Bio-Guided Fractionation Driven by In Vitro α-Amylase Inhibition Assays of Essential Oils Bearing Specialized Metabolites with Potential Hypoglycemic Activity

Type 2 diabetes mellitus (T2DM) is a metabolic disorder characterized by unpaired blood glycaemia maintenance. T2DM can be treated by inhibiting carbohydrate hydrolyzing enzymes (α-amylases and α-glucosidases) to decrease postprandial hyperglycemia. Acarbose and voglibose are inhibitors used in clinical practice. However, these drugs are associated with unpleasant gastrointestinal side effects. This study explores new α-amylase inhibitors deriving from plant volatile specialized metabolites. Sixty-two essential oils (EOs) from different plant species and botanical families were subjected to α-amylase in vitro enzymatic assay and chemically characterized using gas chromatography coupled to mass spectrometry. Several EOs were found to be potential α-amylase inhibitors, and Eucalyptus radiata, Laurus nobilis, and Myristica fragrans EOs displayed inhibitory capacities comparable to that of the positive control (i.e., acarbose). A bio-guided fractionation approach was adopted to isolate and identify the active fractions/compounds of Eucalyptus radiata and Myristica fragrans EOs. The bio-guided fractionation revealed that EOs α-amylase inhibitory activity is often the result of antagonist, additive, or synergistic interactions among their bioactive constituents and led to the identification of 1,8-cineole, 4-terpineol, α-terpineol, α-pinene, and β-pinene as bioactive compounds, also confirmed when they were tested singularly. These results demonstrate that EO oils are a promising source of potential α-amylase inhibitors.


Introduction
The term "diabetes mellitus" groups together a series of metabolic disorders characterized by hyperglycemia that is caused by the impairment of insulin secretion, action, or both. If not treated, chronic hyperglycemia, in association with diabetes, leads to long-term damage, dysfunction and organ failure especially in the eyes, kidneys, heart, nerves, and blood vessels [1]. Diabetes is classified into two broad etiopathogenetic categories: type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM). T1DM is related to hyperglycemia caused by an absolute deficiency of insulin secretion and requires exogenous insulin administration if a patient is to survive. T2DM affects 90 to 95% of diabetic patients with impaired glucose tolerance that is related to diminished tissue response to insulin and is often associated with relative insulin deficiency. A common therapeutic approach for TD2M, especially for patients with unpaired post-prandial blood glucose excursion, is to inhibit the Table 1 reports a list of the 62 EOs screened in this study. They belong to different botanical families (i.e., Annonaceae, Apiaceae, Compositae, Cupressaceae, Ericaceae, Geraniaceae, Lamiaceae, Lauraceae, Myristicaceae, Myrtaceae, Oleaceae, Pinaceae, Piperaceae, Poaceae, Rosaceae, Rutaceae, Santalaceae, Verbenaceae, Zingiberaceae), as described in Figure 1. Table 1. List of the investigated essential oils (EOs), the part of the plant used to obtain them, botanical and common names, and percentage of α-amylase inhibition activity measured by absorbance differences (Method A).

Optimization of the in vitro Enzymatic Test
EOs are poorly soluble in water, and, as in vitro enzymatic tests are carried out in an aqueous buffer solution, the choice of the solvent to be used as a "bridge" to solubilize the EO in the mixture test is the first aspect to be optimized, while avoiding interference with 1) enzymatic activity, 2) UV absorption, and 3) pH variation. Three solvents were tested as solubilizing solvents: ethyl acetate was the only one giving a clear solution when adding 50 μL of the EO solution, while methanol, and ethanol gave opalescent solutions. Absorbance at 540 nm and the pH variation of a 1 mL of buffer solution containing 50 μL of ethyl acetate were measured to evaluate any possible interference. Absorbance was found to be very low (0.080 ± 0.0003, n = 5) and pH variation negligible (from 7.00 to 7.13) and ethyl acetate was therefore selected as the "bridge" solvent. The required EO final concentration in the reaction mixture was set at 0.670 mgmL −1 to match the half maximal inhibitory concentration (IC50) of acarbose (0.601 ± 0.0673 mg mL −1 ). To minimize the amount of ethyl acetate in the reaction mixture, thus avoiding partial interference with the enzymatic activity, 10 μL of a 200 mg

