Î±-Glucosidase Inhibitors from Malbranchea flavorosea - Journal of

Article pubs.acs.org/jnp

α‑Glucosidase Inhibitors from Malbranchea flavorosea Brisa Verastegui-Omaña, Daniela Rebollar-Ramos, Araceli Pérez-Vásquez, Ana Laura Martínez, Abraham Madariaga-Mazón, Laura Flores-Bocanegra, and Rachel Mata* Facultad de Química, Universidad Nacional Autónoma de México, México, Ciudad de México 04510, México S Supporting Information *

ABSTRACT: From an extract prepared from the grain-based culture of Malbranchea f lavorosea two new polyketides, namely, 8chloroxylarinol A (1) and flavoroseoside (2), along with the known compounds xylarinol A (3), xylarinol B (4), massarigenins B and C (5 and 6), and clavatol (7), were isolated. The structures of 1 and 2 were elucidated using spectroscopic methods and corroborated by single-crystal X-ray diffraction analysis. In the case of compound 2 the absolute configuration at the stereogenic centers was established according to the method of Flack. In addition, the X-ray structure of compound 6 is reported for the first time. Compounds 3, 4, and 6 significantly inhibited yeast α-glucosidase. Compound 6 also inhibited the postprandial peak during an oral sucrose tolerance assay when tested in vivo, using normal and NA/STZ-induced hyperglycemic mice.

T

he genus Malbranchea Saccardo (Myxotrichaceae) comprises worldwide species isolated from soil. According to Sigler and Carmichael, the species of Malbranchea belong to one of the following groups: those having curved fertile hyphae and those with straight fertile hyphae.1 Previous studies have confirmed that this genus is a prolific source of novel bioactive compounds. The most relevant metabolites include indole terpenoid alkaloids of the malbrancheamide family with vasorelaxant and calmodulin inhibitor properties;2−5 cytotoxic pyrrole alkaloids6 and steroids;7 terpenoids with antifungal, phytotoxic, and cytotoxic activities;8−10 and antiviral, antioxidant, antibiotic, and vasorelaxant polyketides and related compounds.11−17 Thus, as part of our ongoing search for αglucosidase inhibitors,18,19 we have now examined Malbranchea f lavorosea Sigler & Carmichel obtained from the American Type Culture Collection (ATCC). The search for new αglucosidase inhibitors and other antidiabetic drugs from natural sources is currently very important considering that type II diabetes mellitus is one of the most challenging health problems of the 21st century. According to the International Diabetes Federation around 415 million people were affected worldwide with type-2 diabetes mellitus (T2DM) in 2015; the prevalence is expected to rise beyond 642 million by 2040.20

Compound 1 (8-chloroxylarinol A) was isolated as a colorless crystalline solid. Its molecular formula was determined as C 10H 7 ClO3 by HRMS, requiring seven degrees of unsaturation. The presence of one chlorine atom in the molecule was consistent with the relative abundance of the [M+ + 2] peak with respect to the molecular ion [M+]. The IR spectrum showed typical absorption bands for hydroxy at 3315 cm−1 and a conjugated carbonyl group at 1689 cm−1. The

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RESULTS AND DISCUSSION The defatted extract from moist rice cultures of M. flavorosea inhibited the activity of yeast α-glucosidase (αGHY) with an IC50 of 500 μg/mL. Conventional fractionation of this extract yielded two novel polyketides, namely, 8-chloroxylarinol A (1) and flavoroseoside (2), along with the known compounds xylarinol A (3),21 xylarinol B (4),22 massarigenins B and C (5 and 6),23 and clavatol (7).24 © 2017 American Chemical Society and American Society of Pharmacognosy

Received: October 27, 2016 Published: January 6, 2017 190

DOI: 10.1021/acs.jnatprod.6b00977 J. Nat. Prod. 2017, 80, 190−195

Journal of Natural Products

Article

Table 2. 1H (500 MHz) and 13C (125 MHz) NMR Data for Compound 2 in CD3OD

NMR data (Table 1, Figure S1) were very similar to those of xylarinol A (3); they were consistent with the presence of a Table 1. 1H (500 MHz) and 13C (125 MHz) NMR Data for Compound 1 in CD3OD position

δC

type

δH (J in Hz)

