium sp.
After exposure of the extracts to a simulated gastric fluid, similar results were obtained by Freitas et al. [27], working with leaves of Syzygium sp., and by Pereira et al. [29], working with green and black teas.In both cases, the authors attributed the drop or disappearance of inhibition to the structural or conformational changes of the inhibitor caused by the acid medium.Such hypothesis suggests that although there is a hypoglycemic potential of Syzygium sp.tea, its passage through the gastric environment after ingestion would reduce the action that would occur later in the intestinal environment.However, biological trials are necessary to evaluate in vivo behavior and to validate such hypothesis.

Inhibition of Trypsin.
In α-amylase inhibition, trypsin results showed a mean variation ranging from 6.88% to 13.47% inhibition before the fluid and from 0% to 15.45% after the simulated gastric fluid (Table 1).
Trypsin samples A and B had a decrease in inhibition percentage after gastric fluid, and sample C had an increase in inhibition.Sample D, however, maintained the percentage of inhibition before and after the fluid, and sample E had a slight increase after exposure to the simulated gastric fluid.As it was observed for α-amylase, the trypsin inhibition results also suggest that the phytochemicals content and biological activity may be different in the commercialized samples.
Pereira et al. [30], working with inhibition of digestive enzymes by extracts of Hoodia gordonii, did not detect trypsin inhibition.On the contrary, Marques et al. [31], who evaluated the enzymatic inhibition by hops pellets, reported a high inhibition of trypsin before and after exposure to gastric fluid with values of 67.48% and 89.01%, respectively.e trypsin inhibitors present in plant extracts may lead to a decrease in protein digestibility, which may cause a reduction in the growth rate in animals and also a reduction in weight [31].
e passage of the Syzygium sp.extract through the gastrointestinal tract could cause structural changes and deformations in the inhibitors due to the acidic pH of the stomach, inactivating them [29,31].

Antioxidant Capacity and Total Phenolic Compounds
Content.
e antioxidant capacity and the total phenolic compounds content in the Syzygium sp.samples are shown in Table 2.
e mean percentage of antioxidant activity varied from 51.49% (sample A) to 89.00% (sample D). e total phenolic compounds content ranged from 21.00 mg GAE/L infusion (sample E) to 41.22 mg GAE/L infusion (sample D).
By relating the percentage of inhibition of amylase with antioxidant capacity and total phenolic compounds, it can be observed that sample D showed the highest values of antioxidant capacity and total phenolics with the highest percentage of α-amylase inhibition before exposure to the gastric fluid.On the contrary, sample C, with the second largest antioxidant capacity and third in the total phenolic content, showed no inhibition against α-amylase, before exposure to the simulated gastric fluid.ese findings reinforce the hypothesis of lack of standardization of the samples, which can, therefore, lead them to present different effects when it comes to helping in the treatment of diabetes and obesity.
Adefegha and Oboh [32] demonstrated that the soluble phenolic extract of Syzygium aromaticum possesses α-amylase and α-glucosidase inhibitory activity in a dosedependent manner.For the antioxidant activity, the authors evaluated the reducing power of the DPPH radical, and their results revealed that scavenged DPPH radicals in a dose-dependent manner (104.17-416.67 µg/mL).However, judging by the IC 50 , there was no significant difference in the DPPH radical-scavenging ability of free soluble phenolic extract (212.53 µg/mL) and bound phenolic extract (256.14 µg/mL).Adefegha and Oboh [32] suggested that the antioxidant properties of phenolic compounds could be part of the possible mechanism that induces the antidiabetic effects of the plant.
Ruan et al. [33], when evaluating the antioxidant activity of "jambolão" leaves, showed that the total phenolic content in the methanolic extract of Syzygium cumini was 610.32 ± 9.03 mg/g.e methanolic extract of the leaves was fractionated in water for the analysis of the antioxidant potential.At the concentration of 15.63 μg/mL DPPH, 91.03% of DPPH remnant was obtained, while at the concentrations of 31.25 μg/mL, 62.4 μg/mL, and 125 μg/mL DPPH, the remaining percentages were, respectively, 86.02%, 75.72%, and 56.85%.e authors, Pereira et al. [29], suggested that the antioxidant activity of Syzygium cumini leaves may be related to the presence of phenolic compounds.A significant linear relationship between antioxidant potency, free radical-scavenging ability, and the content of phenolic compounds of leaf extracts supported this observation.

