A considerable portion of the world population uses medicinal plants to treatchronic diseases. In this context, hibiscus (Hibiscussabdariffa L. - Malvaceae) stands out for its supposed hypoglycemicand slimming effect. The present work aimed to carry out enzyme inhibition testsfor aqueous hibiscus extracts, as a possible mechanism of action related to thesupposed slimming and hypoglycemic effects. The inhibition of four digestiveenzymes (α-amylase, α-glucosidase, lipase and trypsin) was tested before andafter exposure to a simulated gastric fluid. In addition, a molecular anchoragestudy was carried out, in order to highlight possible molecular interactionsbetween target and ligand. The results showed that the aqueous extract ofhibiscus, in the proportion 1:10 (w/v), inhibits only α-glucosidase. It wasobserved that cyanidin-3- sambubioside, has interaction with the enzyme withproperties similar to acarbose, which corroborates the possibility of thepresence of an inhibitory effect in the aqueous extract of hibiscus.

A considerable portion of the world population uses medicinal plants to treat chronicdiseases. The use of medicinal plants in diabetes and obesity, sometimes withoutproper scientific proof, has been gaining ground in the treatment of these diseases.In Brazil, as in many countries, pharmacovigilance about medicinal plants is veryfragile, limiting their safety and efficacy[1]. In this context, the hibiscus (Hibiscussabdariffa) has gained prominence in recent years for its supposed andwidespread hypoglycemic and slimming effect.

Based on the above, the present study aimed to carry out tests to inhibit digestiveenzymes by aqueous hibiscus extract, as a possible mechanism of action related tothe supposed slimming and hypoglycemic effects. For this, the inhibition of theenzymes α-amylase, α-glucosidase, lipase and trypsin was tested before and afterexposure to a simulated gastric fluid. In addition, a molecular anchorage study wascarried out, in order to highlight possible molecular interactions between targetand ligand.

The hibiscus samples were collected at the Federal University of Lavras, latitude21º 14’43’’, longitude 44º 59’ 59’’ and altitude 3015,09ft, in October 2015,identified by Herbário ESAL and dehydrated in the greenhouse. The extract wasprepared according to domestic and popular use, from the chalices of the plant,by the method of infusion for 10 minutes in the proportion of 1:10 (w/v).

α-glucosidase was obtained from the fresh porcine duodenum, the swine pancreaticenzymes were: trypsin and lipase type II (MERCK) and α-amylase type VI(SIGMA).

The enzyme activity was performed in a 4-stage kinetic assay. α-glucosidaseaccording to Kwon et al.[2], α- amylase according to Noelting andBernfeld[3], trypsinwas determined by the methodology of Erlanger et al.[4] and the lipase according toSouza[5].

The enzymatic activities were expressed in units (U), which corresponds to theformation of μmol of product per minute under the test conditions. For thispurpose, the absorbance values are converted into µmol of product formed bymeans of standard curves and, in the case of trispsin, by the factor of Erlangeret al.[4],which is based on the substrate's molar extinction coefficient. The value of 1EIU (enzyme inhibition unit) corresponds to the total inhibition of 1U.

Simulated gastric fluid was prepared, according to The United States Pharmacopeia- USP[6], with the aim ofsimulating gastric passage in vitro. The hibiscus extract wasincubated with prepared according to for 1 hour in a water bath, at 37°C. Afterthis period, it was neutralized with sodium bicarbonate and the enzyme activitytests were performed again.

For the molecular anchoring studies, the crystallographic structure of theα-glucosidase protein complexed with the acarbose inhibitor (FIGURE 1) obtained from the“Protein Data Bank” (PDB - codes: 3TOP, resolution 2.88 Å) was used. Thecomputational simulation of molecular anchoring (“docking”) is one of the mostimportant techniques for investigating the molecular interactions between targetand ligand. Molecular anchoring calculations were performed using the MolegroVirtual Docker (MVD) program, which allows determining the most likelyconformation of the ligand in the enzyme. The identification of the ligandconformation is done through the evaluation of several candidates (ligandconformations) estimating the energies of their interactions with theenzyme.

For the study in question, five compounds were selected, according to theliterature, as possible responsible for the inhibitory effect of hibiscusextract, namely: delphinidine-3-sambubioside, cyanidin-3-sambubioside[7-12], citric acid, hydroxycitric acid and hibiscusacid[9,11-15].The three-dimensional structures of these selected compounds were built usingthe PC Spartan Pro program and their partial atomic charges were calculatedusing the semi-empirical method AM1. This procedure is necessary to obtain theinitial conformation of the compounds, which is important for the study ofmolecular anchoring.