HMBC

1 3 4 5 5a 6 7 8 9 9a

61.3 167.7 123.8 139.7 136.4 122.2 130.0 123.0 149.2 118.2

CH2 C CH CH C CH CH C C C

5.27, s

5a, 9, 3

6.38, d (12.1) 7.14, d (12.1)

5a, 5, 3 6, 5a, 3

6.94, d (8.2) 7.41, d (8.2)

8, 5, 9 6, 5a, 9

tetrasubstituted benzene ring possessing one hydroxy and one chlorine substituent, fused to a seven-membered α,β-unsaturated lactone. Thus, in the 1H NMR data the ABC system [δH 6.99 (d, J = 8.1 Hz, H-6), 7.28 (dd, J = 7.9 Hz, H-7), 6.97 (d, J = 7.5, H-8)] of the aromatic ring in compound 3 was replaced by an AB system [δH 6.94 (d, J = 8.2 Hz, H-6), 7.41 (d, J = 8.2 Hz, H-7)] in compound 1, whereas in the 13C NMR data (Figure S2) the main differences were observed in the aromatic carbons [δC 137.0 (C-5a), 120.5 (C-6), 130.1 (C-7), 117.2 (C8), 154.4 (C-9), and 121.5 (9a) in 3; δC 136.4 (C-5a), 122.2 (C-6), 130.0 (C-7), 123.0 (C-8), 149.15 (C-9), and 118.21 (9a) in 1]. All carbon/proton signals and the position of the substituents along the carbocyclic ring were assigned through a comprehensive analysis of the 2D NMR data (COSY, HSQC, NOESY, and HMBC). The proposed structure of compound 1 was established unequivocally by a single-crystal X-ray analysis (Figure 1).

position

δC

type

1 3 4

171.9 77.3 29.6

C CH CH2

4a 5 6 7 8 8a 9 1′ 2′ 3′ 4′ 5′

125.4 149.1 153.3 102.6 157.9 101.9 21.1 102.8 73.9 71.1 88.7 63.1

C C C CH C C CH3 CH CH CH CH CH2

δ (J in Hz) 4.68, m a 2.66, dd (11.5, 16.9) b 3.2, m

6.75, s

1.53, d (6.1) 5.73, d (4.5) 4.18, m 4.30, t (5.2) 4.18, m a 3.23, ddd (3.3, 12.1, 16.8) b 3.71, m (2.9, 12.2, 27.6)

Figure 2. ORTEP drawing of compound 2.

five signals for a β-ribofuranosyl moiety [δH/δC 5.73 (d, J = 4.5 Hz, H-1′)/102.8 (C-1′), δH 4.18 (m, H-2′)/73.9 (C-2′), δH 4.30 (t, J = 5.2 Hz, H-3′)/71.1 (C-3′), δH 4.18 (m, H-4′)/88.7 (C-4′), δHa 3.23 (ddd, J = 3.3, 12.1, and 16.8 Hz, H-5a′)/63.1 (C-5′), and δHb 3.71 (m, J = 2.9, 12.2, and 27.6 Hz, H-5b′)/ 63.1 (C-5′)] in the spectra of 2.26 A single-crystal X-ray analysis of 2 (Figure 2) unequivocally confirmed its structure. Determination of the Flack parameter27 [−0.05(15)] indicated that the absolute structure was identical to that depicted in the refined structure; thus, the configuration at C-3 was R, and the sugar was α-D-ribofuranose. Accordingly, compound 2 was characterized as 6-O-α-ribofuranosyl-(R)-(−)-3,4-dihydro-3methyl-5,8-dihydroxy-1H-2-benzopyran1-one and was given the trivial name flavoroseoside. The structure of compound massarigenin C (6), previously isolated from the aquatic fungus Massarina tunicate,23 was confirmed with an X-ray diffraction analysis; the ORTEP diagram is shown in Figure 3. Once more, calculation of the Flack parameter allowed us to establish the absolute configuration at the stereogenic centers.27 Compounds 3, 4 and 6 showed inhibitory activity when tested against yeast α-glucosidase, with IC50’s of 1.07 ± 0.01, 2.25 ± 0.012, and 1.25 ± 0.015 mM, respectively. In all cases the activity was compared to acarbose (ACAR, IC50 = 0.5 ±