Quantification of Tannin Content, Flavonols, and Flavones and Qualitative Test for Alkaloids.
e presence of  bioactive compounds in leaves of "jambolão" is supposedly related to its beneficial effects for the organism.In this sense, the phytochemical screening of the samples was carried out.e samples had tannin content ranging from 0.0279 ± 0.011 to 0.0369 ± 0.002 mg TAE/mL infusion.e highest content of flavonols and flavones was found in sample D (0.0201 ± 0.0079 mg QE/mL infusion).Additionally, alkaloid was not detected in any of the analyzed samples, but only precipitate formed in the positive control.Although other authors have detected the presence of alkaloids in samples of the genus Syzygium, it is known that several factors may interfere in the phytochemical composition (part of the plant used, harvest season, seasonality, cultivation and climate, and type of the extractor) [34][35][36] and explain the results obtained in the samples tested.
According to a study carried out by Faria et al. [37], the tannin content found in the functional extract (extract rich in anthocyanins extracted from the fruits of "jambolão")-0.2 mg TAE/100 g sample-was lower in comparison with the fruit-3.9mg TAE/100 g sample.High levels of tannins are undesirable in foods because of the astringent taste due to complexation with proteins and other effects that these compounds can cause.
Etxeberria et al. [38] report in their review article that 22 flavonoids were evaluated for their inhibitory activity against α-glycosidase.Among them, isoflavones and luteolin showed lower IC 50 .In addition, luteolin was more efficient than acarbose in inhibiting α-amylase.In contrast, in another study of this review, the authors concluded that the flavonoids tested did not have sufficient efficiency to delay or inhibit the release of glucose in the gastrointestinal tract [38].

Maldi-Tof Spectroscopy.
Analyses were performed in Maldi-Tof spectroscopy to investigate the presence of phenolic compounds known to be α-amylase inhibitors.By analyzing Figures 1(a)-1(e) regarding the Maldi-Tof spectrometry of the Syzygium sp.sheet extracts, it can be seen that there is a peak in the region of molar mass near luteolin (molar mass � 286.24 g/mol) in the five pure samples.Concerning the gastric fluid samples, the peak is not present in Sample A.
As demonstrated in some studies [39,40], phenolic compounds present great inhibition of digestive enzymes, raising hypotheses about their potential use in the treatment of diabetes and obesity.However, most of these works are limited to enzyme activity trials, and the possible mechanisms of these inhibitions are not described.Among the phenolics, the flavonoid luteolin (Figure 2) stands out in which inhibitory potentials of α-glycosidase and α-amylase have been demonstrated [38,39].Luteolin has been described as an important α-amylase inhibitor present in seeds of Syzygium cumini [39].However, its presence in leaves of Syzygium sp., and particularly in commercial tea, has not been reported yet.
In addition to luteolin, Karthic et al. [39] and Poongunran et al. [28] describe in their studies the presence of other α-amylase and α-glycosidase inhibitors in seeds and leaves of Syzygium cumini, respectively.ey are betulinic acid, ursolic acid, and oleanolic acid, all with molecular weight of 456.7 g/mol.However, in the present study, no peaks were found in this region in any of the samples.
Although the presence of luteolin has been suggested (except for sample A with fluid), the fact that it is a complex matrix may be the responsible for the variation of the profile of enzymatic inhibition between the samples, either by inactivation or stimulation.is variation might be associated with the lack of standardization of samples.

Enzymatic Kinetics.
Regarding the kinetic study, all samples showed uncompetitive inhibition against α-amylase as shown in Table 3.
e values of V max and K m obtained were 0.430 and 6.67, respectively, for all samples.In this type of inhibition, the inhibitor only binds to the enzyme-substrate (ES) complex.
e free enzyme does not interact with the inhibitor; i.e., the mixed inhibitor binds only to the ES complex of the enzyme leading to an inactive complex, ESI.Inhibition is not reversed by the increase in substrate concentration.e result is the apparent modification (decrease) of K m and V max [41].
In a study by Ponnusamy et al. [42], the aqueous extracts of the Syzygium cumini seed exhibited concentrationdependent (competitive) inhibition.In contrast, Tong et al. [12] demonstrated that water-soluble tannins isolated from the Eugenia jambolana methanolic tea inhibited α-amylase in a noncompetitive manner.