Optimization of the In Vitro Enzymatic Test
EOs are poorly soluble in water, and, as in vitro enzymatic tests are carried out in an aqueous buffer solution, the choice of the solvent to be used as a "bridge" to solubilize the EO in the mixture test is the first aspect to be optimized, while avoiding interference with 1) enzymatic activity, 2) UV absorption, and 3) pH variation. Three solvents were tested as solubilizing solvents: ethyl acetate was the only one giving a clear solution when adding 50 µL of the EO solution, while methanol, and ethanol gave opalescent solutions. Absorbance at 540 nm and the pH variation of a 1 mL of buffer solution containing 50 µL of ethyl acetate were measured to evaluate any possible interference. Absorbance was found to be very low (0.080 ± 0.0003, n = 5) and pH variation negligible (from 7.00 to 7.13) and ethyl acetate was therefore selected as the "bridge" solvent. The required EO final concentration in the reaction mixture was set at 0.670 mgmL −1 to match the half maximal inhibitory concentration (IC 50 ) of acarbose (0.601 ± 0.0673 mg mL −1 ). To minimize the amount of ethyl acetate in the reaction mixture, thus avoiding partial interference with the enzymatic activity, 10 µL of a 200 mg mL −1 solutions in ethyl acetate of each investigated EO were added to the enzymatic assay reaction mixture.
The high volatility of EO components is the second aspect to be considered. The parameters to be defined in this respect are the headspace volume of the vial containing the reaction mixture, and whether the biological test must be run in sealed vials. The experiments demonstrated that the reaction must be carried out in closed vials to minimize the headspace and limit the loss of bioactive volatile compounds in the headspace/environment (data not shown). A 4.0 mL vial was used for all of the in vitro inhibition experiments as the total volume of the reaction mixture was 3.0 mL.

In Vitro α-Amylase Inhibition Test
The inhibitory activity against α-amylase of the EOs listed in Table 1 was tested in vitro using a spectrophotometric assay (carried out in triplicate for each EOs) to evaluate their antidiabetic/hypoglycemic properties. Acarbose was chosen as the α-amylase inhibitor positive reference. The percentage α-amylase inhibition data are reported in Table 1. They were determined by measuring the difference in absorbance as reported in the Experimental Section (see par. Section 3.2). The most active EOs were Eucalyptus radiata, Myristica fragrans, and Laurus nobilis, which are characterized by inhibition activities higher than, or very similar to, that of acarbose, 65%, 59%, and 51%, respectively. Within the Lauraceae family, in addition to laurel EO, Cinnamomum camphora (20%) showed interesting activity, although it was lower than that of acarbose. Similar considerations can be made for other botanical families, with several species presenting interesting inhibition activity, i.e., Myrtaceae (Corymbia citriodora 44%, Eucalyptus globulus 34%, Melaleuca viridiflora 28%, Myrtus communis 20%), Lamiaceae (Mentha arvensis 39%, Mentha x piperita 33%, and 40% for leaf and leaf-twig EOs, respectively), and Compositae (Artemisia vulgaris 48% and Matricaria chamomilla 32%).
The percentage of inhibition of the most active EOs was confirmed by comparing the amounts of maltose produced during the enzymatic reaction in absence and in presence of the possible inhibitor. A calibration curve was built by plotting the optical density at 540 nm of solutions of known concentration of maltose previously reacted with 3,5-dinitrosalicylic acid (DNSA) in function of maltose concentration. Knowing the final absorbance at 540 nm, the effective amount of maltose liberated during the enzymatic reaction was determined by interpolation from the regression line of A 540nm on maltose concentration (y = −0.048 + 1.4x, r 2 = 0.9985, 95% confidence interval for the slope 1.3 < β < 1.5; SEb = 0.10, t0.005(2), 3 = 3.18). The results obtained using the two approaches were compared, in particular for the three most active EOs, adopted as reference, i.e., laurel, nutmeg, and eucalyptus ( Table 2). Percentage inhibition values were found to match perfectly, as the variation coefficient (% CV) was always below 0.8%. Table 2. Comparison between the percentage of inhibition activity determined by absorbance differences (Approach A) and that obtained by applying the maltose calibration curve (Approach B). Twenty-three EOs belonging to different botanical families were found to be inactive in inhibiting α-amylase at the tested concentration under the applied experimental conditions: Cinnamomum zeylanicum and Cinnamomum cassia, several species belonging to Lamiaceae (i.e., lemon balm, clary sage, thyme, and patchouli), fennel and clove, among others.

