Characterization and Potential Antidiabetic Activity of Proanthocyanidins from the Barks of Acacia mangium and Larix gmelinii

Proanthocyanidins in ethanol extracts from the barks of Acacia mangium and Larix gmelinii were analyzed by gel permeation chromatography, MALDI-TOF/TOF MS, and HPLC/MS. -e inhibitory effects of proanthocyanidins and acid-catalyzed hydrolysis of proanthocyanidins against carbolytic enzymes were also tested. A significant relationship between carbolytic enzymes inhibition and degree of polymerization was established, showing that the degree of polymerization is a major contributor to the biological activity of the proanthocyanidins from both types of woody plant bark. -e results indicate that proanthocyanidins from the barks of A. mangium and L. gmelinii have potential antidiabetic properties.


Introduction
Proanthocyanidins (PAs), also known as condensed tannins, are widely distributed in almost all plant-based foods and beverages and are comprised of oligomerized/polymerized flavan-3-ol monomer units [1] with molecular weights between 500 and 30000 Da [2].e monomeric flavanols differ in their hydroxylation patterns and stereochemistry at C-3. e most common monomers are the diastereomers (+)-catechin/ (−)-epicatechin, (−)-gallocatechin/(−)-epigallocatechin, and (+)-afzelechin/(−)-epiafzelechin, and their respective oligomeric components and polymers are called procyanidins, prodelphinidins, and propelargonidins [2].Flavanol monomers are usually linked by C4-C6 or C4-C8 bonds (B-type PAs).In some plants, compounds with an additional C2-C7 ether linkage can occur (A-type PAs).e degree of polymerization (DP) varies over a broad range, from dimers up to about 200 monomeric units [3].For extracts rich in procyanidins, propelargonidins, and prodelphinidins, their DP and types of flavanols can be analyzed by using the method of acid-catalyzed hydrolysis in the presence of excess phloroglucinol, and the reaction mechanism is displayed in Figure 1 [4].For extracts rich in some uncommon PAs such as profisetinidin and prorobinetinidin, their DP are usually analyzed by using MALDI-TOF/MS and gel permeation chromatography for that interflavanyl bond of profisetinidin and prorobinetinidin is stable in acid hydrolysis [5].Owing to the special antioxidant properties and other physiological activities of PAs, known to reduce risk factors associated with certain types of diseases, the chemical connectivity and biological activities of PAs have been extensively studied [1,[6][7][8].
Acacia mangium bark extracts (ABE) and Larix gmelinii bark extracts (LBE) can be used to produce tanned leather base on the PAs-protein interaction, which transforms biodegradable raw hide into leather [9][10][11].Moreover, many scholars have attempted to explain their chemical structures and biological activities of PAs [6,9,10,12].Large quantities of L. gmelinii and A. mangium barks, which are rich in PAs, are currently being wasted in China.In early stage, we have detected the inhibitory e ects of Acacia mearnsii PAs on carbolytic enzymes, results proved that Acacia mearnsii PAs with the low DP ranging from 1 to 11 exhibited a stronger inhibition against α-glucosidase and a mild inhibition on α-amylase, we could just nd that PAs inhibited α-amylase might dominantly due to its DP, and we could not conclude if the DP of PAs play a predominant role on α-glucosidase inhibition [5].erefore, the purpose of this study was to characterize PAs in crude ethanol extracts from the barks of L. gmelinii and A. mangium and determine the inhibition of PAs and acid-catalyzed hydrolysis of PAs on carbolytic enzymes.By using these methods to further verify our previous deduction, the relationship between inhibitory e ects on α-glucosidase and DP was found.At the same time, resources might be utilized comprehensively.

Sample Preparation.
e dried barks were ground in a small industrial pulverizer, and the powder was passed through a 5-mesh sieve with 4 mm openings.en, 25 g of the resulting power was defatted twice with hexane (1 : 5, w/v) and stirred at 300 rpm.Defatted barks were resuspended in 50% ethanol (375 mL), and then ultrasonicassisted extraction was performed for 30 min at 50 °C.Two extractions were performed for each sample, the combined solution was evaporated, and remaining aqueous phase was lyophilized to afford ABE and LBE. e acquired extracts were freshly dissolved in dimethyl sulfoxide as a 50 mg/mL as the stock solution and diluted with water or sodium phosphate buffer for total polyphenol content (TPC) and carbolytic enzymes inhibition analyses.