Compound 2 was obtained as a crystalline, white solid. Its structure elucidation was based on NMR methods (Table 2) and X-ray analysis (Figure 2). The 13C NMR spectrum (Figure S4) exhibited 15 signals (Table 2) in agreement with the molecular formula C15H18O9, which requires seven degrees of unsaturation. The NMR data showed notable similarity to (R)(−)-3,4-dihydro-3-methyl-5,6,8-trihydroxy-1H-2-benzopyran1one, previously isolated from a fungus of the genus Cryptosporiopsis.25 The relevant difference was the presence of 191



Article

These results are consistent with the α-glucosidase inhibition activity of this compound in vitro, although other mechanisms might be involved in its antihyperglycemic action. Docking studies were performed with compound 6 using protein models of human N-terminal and C-terminal subunits of maltase glucoamylase (hNt-MGAM, PDB code 2QMJ, and hCt-MGAM, PDB code 3TOP, respectively), human Nterminal sucrose isomaltase (hNt-SI, PDB code 3LPP), and yeast isomaltase (y-IM, PDB code 3A4A).28 Prior to docking, all bound ligands were removed from the crystallized proteins and subsequently docked with 6. In addition, ACAR was docked with Nt-MGAM and y-IM for validation purposes. A binding pocket search for the mammalian enzymes was performed using the recently developed algorithm Autosite from the MGLTools2 Package29 (Figure S13). Docking of ACAR to Nt-MGAM resulted in a pose coinciding with that of the resolved crystal structure, supporting the validity of the method used.18 However, compound 6 bound to the enzyme with an estimated free energy of binding (EFEB) of −6.1 kcal/ mol, far from the catalytic site (Figure S14). The interaction involved hydrophobic contacts with Trp194, Val244, Ser40, Lys195, and hydrogen bonding (HB) with Arg254 and Thr196 (Figure S14). When docked to Ct-MGAM (3TOP), the EFEB (−6.5 kcal/mol) of 6 was lower (Figure S15), binding also far from the catalytic pocket; in this case the interacting forces were mainly by hydrophobic contacts and HB with residues Phe1289 and 1404. Finally, docking to human Nt-SI (EFEB = −6.2 kcal/mol) showed that the main contacts were also hydrophobic with Asp726, Ala727, Gly761, Tyr762, Gly788, and HB through with Arg759, Gly760, and Glu789 (Figure S16). With y-IM, the results predicted that 6 binds also in a site different from the catalytic domain [EFEB = −6.55 kcal/mol], interacting through hydrophobic forces with Ser298, Leu 297, His295, and Ile272 and HB between Ala292 and Asn259


0.014 mM). Unfortunately, the yield of 1, 2, 5, and 7 precluded their biological testing. Compound 6 was also tested in vivo using an oral sucrose tolerance test (OSTT) in healthy and hyperglycemic mice (Figure 4). This compound was selected for testing in vivo due to the availability of the sample. Oral administration of 6 reduced significantly the postprandial peak in a non-dosedependent manner during an OSTT using normal and NA-STZ (50−130 mg/kg)-induced hyperglycemic mice. In all cases, the effect was comparable to that of the positive control acarbose, thus revealing the antihyperglycemic potential of compound 6.

Figure 4. Effect of compound 6 (3.2−31.6 mg/kg, p.o.) on blood glucose levels in normoglycemic (A and B) and NA-STZ-hyperglycemic mice (C and D) during an OSTT. AUC: area under the curve (obtained from temporal curves); VEH: vehicle; ACAR: acarbose. Each point or bar represents the mean ± SD for 6 mice in each group. *p < 0.05, **p < 0.01, and ***p < 0.001 represent significantly different two-way ANOVA followed by Bonferroni’s test for comparison with respect to vehicle control at the same time (A and C) or ANOVA followed by Dunnett’s test for comparison with respect to vehicle control (B and D). 192