Essential Oil Composition
All EOs, both active and inactive, were analyzed using gas chromatography, with flame ionization (FID) and mass spectrometry (MS) detectors, to characterize their composition and evaluate the relative percentage abundance of hydrocarbons and oxygenated compounds for correlation with the α-amylase inhibition activity. Table 3 reports the percentage of hydrocarbons and oxygenated compounds for each active EO, together with the most abundant compounds that characterize their composition. Table 3. List of the most abundant specialized metabolites and hydrocarbon and oxygenated compounds percentage composition of those EOs displaying α-amylase inhibitory activity. (The complete chemical composition of each investigated EO is reported in Table S1 of Supplementary Material). Bold highlighted the most abundant fraction.

Species Hydrocarbon Compounds
Oxygenated Compounds

List of the Most Abundant Components
Artemisia

Species Hydrocarbon Compounds
Oxygenated Compounds

Bio-Guided Assay Fractionation of Eucalyptus radiata and Myristica fragrans
Eucalyptus radiata and Myristica fragrans were selected from the most active EOs for further bio-guided assay fractionation; the first is representative of a class of EOs with a composition that is prevalently oxygenated monoterpenes, and the latter of a class of EOs that mainly consists of terpenic hydrocarbons. While inhibiting α-amylase to nearly the same extent, Eucalyptus radiata and Myristica fragrans EOs have significantly different chemical compositions, and their bioassay-oriented fractionation should thus provide good insight into the components that may be responsible for their biological activity.
The pure oxygenated and hydrocarbon fractions of the investigated EOs were isolated using automated flash chromatography and were individually subjected to α-amylase inhibitory assays. The amount of the EO (960 mg) to be fractionated was selected so that a suitable amount of each fraction could be recovered for the next biological testing.
Flash chromatography separations were carried out on pre-packed 50 µm silica gel cartridges using a gradient of 0-20% ethyl-acetate in petroleum ether. All of the hydrocarbons eluted during the isocratic step of the gradient with 100% petroleum ether, and the complete fractionation of the pure material was completed within 20 minutes, regardless of the EO being treated. The yields of the oxygenated and hydrocarbon fractions were 123.6 mg and 718.1 mg, respectively, for the Myristica fragrans EO, and 744.7 mg and 82.4 mg, respectively, for Eucalyptus radiata. Each isolated fraction was analyzed using GC-MS and GC-FID to assess the chemical composition of the fraction and its chemical homogeneity. Figure 2 reports the GC-MS patterns of the total Eucalyptus radiata and Myristica fragrans EO together with those of the corresponding hydrocarbon and oxygenated fractions. Table 4 reports the composition of total Eucalyptus radiata and Myristica fragrans EOs, and of their hydrocarbon and oxygenated fractions, as obtained by flash chromatography.  Table 4.   Table 4.  [18,19]. However, a proper comparison was not possible due to the absence of the positive control (i.e., acarbose) in the referenced publications. The bioactivity of other tested EOs, namely Cinnamomum camphora, Eucalyptus globulus, Hyssopus officinalis, Laurus nobilis, and Melaleuca viridiflora, can thus partially be ascribed to the presence of 1,8-cineole, which is the major compound in all of the above mentioned EOs. However, linear regression analyses (data not shown) did not reveal a significant positive linear relationship between the amount of 1,8-cineole in an EO and the observed percentage of α-amylase inhibition. This is probably because of combination effects with other EO components having an influence on 1,8-cineole overall activity. The hydrocarbon and oxygenated fractions of Myristica fragrans EO inhibited α-amylase by 11.1% and 15.0%, respectively. Unlike Eucalyptus radiata EO, the chemical composition of this EO mainly consists of hydrocarbons, namely sabinene, α-pinene, β-pinene, and limonene, whose relative percentage areas are 27.0%, 23.0%, 13.0%, and 10.0%, respectively, and that are present in the corresponding isolated fraction in similar percentages (i.e., 26.3%, 21.8%, 20.4%, and 15.3%, respectively). When tested as pure standards, at a concentration of 0.670 mg mL −1 , sabinene and limonene were inactive, while αand β-pinene inhibited enzymatic activity by 32 ± 1% (IC 50 1.05 ± 0.0252 mg mL −1 ) and 29 ± 1% (IC 50 1.17 ± 0.0233 mg mL −1 ), respectively. This result suggests that they are responsible for the observed bioactivity of the hydrocarbon fraction, but that their contribution is only partial compared to that of the whole EO. The oxygenated fraction mainly contains 4-terpineol which, similarly to its structural isomer α-terpineol, inhibits α-amylase by 40 ± 2% (IC 50 0.838 ± 0.0335 mg mL −1 ) when tested individually at a concentration of 0.670 mg mL −1 . Despite the evidence that has emerged from these experiments, further studies into the mechanism of enzymatic inhibition of both Myristica fragrans EO and its respective bioactive components are required to understand how they influence the overall EO activity.
The results obtained from the bioassay-oriented fractionation of Myristica fragrans EO have provided indications that allow us to, at least partially, understand the inhibition percentage results of other screened EOs. In addition to Myristica fragrans, the investigated EOs that contain high amounts of α and β-pinene are Cupressus sempervirens, Juniperus communis, Pinus mugo, and Pinus sylvestris EOs. However, unexpectedly, only the Myristica fragrans and Cupressus sempervirens EOs significantly inhibited α-amylase, while the others were inactive. In terms of chemical composition, Myristica fragrans and Cupressus sempervirens EOs mainly differ from the others because they are both free from trans-β-caryophyllene. Pearson's correlation coefficient was thus measured to define whether an association exists between the presence of trans-β-caryophyllene and biological activity. The results suggest that the variables are indeed negatively related (r = −0.4583), and the correlation was sufficiently high to warrant the rejection of the null hypothesis of zero correlation (t = −2.871, df = 31, p-value = 0.007).