HPLC/MS Analysis.
e HPLC/MS analysis was conducted as our previous work with some modifications [5].
e modification was that we used an Agilent 1260 Diode Array Detector HPLC platform connected with a Agilent 6130 MS to tentatively identify PAs using the combination of MS and UV-visible spectra.
e parameter setting was detailed in our previously published protocols [5].Identities of the compounds were determined by comparing the observed [M + Na] + with theoretical values calculated using the following formula [13]: where EC, R, F, EGC, and GAL correspond to the number of (epi)catechin, robinetinidol, fisetinidol, (epi)gallocatechin, and galloyl moieties, respectively, and A and B represent the number of A and B linkages.
Gel permeation chromatography (GPC) was performed to verify the molecular weight (MW) of PAs in the extracts.
e GPC analysis was conducted using a Waters 1515 HPLC system with a UV detector at 280 nm.A 10 µm Styragel HT 3 column (i.d., 300 × 7.8 mm; Waters, Florida, USA) and a 10 µm Styragel HT 4 column (i.d., 300 × 7.8 mm; Waters) were connected in series.e extracts were firstly dissolved in tetrahydrofuran (about 5 mg/mL), separations and analysis were conducted as our previous protocols [14].

Total Polyphenol Content (TPC).
e TPC was measured using the Folin-Ciocalteu assay as our previously published methods [14,15].Results are presented as milligrams gallic acid equivalent (GAE)/g of dried extract.

Acid Catalysis of PAs in the Presence of Excess
Phloroglucinol.ABE or LBE (100 mg) was dissolved in 20 mL of freshly prepared methanol solution consisting of 0.2 N HCl, 50 g/L phloroglucinol, and 10 g/L ascorbic acid [16].e solution was maintained at 55 °C for 30 min to allow the reaction to proceed, and the reaction stopped by adding an equal volume of 200 mM aqueous sodium acetate.
en, the organic solvent was removed by evaporation.Finally, the acid-catalyzed hydrolysis PA solutions were lyophilized.e lyophilized samples were dissolved in dimethyl sulfoxide as a 250 mg/mL stock solution and diluted in sodium phosphate buffer for carbolytic enzymes inhibition analyses.

Carbolytic Enzyme
Inhibition. α-Amylase and α-glucosidase are the important enzymes associated with type 2 diabetes mellitus, and consequently, inhibition of these enzymes is postulated to be a preventive treatment among currently available antidiabetic therapeutic methods [17].
e inhibitory effects of both bark extracts on α-amylase were performed using turbidity measurements, and details of the procedures were provided in our previously published methods [5].e percentage of inhibition was calculated using the following equation: where AUC S is the area under the inhibitory curve and AUC C is the area under the control curve.IC 50 can be defined as the concentration of inhibitor that produces 50% inhibition of enzyme activity under a specified condition and was determined by linear interpolation of the percentage of inhibition using an inhibitor concentration curve.e inhibitory effects on α-glucosidase were assayed using our previously described method [5,14].e absorbance was read at 405 nm, and results were calculated using the following equation: where A C denotes the control sample absorbance and A S denotes the sample absorbance.Results are expressed as the sample concentration (μg/mL) required to inhibit 50% of the enzyme activity (IC 50 ).
Journal of Chemistry 2.8.Statistical Analysis.Samples were analyzed in triplicate.All data are expressed as mean ± one standard deviation (SD).Statistical analyses were performed using Origin software (OriginLab, Northampton, MA, USA)., respectively [18].We did not detect any PAs with DP greater than 3 in both bark extracts, which may be due to the fact that ESI works poorly for detecting PAs with a higher molecular weight [13].All the PAs detected in Table 1 were B-type linkages and some of them were isomers.Chemical connectivity of the various isomers was not determined.