After the construction of the 3D structures of the compounds, they weretransferred to the MVD program, where each ligand was anchored in the activesite of the enzyme α-glucosidase. In this step, the identification of theligand's interaction modes is interactive, evaluating a number of solutions(conformation and orientation of the ligand) and estimating the energy of itsinteractions with the protein.

The results of the inhibition of digestive enzymes by the aqueous hibiscus extract,before and after its exposure to the simulated gastric fluid, are shown inTABLE 1.

As noted, there was inhibition only of the enzyme α-glucosidase, of 128.3 EIU or57.14%, in the 1:10 dilution (w/v) of the aqueous extract of hibiscus.

From the bibliographic survey of the main molecules present in the extract and in theselection of the five most cited (delphinidine-3-sambubioside,cyanidin-3-sambubioside, citric acid, hydroxycitric acid and hibiscus acid), thestudy was carried out by molecular anchoring in active site of the α-glucosidaseenzyme. A cavity prediction algorithm based on a 3D box was used to generate theα-glucosidase binding site using the MVD program. The cavity volume was 259.07 Å3and is shown in FIGURE 1 togetherwith the inhibitor acarbose (compound crystallized at the active site).

The five compounds were anchored in the active site of α-glucosidase and compared tothe active ligand (acarbose). The energies of intermolecular interaction andhydrogen ligand-protein binding were calculated to better understand what are thevariations between the modes of interaction of the compounds in the active site ofthe enzyme and to verify which factors are responsible for the activity of thecompounds. All of these interactions are highlighted in FIGURE 2.

TABLE 2 shows the values ofprotein-ligand intermolecular interaction.

The acarbose redocking study on the α-glucosidase active site (FIGURE 3) was carried out with the aimof validating the calculation methodology used for the five compounds studied, andalso to know the main interactions that this inhibitor (already used in therapy)performs at the active site of the enzyme, comparing with the results obtained forcyanidin-3-sambubioside.

Even after the passage of the extract through the simulated gastric fluid,α-glucosidase inhibition was maintained in vitro. The percentage ofinhibition found, falls within the range considered ideal (40% - 85%) of goodα-glucosidase inhibitors according to Kwon et al.[2]. The inhibition of α-glucosidaseprovides a decrease in caloric availability, in addition to contributing to a dropin postprandial hyperglycemia, due to the lower intestinal absorption ofcarbohydrates, which may suggest one of the possible mechanisms of action ofhibiscus.

Buchholz and Melzig[16] conducted ascreening with a range of medicinal plants relating them to the inhibition of lipaseand α-amylase. In the study, the methanolic extract of H.sabdariffa and that of several other plants were used. H.sabdariffa extract was the most effective lipase inhibitor (IC50: 35.8± 0.8 µg/mL) and good α-amylase inhibitor (IC50: 29.3 ± 0.5 µg/mL) which, as opposedto that found in our studies working with the aqueous extract, may suggest new formsof preparation, more effective and with greater therapeutic power.

In another study, Shadhan et al.[17] when evaluating the inhibition of α-glucosidaseby methanolic extract of H. Sabdariffa, observed that the enzymaticinhibition reveals a gradual pattern with increasing concentrations of extract andmethanolic fractions, which is very similar to that characterized for acarbose(positive control of the study). In addition, it was observed that the ethyl acetatefraction showed high inhibitory activity when compared to acarbose at the sameconcentration. In the study, a very similar inhibition was detected between theethyl acetate fraction and acarbose, a drug used worldwide, reinforcing therelevance of research on the therapeutic potentials of hibiscus as an aid in thecontrol of body weight and blood glucose.

The analysis of the results raises some questions. The first is the preparation ofthe extract, showing that different solvents extract different molecules. Thus,cultivation and the extraction method are factors that directly influence theeffectiveness of the plant, requiring standardization for greatereffectiveness[18].

On the other hand, inhibition of α-glucosidase alone is more interesting for thehypoglycemic function, given the decrease in some side effects, such as thepancreatic hypertrophy observed by Silva and Silva[19] caused by inhibition of trypsin, and fermentationabnormal bacterial digestion of undigested carbohydrates in the colon by α-amylaseinhibition, described by Kwon et al.[2].