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(81 mg) was subjected to preparative RP-HPLC using a gradient of MeCN−0.1% aqueous formic acid (85:15 to 100:0 in 15 min) to yield 3 (20.0 mg, tR 8.4 min) and 5 (16.8 mg, tR 7.30 min). 8-Chloroxylarinol A (1): (C10H7ClO3) colorless crystals; mp 151− 152 °C; UV λmax 200.4, 274.8 nm; IR (FTIR) νmax 3315, 1689, 1577, 1278 cm−1; 1H and 13C NMR in Table 1; HRESIMS m/z 210.6153 [M + H]+, calcd 210.6151. Flavoroseoside (2): (C15H18O9) colorless crystals; mp 245 °C (dec); UV λmax 205.1, 229.8, 266.5, 330.8 nm; IR (FTIR) νmax 3370, 2928, 1630, 1648, 1291, 1204 cm−1; 1H and 13C NMR in Table 2; HRESIMS m/z 342.2982 [M + H]+, calcd 342.2984. X-ray Crystal Structure Analysis of Compound 1. Single crystals suitable for X-ray analysis were obtained by recrystallization from CH2Cl2−MeOH (8:2). A colorless crystal of approximate dimensions of 0.383 × 0.160 × 0.076 mm3 was mounted on a glass fiber. All measurements were made on a Bruker D8 Venture diffractometer spectrometer (Billerica, MA, USA) with Cu Kα radiation (λ = 1.541 78 Å) at 180 K. The structure was solved by the SHELXS-2013 method and refined using full-matrix least-squares on F2. Crystallographic data for 1 have been deposited with the Cambridge Crystallographic Data Centre (CCDC) with the accession number 1501989. These data are available, free of charge, from the CCDC via http://www.ccdc.cam.ac.uk/data_request/cif. Crystal data for 1: C10H7ClO3, MW 210.61, orthorhombic, space group Pna21 with unit cell parameters a = 16.6863(7) Å, b = 4.1596(2) Å, c = 12.8685(6) Å, α = 90°, β = 90°, γ = 90°, Z = 4, T = 180(2) K, volume = 893.18(7) Å3, μ(Cu Kα) = 3.608 mm−1, F(000) = 432, density(calcd) = 1.566 Mg/m3. Intensity data were collected in the range of 5.302−68.215° using a ω scan; 7356 reflections collected, 1610 independent reflections [R(int) = 0.1508] were considered, observed, and used in the calculations. The final R1 indices were 0.0437 [I > 2σ(I)]. The final wR2 values were 0.1113 [I > 2σ(I)], with a data/restraints/parameters ratio of 1610/2/132. The final R1 values were 0.0487 (all data). The final wR2 values were 0.1338 (all data). The absolute structure parameter was 0.02(4). X-ray Crystal Structure Analysis of Compound 2. Single crystals suitable for X-ray analysis were obtained by recrystallization from CH2Cl2−MeOH (1:1). A colorless crystal with approximate dimensions of 0.360 × 0.113 × 0.081 mm3 was mounted on a glass fiber. All measurements were made on the same diffractometer with Cu Kα radiation (λ = 1.541 78 Å) at 180 K. The structure was solved by the SHELXS-2013 method and refined using full-matrix leastsquares on F2 and refined to a discrepancy index of 3.3%. The data set was also used to calculate the Flack parameter. Crystallographic data for 2 have been deposited with the Cambridge Crystallographic Data Centre (CCDC) with the accession number 1501990. These data are available, free of charge, from the CCDC via http://www.ccdc.cam.ac. uk/data_request/cif. Crystal data for 2: C15H18O9, MW 342.29, orthorhombic, space group P212121 with unit cell parameters a = 5.1645(4) Å, b = 13.0453(9) Å, c = 22.1270(16) Å, α = 90°, β = 90°, γ = 90°, Z = 4, T = 150(2) K, volume = 1490.75(19) Å3, μ(Cu Kα) = 1.099 mm−1, F(000) = 720, density(calcd) = 1.525 Mg/m3. Intensity data were collected in the range of 3.934−68.492° using a ω scan; 11 414 reflections collected, 2743 independent reflections [R(int) = 0.1077] were considered, observed, and used in the calculations. The final R1 indices were 0.0406 [I > 2σ(I)]. The final wR2 values were 0.1064 [I > 2σ(I)], with a data/restraints/parameters ratio of 2743/5/233. The final R1 values were 0.0428 (all data). The final wR2 values were 0.1093 (all data). The absolute structure parameter was −0.05(15). X-ray Crystal Structure Analysis of Compound 6. Single crystals suitable for X-ray analysis were obtained by recrystallization from CH2Cl2−MeOH (2:8). A colorless crystal having approximate dimensions of 0.403 × 0.256 × 0.188 mm3 was mounted on a glass fiber. All measurements were made on a Bruker D8 Venture diffractometer with Cu Kα radiation (λ = 1.541 78 Å) at 180 K. The structure was solved by the SHELXS-2013 method and refined using full-matrix least-squares on F2 and refined to a discrepancy index of 3.3%. The data set was also used to calculate the Flack parameter. Crystallographic data for 6 have been deposited with the Cambridge

(Figure S17). The overall docking results were consistent with the in vitro α-glucosidase inhibitory properties of compound 6 and predicted that its inhibitory action could be exerted throughout an allosteric interaction with the enzymes. In summary, M. f lavorosea biosynthesizes simple polyketides with potential for the development of antihyperglycemic drugs. The metabolic profile of this fungus is different from the other Malbranchea species analyzed so far. The chemotaxonomic relevance of this finding remains an open question, which will be answered as soon as more species of the genus are investigated.