MALDI-TOF/TOF MS and GPC Analysis of PAs.
Because larger PAs were found difficult to be detected by ESI, we used MALDI-TOF/TOF MS to identify PAs with a DP greater than three.Figures S3 and S4 show the MALDI-TOF positive-ion reflectron mode mass spectra of PAs in ABE and LBE recorded as sodium adduct ions.
As shown in Table 2, all PAs in LBE appeared to consist of (epi)catechin and (epi)gallocatechin, linked through C 4 -C 6 or C 4 -C 8 bonds.However, PAs from ABE were different and appeared to consist of robinetinidol, fisetinidol, and gallocatechin, possessing C 4 -C 6 or C 4 -C 8 linkage.However, we could not deduce the order of linkages.A total of 16 PAs were detected in the extracts (Table 2), corresponding to a wide variety of structures, including trimers to heptamers of procyanidins, prodelphinidins, profisetinidins, and prorobinetinidins with only B-type linkages, one of them galloylated.We did not detect any PAs with a DP greater than seven by MALDI-TOF/TOF MS, perhaps because Na works poorly for PAs with a DP greater than eight [13].
To further determine the properties of PAs in extracts, GPC was used to analyze MW.Results are shown in Figure 3.
e maximum MW of 3100 Da was observed among PAs from ABE, which was higher than those of LBE.

Determination of TPC.
e values of TPC varied from 340 (LBE) to 415 (ABE) mg GAE/g of the dried extract.e results indicated that both extracts from woody plant barks are rich in PAs and possess a high polyphenol content.

α-Amylase and α-Glucosidase Inhibition.
As shown in Figure 4, LBE at 40 μg/mL and ABE at 30 μg/mL can cause obvious inhibitory effects on α-amylase.When increasing the concentration to 50 μg/mL, more than 70% inhibition can be achieved.From the dose-response curves (Figure 5(a)), the two plant extracts clearly display inhibitory activity against α-amylase, with IC 50 values of 29.7 ± 2.5 μg/mL (LBE) and 19.1 ± 3.4 μg/mL (ABE).Dose-response curves for α-glucosidase inhibition are presented in Figure 5(b).For both extracts, inhibition against α-glucosidase is clearly observed, with similar IC 50 of about 10.1 ± 1.9 μg/mL (LBE) and 22.0 ± 1.7 μg/mL (ABE).As a reference, inhibition by acarbose displayed in Figure 6 was measured under the same conditions, and the IC 50 values were calculated as 8.25 ± 4.3 and 164.21 ± 3.5 μg/mL for α-amylase and α-glucosidase, respectively.ese values are very similar to those presented in previously published works [5,19,20].For convenient comparison, both plant extracts exhibited mild α-amylase inhibition activity and strong α-glucosidase inhibition activity, which could potentially prevent abnormal bacterial fermentation of undigested carbohydrates in the colon [21].
When acid-catalyzed hydrolysis of PAs occurs in the presence of excess phloroglucinol, PAs are decomposed into subunit compositions including terminal subunits, such as flavan-3-ol monomers (catechin or epicatechin), and extension subunits [4].We also detected inhibitory effects against α-amylase and α-glucosidase.e results are presented in Figures 7 and 8.After acid-catalyzed hydrolysis of the PAs of LBE, no inhibition against α-amylase at high concentrations (5 mg/mL) was observed; however, the starch degradation rate improved, perhaps owing to an increase in the mass transfer effect of some compounds in the sample which increases the likelihood of contact between the α-amylase and starch.Furthermore, the results suggest that DP effectively contributes to the inhibition of α-amylase.Similar results were previously reported.More specifically, the addition of gelatin to bind and precipitate PAs greatly diminishes inhibitory activity against α-amylase [20,22].
e study also implicated PAs as active substances in LBE.In our study, PAs of acid-catalyzed hydrolysis from ABE showed weaker inhibitory effects against α-amylase, 4 Journal of Chemistry including no inhibition at 0.5 mg/mL and approximately 62% inhibition at 1.8 mg/mL. is phenomenon may be the result of PAs degradation in this chemical environment, rendering them unable to fully catalyze.Moreover, pro setinidin, prorobinetinidin, and prodelphinidin were detected in A. mangium using MALDI-TOF/MS [18].e absence of 5-hydroxy groups in the chain extender units of pro setinidins and prorobinetinidins leads to stable inter avanyl bonds, which prevent acid hydrolysis [5,7].After acid-catalyzed hydrolysis of PAs in the presence of excess phloroglucinol, inhibitory effects against α-glucosidase were similar.Consequently, we hypothesize that the monomers themselves may possess certain inhibitory e ects against α-glucosidase.
Here, we con rmed our prediction by determining the inhibitory e ects of epicatechin on α-glucosidase.e results    Journal of Chemistry are displayed in Figure 9. Epicatechin exhibited inhibitory activity against α-glucosidase with an IC 50 of approximately 200 ± 3.2 μg/mL.e result indicates some inhibitory e ects of the monomer against α-glucosidase.However, epicatechin cannot be used to con rm prediction that PAs become depolymerized by acid in the presence of excess phloroglucinol, releasing terminal subunits such as avan-3-ol monomers (catechin and epicatechin) and extension subunits such as electrophilic avan-3-ol intermediates.e electrophilic intermediates can be trapped by nucleophilic reagents to generate analyzable adducts (Figure 1) [4].Additionally, avonoids often exhibit synergistic e ects on biological activities [23].Based on the inhibitory e ects of epicatechin against α-glucosidase, the result of DP not playing the predominant role for α-glucosidase inhibition was proved, which may also be a ected by the nature of inter avanyl bonds of PAs.demonstrate successful carbolytic enzyme inhibition by PAs.Furthermore, DP of PA was shown to play an important role in the inhibitory e ect on α-amylase and α-glucosidase, but not a predominant role for α-glucosidase inhibition.eir potential antidiabetic e ects were tentatively investigated.However, further research is still needed to investigate the biological e ects of these bioactive compounds in vivo.