In another study of enzymatic inhibition, among the few produced with aqueous extractof Hibiscus sabdariffa, Ademiluyi and Oboh[20], observed the inhibition ofα-glucosidase (IC50: 187.9 ± 10.2 µg / ml) and α-amylase (IC50: 25.2 µg / ml ± 2.4µg / ml). With that, it is returned again to the fact of the great variation ofresults, either by the scarcity of researches with the extract in its most used formdomestically, or by the lack of methodological standardization.

A review paper published by Da-Costa-Rocha et al.[21], attributed to anthocyanins,flavonoids and organic acids as being the main molecules with pharmacologicalpotential present in Hibiscus sabdariffa. Among the anthocyanins,delphinidin-3-sambubioside (delphinidin-3-O-(2-ObD-xylopyranosyl)-bD-glucopyranoise), named hibiscin, andcyanidin-3-sambubioside (cyanidin-3 -O- (2-ObD-xylopyranosyl)-bD-glucopyranoside),called gossy picyanin, from the calyx of the plant[7-12].

Regarding organic acids, Hida et al.[13], demonstrated that hydroxycitric acid is the mainconstituent of this class found in the calyx of the plant. In addition to this, twoother components are present in large quantities in the goblets of Hibiscussabdariffa and can be related to their enzymatic inhibition, beingcitric acid and hibiscus acid[9,11,12,14,15].

Considering the five molecules analyzed in the present study(delphinidine-3-sambubioside, cyanidin-3- sambubioside, citric acid, hydroxycitricacid and hibiscus acid) and as can be seen in Table2, the compound cyanidin-3-sambubioside was the one that presented a morestable value of intermolecular interaction energy, that is, it has a greaterinteraction with the active site of α-glucosidase and, consequently, a greaterinhibitory potential. In addition, it was the one that was best positioned insidethe active site, presenting a more stable hydrogen bonding energy than the othercompounds, -27.90 kcal mol-1. In other words, it presented a total of sixteen“hydrogen bonding” interactions between groups containing electronegative atoms inthe amino acid residues of the active enzyme site, such as Gln1158; Pro1159;Lys1460; Asp1157; Asp1526; Arg1510 with alcoholic and phenolic hydroxyls incyanidin-3-sambubioside.

The RMSD value between the acarbose structures superimposed on the active site was0.99 Å (FIGURE 3A). According tothe literature, an RMSD less than 2.00 Å is considered satisfactory, thuscorroborating the protocol used for molecular anchoring calculations. The energiesof intermolecular interaction and hydrogen bonding obtained in the redocking were-219.80 and -28.41 kcal mol-1, respectively. It was observed that acarbose performshydrogen interactions similar to cyanidin-3-sambubioside at the active site of α-glucosidase, with the amino acid residues Asp1157; Asp1526 and Arg1510. That is,with molecular anchoring studies it is possible to suggest thatcyanidin-3-sambubioside may be the molecule responsible for the invitro inhibition of α-glucosidase observed.

Finally, after in-vitro scientific verification of the significantinhibition of alpha-glucosidase against the aqueous extract of Hibiscussabdariffa, it is extremely important to raise public awareness aboutthe range of possible adverse effects resulting from the use of the plant. Morecommonly, gastrointestinal effects such as abdominal pain, flatulence and diarrheahave been observed[22]. Also, dueto its hypoglycemic action, dose-dependent hypoglycemic effects can be deducted fromthe study, which can be quite harmful. Furthermore, in another study, it was foundthat 14% of respondents claimed to be aware of cases of hypotension as a result ofexcessive use of Hibiscus sabdariffa teas[23].

In view of this, and in view of the absence of in vivo studies todetermine adequate and safe doses, caution is essential when using the plantextract, since, even with beneficial actions found, the harmful effects of possibleadverse effects can cover the expected benefits.

The results showed that the aqueous extract of hibiscus, in the proportion 1:10(w/v), inhibits only α- glucosidase among the enzymes tested. The enzyme inhibitionis maintained even after the extract passes through the simulated gastric fluid.From the molecular anchorage studies between α-glucosidase and the compoundsevaluated, it is concluded that cyanidin-3-sambubioside, has interaction with theenzyme with properties similar to that of acarbose, a drug that is already widelyused, which shows the inhibitory effect by the aqueous extract of hibiscusfound.

The results obtained in the inhibition of α-glucosidase in vitrowere richly elucidated by the theoretical assay, demonstrating the high therapeuticpotential of hibiscus and the need for further studies with the objective ofstandardizing the extract, definition of possible therapeutic dose, in addition toaccurate assessment efficiency and safety.