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EXPERIMENTAL SECTION

General Experimental Procedures. Melting points were determined on a Fisher-Johns apparatus and are uncorrected. IR spectra were recorded using a PerkinElmer 400 FT-IR spectrophotometer (Waltham, MA, USA). NMR spectra, including bidimensional, were recorded in CD3OD, DMSO-d6, or CDCl3 solution on a Bruker Avance III HD spectrometer (Billerica, MA, USA) at either 500 MHz (1H) or 125 MHz (13C) or a Varian VNRMS (Palo Alto, CA, USA) at 400 MHz (1H) or 100 MHz (13C), using tetramethylsilane as an internal standard. Preparative HPLC was carried out with a Waters instrument (Milford, MA, USA) equipped with a 2535 pump and a 2998 photodiode array detector, using a Gemini 5u C18 110A AXIA packed column (21.1 × 250 mm) and different gradient systems of MeCN and 0.1% aqueous formic acid, at a flow rate of 21.24 mL/min. Control of equipment, data acquisition and processing, and management of chromatographic information were performed by the Empower 3 software package (Waters). Column chromatography (CC) was carried out on Sephadex LH-20 (General Electric). Flash chromatography was carried out with a Teledyne CombiFlashRf +Lumen (Thousand Oaks, CA, USA) chromatograph equipped with a photodiode array detector and an evaporative light scattering detector, using a RediSepRf high-performance GOLD silica gel column and eluting with a gradient of hexane, chloroform, and methanol. Thinlayer chromatographic (TLC) analyses were performed on silica gel 60 F254 plates (Merck, Kenilworth, NJ, USA), and visualization of the plates was carried out using a Ce2(SO4)3 (10%) solution in H2SO4. Fungal Material. M. f lavorosea was purchased from the ATCC (No. 34529). The lyophilized material was resuspended in 5 mL of sterile water and left overnight. Petri dishes containing potato-dextrose agar (PDA) were inoculated using 200 μL of the previous suspension. Stock cultures of the fungus are stored in PDA and monthly subcultured for preservation. Fermentation, Extraction, and Isolation. Seed cultures of the fungus were prepared using potato-dextrose broth (PDB) media and incubated at room temperature for 15 days at 200 rpm. Next, M. f lavorosea was grown on Erlenmeyer flasks containing 85 g of rice and 200 mL of water (5 × 500 mL). After 45 days of fungal growth, the culture media was extracted exhaustively with 8:2 CH2Cl2−MeOH (4 × 500 mL), and the resulting extract was evaporated in vacuo to yield 21.7 g of a brown, oily residue. The total extract was suspended in 150 mL of a mixture of MeCN−MeOH (1:1) and partitioned with n-hexane (8 × 150 mL). The combined MeCN−MeOH fractions were dried under vacuum, and the resulting residue (5.9 g) was dissolved in MeOH and subjected to flash chromatography using a gradient of nhexane−CHCl3 (0.0−10.0 min, 100:0 → 0:100) and CHCl3−MeOH (10.0−17.5 min, 100:0, 17.5−25.0 min, 100:0 → 98:2; 25.0−32.5 min, 98:2 → 95:5; 32.5−45.0 min, 95:5 → 90:10; 45.0−65.0 min, 90:10 → 80:20, 65.0−90.0 min, 80:20 → 0:100). The separation yielded 16 primary fractions (FA−P). Fraction FI, eluted with CHCl3−MeOH (9.0:1.0), yielded compound 6 (150 mg). Fraction FF (104 mg), eluted with CHCl3−MeOH (9.8:0.2), was subjected to preparative RP-HPLC using a gradient of MeCN−0.1% aqueous formic acid (35:65 to 100:0 in 12 min) to yield 1 (2.5 mg, tR 6.7 min), 2 (5 mg, tR 9.16 min), and 4 (7 mg, tR 6.4 min). Fraction FK (160 mg) was subjected to preparative RP-HPLC using a gradient of MeCN−0.1% aqueous formic acid (35:65 to 100:0 in 12 min) to yield 7 (2 mg, tR 8.25 min). Fraction FH 193