Molecular Docking.
Molecular docking was performed in order to theoretically assess the interactions between the pancreatic α-amylase and the luteolin compound.is approach predicts the conformation of a ligand within the active site of a receptor and the mode of binding with less energy between the ligand-receptor complex.Figure 3 shows the predicted lowest binding mode resulting from the docking simulations of α-amylase complexed with luteolin.In Figure 3, eight hydrophobic interactions and three hydrogen bonds between luteolin and the enzyme residues were identified.Regarding the hydrogen bonds, two of them (His201 and His299) appear in the X-ray crystal structure between α-amylase and the carbohydrate inhibitor, which is double the size of luteolin and has four histidines coordinating the protein-inhibitor hydrogen bond interactions [43].e carboxylic oxygens of the catalytic residues Glu233 and Asp300 form hydrogen bonds in the X-ray complex; therefore, in the predicted α-amylase-luteolin complex, they are present as hydrophobic interactions.e third residue of the catalytic triad Asp197 is located on the opposite side of the inhibitor binding cleft (according to the crystallographic structure) and does not have a close interaction with luteolin.However, hydrophobic stacking of aromatic residues with the inhibitor surface occurs in a similar manner in both structures.
AutoDock free-energy scoring function was used to estimate the complex binding energy (ΔG).e best-ranked α-amylase-luteolin complex from Figure 3 resulted in a stable structure (ΔG � −8.2 kcal•mol −1 ), when compared to the binding free energy calculated for the X-ray complex Although not always the most stable system leads to active conformation, the best-ranked conformation of α-amylase-luteolin complex resulted in a similar binding free energy as the native complex α-amylase-acarbose when performing redocking experiments.e use of the molecular docking tools in theoretical studies has become increasingly attractive and relevant to the understanding of protein-ligand interactions in order to support experimental results and predict binding modes.In this study, molecular docking simulations were performed to investigate the luteolin mechanism of interaction with α-amylase as a comparison with the carbohydrate enzyme inhibitor present in the experimental structure.Competitive inhibition could be expected as molecular docking analysis predicted that both acarbose and luteolin bind at the same α-amylase active site with similar binding free energy.erefore, it is hypothesized that there are other inhibitory compounds present in the aqueous extracts of Syzygium sp., as shown by the enzymatic kinetics assay.us, it is possible that, in addition to luteolin, other inhibitory compounds are  present, or that, corroborating the other findings, and the complex matrix of each extract is also responsible for the variation of the inhibitory profile.

Conclusion
e aqueous extracts of Syzygium sp., obtained by 1 : 200 infusion, present low percentages of inhibition of the digestive enzymes, suggesting a decrease in the potential hypoglycemic effect at this dilution.us, considering the different profiles of enzymatic inhibition, it is suggested that the phytochemical content and biological activity of samples may be distinct, in spite of their commercialization as "jambolão."Although the antioxidant activity of the tea was significant, the contents of phenolic compounds, flavonoids, flavones, alkaloids, and tannins were not expressive at the 1 : 200 dilution.
e results regarding the Maldi-Tof spectroscopy revealed a peak around 284 in the mass region (m/ z), suggesting the presence of luteolin.It was proposed that luteolin binds with α-amylase enzyme in a similar manner as the carbohydrate inhibitor present in the X-ray structure.
Even though both inhibitors bind with the catalytic active site, enzymatic kinetics trials demonstrated uncompetitive inhibition, indicating that there are other enzymatic inhibitors present.
erefore, the complex matrix of the Syzygium sp.extract may also be responsible for the variation of the inhibitory profile of the samples.
Considering that the phytochemical content and biological activity may be distinct among samples, the use of such extract by the population to aid in the treatment of diabetes and obesity is still not recommended.Additional in vivo studies should be conducted to elucidate the efficacy and safety of the use of extracts of Syzygium sp.

Data Availability
e data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
e authors declare that they have no conflicts of interest.

Figure 3 :
Figure 3: (a) Docking pose predicted between the pancreatic α-amylase (grey) and luteolin (yellow) with the active catalytic site enhanced.Amino acid residue side chains interacting via hydrogen bond with luteolin are highlighted.(b) 2-dimensional diagram projection of the hydrogen bonds and hydrophobic interactions shared between the enzyme amino acid residues and the ligand luteolin.

Table 1 :
Mean percentages of inhibition of digestive enzymes by aqueous extracts of Syzygium sp.obtained by infusion at a ratio of 1 : 200 (w/v) before and after exposure to a simulated gastric fluid for 5 enzyme trials.

Table 2 :
Antioxidant capacity and total phenolic content of the aqueous extracts of Syzygium sp.obtained by infusion at a ratio of 1 : 200.

Table 3 :
Enzymatic kinetics of the aqueous extracts of Syzygium sp.obtained by infusion at a ratio of 1 : 200.