Conclusions
Data Availability e data used to support the ndings of this study are included within the article.

Figure 1 :
Figure 1: Reaction mechanism of acid-catalyzed hydrolysis in the presence of excess phloroglucinol.

Figure 5 :
Figure 5: Dose-response curves of α-amylase (a) and α-glucosidase (b) inhibitory activities of ABE and LBE.Data are presented as mean ± one standard deviation.

eFigure 6 :
Figure 6: (a) Kinetic curves of starch hydrolysis by α-amylase in the presence of acarbose.Dose-response curves of acarbose for inhibitory activities against α-amylase (b) and α-glucosidase (c).Data are presented as mean ± one standard deviation.

Figure 7 :
Figure 7: Kinetic curves of starch hydrolysis by α-amylase under di erent concentrations of acid-catalyzed hydrolysis of proanthocyanins from ABE and LBE.

Figure 8 :
Figure 8: Dose-response curves for α-glucosidase inhibitory activity at di erent concentrations of acid-catalyzed hydrolysis of proanthocyanins from ABE and LBE.Data are expressed as mean ± one standard deviation.

Figure 9 :
Figure 9: Dose-response curve for α-glucosidase inhibitory activity of epicatechin.Data are expressed as mean ± one standard deviation.
As shown in Table1, 12 components were found to be present in the extracts and tentatively identified as monomers, dimers, and trimers.Since catechin and epicatechin (Figure2(a)) were detected in LBE, the peaks associated with the LBE group were separated by intervals of m/z 288, corresponding to the incremental mass of (epi) catechin extension.Peaks associated with the ABE groups were separated by intervals of m/z 272, m/z 288, and m/z 304, corresponding to the incremental mass of (epi)fisetinidol (Figure 2(b)), (epi)robinetinidol (Figure 2(c)), and (epi) gallocatechin (Figure 2(d))

Table 1 :
Components identified in ABE and LBE by LC/MS.Boxes with × indicate that the compound was identified in the sample.Catechin and epicatechin were supported through analysis of standard solutions.

Table 2 :
PAs from ABE and LBE detected by MALDI-TOF/TOF MS.