Data Precision
The precision of the in vitro α-amylase inhibition test was evaluated in terms of repeatability (by repeating the enzymatic inhibition assay three times in the same day) and intermediate precision (by repeating the enzymatic inhibition assay three times every four weeks over a period of six months). Results were very satisfactory and never exceeded 8.1% for repeatability and 11.8% for intermediate precision. Table 5 reports the percentage relative standard deviation (RSD%) for inhibition tests carried out with acarbose (reference) and with laurel, eucalyptus, and nutmeg EOs. Similar precision values were obtained for all the tested EOs.

Reagents
Sixty-one EOs from different botanically authenticated species were supplied by Witt Italia SpA. Mentha x piperita EO (leaves) was obtained from fresh leaves (provided by Dr. Franco Chialva, Chialvamenta), and submitted to steam distillation. Table 1 reports the list of the EOs investigated, including botanical and common name, botanical family, and the part of the plant from which the EO was obtained. At least three different batches were considered for each EO. Pure standard samples of 4-terpineol, α-terpineol, 1,8-cineole, α-pinene, β-pinene, sabinene and limonene (purity > 98%) were purchased from Merck. The solvents were all HPLC-grade and obtained from Carlo Erba. Phosphate saline buffer, α-amylase from Aspergillus oryzae, maltose, acarbose, potato starch, and 3,5-dinitrosalicilic acid were also obtained from Merck.