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Crystallographic Data Centre (CCDC) with the accession nnumber 1501991. These data are available, free of charge, from the CCDC via http://www.ccdc.cam.ac.uk/data_request/cif. Crystal data for 6: C22H26O11, MW 466.43, monoclinic, space group C2 with unit cell parameters a = 20.881(3) Å, b = 7.2139(11) Å, c = 7.1436(11) Å, α = 90°, β = 95.201(4)°, γ = 90°, Z = 2, T = 150(2) K, volume 1071.7(3) Å3, μ(Cu Kα) = 0.996 mm−1, F(000) = 492, density(calcd) 1.445 Mg/m3. Intensity data were collected in the range of 4.252−68.255° using a ω scan; 6426 reflections collected, 1971 independent reflections [R(int) = 0.1126] were considered, observed, and used in the calculations. The final R1 indices were 0.0501 [I > 2σ(I)]. The final wR2 values were 0.1291 [I > 2σ(I)], with a datarestraints-parameters ratio of 1971/5/160. The final R1 values were 0.0503 (all data). The final wR2 values were 0.1293 (all data). The absolute structure parameter was 0.2(3). Enzymatic Assay. The fungal extract, compounds 3, 4, and 6, and acarbose (positive control) were dissolved in MeOH or phosphate buffer solution (PBS, 100 mM, pH 7). Aliquots of 0−20 μL of testing materials (triplicated) were incubated for 10 min with 20 μL of 1 U/ mL from Saccharomyces cerevisiae (αGHY) enzyme in PBS. After incubation, 10 μL of p-nitrophenyl-α-D-glucopyranoside (pNPG, 5 mM) were added and incubated a further 30 min at 37 °C,18 and the absorbances were determined at 415 nm. The inhibitory activity was determined as percentage in comparison to the blank according to the following equation:

compound 6 at doses of 3.2, 10, and 31.6 mg/kg. Thirty minutes after administering the treatments, an oral sucrose (3 g/kg) load was given to each animal. Blood glucose levels were determined at 30, 60, 90, and 120 min postadministration of the carbohydrate load. The percentage of glycemic variation (%) was determined with respect to the basal level as follows:

⎡ (G − Gi) ⎤ % variation of glycemia = ⎢ t ⎥ × 100 Gi ⎣ ⎦ where Gi is the basal glycemia and Gt is the different glycemia values after treatment administration.30 Statistical Analysis. Data are expressed as the mean ± standard error of the mean. Statistical significance differences were ascertained by means of two-way ANOVA followed by Bonferroni’s test for comparison with respect to vehicle control at the same time or ANOVA followed by Dunnett’s test for comparison with respect to vehicle control. GraphPad Prism software (version 5.0) was used for statistical analysis. Docking Studies. Chemical structures of 6 and acarbose were constructed using the Spartan’10 software (Wavefunction Inc., Irvine CA, USA), and the conformer distribution search was performed by means of the molecular mechanics force field implemented in the same program. The minimum energy structures were then geometrically optimized using density functional theory at the B3LYP/DGDZVP level of theory using Gaussian 09 software (Gaussian Inc., Wallingford, CT, USA). The minimized structures were prepared for docking simulations using Autodock Tools package v1.5.4.28 Addition of Gasteiger charges and number of torsions were set for ligands. For the macromolecules, all hydrogens (polar and nonpolar) and Kollman charges were added. Molecular docking studies were performed with AutoDock Vina v1.1.2 (mammalian enzymes) or AutoDock 1.5.4 (yIM).29 First, a blind docking was performed in order to establish the common site of interaction of 6 with the three mammalian αglucosidases, the human N-terminal and C-terminal subunits of human maltase-glucoamylase (2QMJ and 3TOP, respectively), human Nterminal sucrase-isomaltase (3LPP), and yeast isomaltase (y-IM). For the mammalian enzymes the search space for this preliminary docking was defined as a box size of 66 × 70 × 72 Å in the x, y, and z dimensions, with a grid spacing of 1.0 Å and the macromolecule set as the center of the box. The default parameters of exhaustiveness and number of modes were not changed. Next, a refined docking was performed with a smaller box of searching space (20 × 21 × 19 Å, and 1.0 Å of grid spacing). In the case of y-IM the initial box size was 126 × 126 × 126 Å in the x, y, and z dimensions, and the smaller size box was 40 × 40 × 40 Å in the x, y, and z dimensions. The conformational state poses from the docking simulations were analyzed using AutodockTools software, which also identified the H-bonds and van der Waals interactions. The resulting docked conformations showing the lowest binding energy were taken as the predicted protein−ligand complexes. Preparation of the figures was achieved with the PyMOL visualization tool (The PyMOL Molecular Graphics System, version 1.7.4 Schrödinger, LLC)31 and LigPlotPlus.32