In Vitro α-Amylase Inhibition Test
The in vitro inhibition test adopted in this study was modified from that of Sigma-Aldrich [20]. Three different solvents (i.e., methanol, ethanol, and ethyl acetate) were tested to select the best one to be used as "bridge" to solubilize the EOs into the test mixture. 15 mg mL −1 solutions of tangerine and chamomile EOs were prepared for each investigated solvent. For each solution, 50 µL were added to 1 mL of buffer solution and UV absorption at 540 nm and pH variation were assessed. The enzyme solution was prepared daily at a concentration of 33.3 µg/mL in a 0.02 M phosphate buffer (pH 6.9). The starch solution (1% w/v) was obtained by stirring 1 g of potato starch in the same buffer at 90 • C for 15 min. Then, 200 mL of the 3,5-dinitrosalicylic reagent was prepared as follows: first, 2.18 g of 3,5-dinitrosalicylic acid (DNSA) was dissolved in 40 mL NaOH 2 M and 100 mL of deionized water; 60 g of potassium citrate (Rochelle salt) was then added and the volume was made up to 200 mL with deionized water; the solution was finally heated to 40 • C to dissolve the potassium citrate. The reactant solution was stored in the dark and protected from CO 2 [21]. A 200 mg mL −1 solution in ethyl acetate was prepared and stored at 4 • C for each investigated EO, each standard sample, each isolated fraction, and for acarbose.
Sample mixtures were prepared as follows: 10 µL of each EO solution (corresponding to an absolute amount of 2 mg) was added to 1 mL of enzyme solution (corresponding to 1 unit of α-amylase) and pre-incubated for 5 min at 25 • C under constant stirring. Then, 1 mL of the starch solution was added and incubated for 3 min at 25 • C. Finally, 1 mL of the DNSA solution was added and left to react for 15 min at 90 • C. The mixture was then cooled and diluted with 9 mL of deionized water and the absorbance was measured at 540 nm. The standard test containing the non-inhibited enzyme was prepared as described above, with 10 µL of pure ethyl acetate instead of the EO solution. The final concentration of EOs and acarbose in the reaction mixture was 0.67 mg mL −1 (i.e., 2 mg in terms of absolute amount), which corresponds to IC 50 for acarbose.
The α-amylase inhibition activity of each investigated EO was measured using two different approaches. The first approach (Method A) relies on difference in absorbance and α-amylase inhibition activity is measured by applying the following Equation (1): where A Standard is the absorbance at 540 nm when the enzyme was not inhibited, A Standard blank is the absorbance of the standard blank solution containing the same reagents as the standard mixture, but with 1 mL of buffer instead of the enzyme, A Sample is the absorbance at 540 nm in the presence of the investigated inhibitor, and A Sample blank is the absorbance of the sample blank solution prepared with the same reagents as the sample mixture, including the 10 uL of the EO solution, but with 1 mL of buffer instead of the enzyme. Each blank underwent the same steps as the corresponding sample. A blank of each EO was performed to account for possible variations in absorbance due to differences in EO chemical composition. Acarbose was used as the positive reference; positive reference inhibition tests were carried out using 10 µL of acarbose solution at a concentration of 200 mgmL −1 in ethyl acetate (corresponding to an absolute amount of 2 mg). The second approach (Method B) measures amylase inhibition activity considering the differences in the amount of maltose generated by α-amylase activity via the reduction of 3,5-dinitrosalicylic acid to 3-amino-5-nitrosalicylic acid, by applying the following Formula (2): where C maltose sample is the concentration of maltose produced by α-amylase in the presence of EO or acarbose, and C maltose Standard is the concentration of maltose produced by the non-inhibited α-amylase.

Maltose Calibration Curve
Maltose solutions were prepared in 0.02 M phosphate buffer (pH 6.9) at seven different concentrations: 1.0, 0.75, 0.50, 0.25, 0.20, 0.15, and 0.10 mg mL −1 . Then, 1.0 mL of each solution was mixed with 1.0 mL of buffer solution and 1 mL of the DNSA solution and the mixture was left to react for 15 min at 90 • C. Once the reaction was finished, the mixture was cooled and diluted with 9 mL of deionized water and the absorbance was read at 540 nm. The maltose calibration curve was built by plotting the absorbance at 540 nm as a function of maltose concentration (mg mL −1 ).

Flash Column Chromatography
EO fractionation was carried out on a flash column chromatography system PuriFlash 450 by Sepachrom, equipped with both UV and Evaporative Light Scattering (ELSD) detectors. Stationary phase: Sepachrom silica Daily 50 µm; mobile phase: petrolether (A) and ethyl acetate (B), flow 25 mL min −1 . Linear gradient elution was adopted from 100% of A to 80% of A and 20% of B in 20 min.

Conclusions
This study explores the α-amylase inhibition ability of 62 EOs of different composition and belonging to different plant families. Under the applied experimental conditions, three EOs showed high inhibitory capacity (Eucalyptus radiata, Laurus nobilis, and Myristica fragrans) that was comparable or slightly higher than that of acarbose, which was chosen as the positive control. The results showed that the EO components seem to act synergistically, as the total EO percentage inhibition activity is higher than those of both the combined isolated fractions and pure compounds. Moreover, an interesting number of both hydrocarbon and oxygenated compounds. were characterized as good α-amylase inhibitors, with percentage inhibitions of about 30% (i.e., 1,8-cineole, 4-terpineol, α-terpineol α-pinene, and β-pinene). These results demonstrate that EO oils are a promising source of potential α-amylase inhibitors.