⎛ A ⎞ % αGHY = ⎜1 − 415b ⎟× 100% A415c ⎠ ⎝ where % αGHY is the percentage of inhibition, A415c is the corrected absorbance of the samples under testing (A415 end − A415 initial), and A415b is the absorbance of the blank (A415 end blank − A415 initial blank). All assays were performed in triplicate. The IC50 was calculated by regression analysis, using the following equation:

A100

% Inhibition = 1+

s

( ) I IC50

where A100 is the maximum inhibition, I is the inhibitor concentration, IC50 is the concentration required to inhibit activity of the enzyme by 50% ± SD, and s is the cooperative degree.18 Experimental Animals. ICR male mice, age 3−4 weeks (25−30 g body weight), were obtained from Envigo Mexico RMS (CDMX). Animals were kept in an environmentally controlled room maintained at 22 ± 1 °C with an alternating 12 h light/dark natural cycle, with free access to standard rodent pellet diet (Teklad 2018S, Envigo) and water ad libitum until the beginning of each experiment. All animal experimental protocols followed the recommendations of the Mexican Official Norm for Animal Care and Handling (NOM-062-ZOO-1999) and were in conformity with International Ethical Guidelines for the care and use of laboratory animals. The Ethical Committee for the Use of Animals in Pharmacological and Toxicological Testing, Facultad de ́ Quimica, UNAM (FQ/CICUAL/132/16), approved the protocol on March 8, 2016. Nicotinamide−Streptozotocin (NA-STZ)-Induced Experimental Hyperglycemia in Mice. The experimental hyperglycemia in mice was achieved by giving a single intraperitoneal (i.p.) administration of NA (50 mg/kg) dissolved in saline solution.31 Thirty minutes later, STZ (130 mg/kg) dissolved in 0.1 M citrate buffer (pH 4.5) was administrated i.p. to all animals. One week later, blood glucose levels were measured using a commercial glucometer (One Touch Ultra 2, Johnson & Johnson, New Brunswick, NJ, USA); mice having a blood glucose of ≥200 mg/dL were considered hyperglycemic and selected for the study. Oral Sucrose Tolerance Test. Normal and hyperglycemic mice were fasted 4 h before the experiment with free access to drinking water. Mice were divided into five groups (I−V) of six mice each. The animals of group I were administered with VEH (saline solution with 0.05% tween 80), while group II was fed with reference drug ACAR (acarbose 5 mg/kg). Groups III−V were administered orally with

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00977. Crystallographic data (CIF) Crystallographic data (CIF) Crystallographic data (CIF) One-dimensional NMR spectra of compounds 1−6; Xray crystallographic data of 1, 2, 6, and 7; and binding models of compound 6 to human Nt-MGAM (PDB code 2QMJ), Ct-MGAM (PDB code 3TOP), and Nt-SI (PDB code 3LPP) and to yeast IM (PDB code 3A4A) (PDF) 194



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AUTHOR INFORMATION

Corresponding Author

*Phone (R. Mata): +52-555-622-5289. E-mail: rachel@unam. mx. ORCID

Rachel Mata: 0000-0002-2861-2768 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by grants from CONACyT 219765, INFRA-252226 and -205195, and PAIP-UNAM 5000-9140. We thank R. del Carmen, I. Rivero-Cruz, G. Duarte, M. Gutiérrez, M. Monroy, and R. I. del Villar for their valuable technical assistance. B.V.-O. and A.L.M. acknowledge the fellowship from DGAPA-UNAM to pursue postdoctoral research. We are indebted to Dirección General de Cómputo ́ de Información y Comunicación (DGTIC), y de Tecnologias UNAM, for providing the resources to carry out computational calculations through the Miztli supercomputing system.

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