α-Glucosidase and Protein Tyrosine Phosphatase 1B Inhibitors from Malbranchea circinata

Article abstract of DOI:10.1021/acs.jnatprod.9b01108

During a search for new α-glucosidase and protein tyrosine phosphatase 1B inhibitors from fungal sources, eight new secondary metabolites, including two anthranilic acid-derived peptides (1 and 2), four glycosylated anthraquinones (3-6), 4-isoprenylravenelin (7), and a dimer of 5,8-dihydroxy-4-methoxy-α-tetralone (8), along with four known compounds (9-12), were isolated from solid rice-based cultures of Malbranchea circinata. The structural elucidation of these metabolites was performed using 1D and 2D NMR techniques and DFT-calculated chemical shifts. Compounds 1-3, 9, and 10 showed inhibitory activity to yeast α-glucosidase (αGHY), with IC₅₀ values ranging from 57.4 to 261.3 μM (IC₅₀ acarbose = 585.8 μM). The effect of 10 (10.0 mg/kg) was corroborated in vivo using a sucrose tolerance test in normoglucemic mice. The most active compounds against PTP-1B were 8-10, with IC₅₀ values from 10.9 to 15.3 μM (IC₅₀ ursolic acid = 27.8 μM). Docking analysis of the active compounds into the crystal structures of αGHY and PTP-1B predicted that all compounds bind to the catalytic domains of the enzymes. Together, these results showed that M. circinata is a potential source of antidiabetic drug leads.

Full text of DOI:10.1021/acs.jnatprod.9b01108

Article
pubs.acs.org/jnp
Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX
α‑Glucosidase and Protein Tyrosine Phosphatase 1B Inhibitors from
Malbranchea circinata
§
Manuel Rangel-Grimaldo,†,‡ Martha L. Macías-Rubalcava,⊥ Martin Gonzalez-Andrade,
́
Huzefa Raja,∥ Mario Figueroa,† and Rachel Mata*,†
†
⊥
§
́
́
Facultad de Química, Instituto de Química, and Facultad de Medicina, Universidad Nacional Autonoma de Mexico, Ciudad de
́
́
Mexico 04510, Mexico
∥Department of Chemistry and Biochemistry, University of North Carolina Greensboro, Greensboro, North Carolina 27402, United
States
S
* Supporting Information
ABSTRACT: During a search for new α-glucosidase and
protein tyrosine phosphatase 1B inhibitors from fungal
sources, eight new secondary metabolites, including two
anthranilic acid-derived peptides (1 and 2), four glycosylated
anthraquinones (3−6), 4-isoprenylravenelin (7), and a dimer
of 5,8-dihydroxy-4-methoxy-α-tetralone (8), along with four
known compounds (9−12), were isolated from solid rice-
based cultures of Malbranchea circinata. The structural
elucidation of these metabolites was performed using 1D
and 2D NMR techniques and DFT-calculated chemical shifts.
Compounds 1−3, 9, and 10 showed inhibitory activity to
yeast α-glucosidase (αGHY), with IC50 values ranging from 57.4 to 261.3 μM (IC50 acarbose = 585.8 μM). The effect of 10
(10.0 mg/kg) was corroborated in vivo using a sucrose tolerance test in normoglucemic mice. The most active compounds
against PTP-1B were 8−10, with IC50 values from 10.9 to 15.3 μM (IC50 ursolic acid = 27.8 μM). Docking analysis of the active
compounds into the crystal structures of αGHY and PTP-1B predicted that all compounds bind to the catalytic domains of the
enzymes. Together, these results showed that M. circinata is a potential source of antidiabetic drug leads.
he genus Malbranchea contains about 30 species, which
extract showed activity against α-glucosidases and the protein
Tare saprotrophic fungi isolated from decaying vegetation, tyrosine phosphatase 1B (PTP-1B). The latter is critical for
soil, or animal dung worldwide.1 The members of this genus
insulin signaling due to its ability to dephosphorylate and
inactivate the insulin receptor, and inhibition of PTP-1B is an
attractive target to improve insulin sensitivity in different cell
types.13−15 α-Glucosidase inhibitors are currently used in the
treatment of patients with T2DM since these agents delay the
absorption of carbohydrates from the small intestine and lower
the effect on postprandial blood glucose and insulin levels. In
this work we describe the isolation and structure elucidation of
two anthranilic acid derivatives (1 and 2), four glycosylated
anthraquinones (3−6), a prenylated xanthone (7), and a dimer
of 5,8-dihydroxy-4-methoxy-α-tetralone (8), along with four
known compounds (9−12). All isolates were tested for their
inhibitory properties against both α-glucosidase and PTP-1B.
produce an array of bioactive compounds including eremo-
phyllanes;2 the malbrancheamides,2−4 a unique family of
indole terpenoid alkaloids with vasorelaxant and calmodulin
inhibitor properties whose biosynthetic pathway was elegantly
elucidated through complementary approaches;5 pyrrole
alkaloids and modified steroids with cytotoxic effects;6
terpenoids with antifungal, phytotoxic, and cytotoxic activ-
ities;7−9 and polyketides and peptides with antidiabetic
properties.10,11
According to the International Federation of Diabetes, over
the past decade the global prevalence of type-2 diabetes
mellitus (T2DM) has nearly quadrupled since 1980,
particularly in low- and middle-income countries.12 In this
scenario, it is essential to provide new therapeutic alternatives,
including those arising from fungal sources. Hence, we
previously reported that assay-guided fractionation of solid-
substrate cultures of Malbranchea flavorosea led to the isolation
of some polyketides and peptide-type compounds with in vitro
and in vivo α-glucosidase inhibitory properties.10,11 Here,
Malbranchea circinata Sigler & Carmichel (Myxotrichaceae)
from the American Type Culture Collection (strain no. 34526)
was selected for investigation because its rice-based culture
RESULTS AND DISCUSSION
■
The defatted extract from moist rice cultures of M. circinata
inhibited the activity of the α-glucosidase from yeast (IC50
=
360.7 0.2 μg/mL) and PTP-1B (IC50 = 5.3 0.3 μg/mL).
Special Issue: Special Issue in Honor of Jon Clardy
Received: November 7, 2019
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.9b01108
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Journal of Natural Products
Article
Chart 1
1
Table 1. H (700 MHz) and 13C (175 MHz) NMR Data of Compounds 1 and 2 in CD3OD
1
2
1
1
position
δC
type
δH, mult. (J in Hz)
HMBC H→13C
δC
type
δH, mult. (J in Hz)
HMBC H→13C
1
2
3
4
4a
5a
5b
6
7
8
126.6
127.6
135.0
126.8
147.3
154.5
133.8
128.5
127.6
131.1
126.1
136.3
170.8
134.2
116.9
130.9
117.8
152.6
121.7
161.0
120.5
CH
CH
CH
CH
C
8.31, dd (8.0, 1.5)
3,4,4a,17
4,4a,17,17a
1,4a, 17a
126.6
127.7
135.1
126.9
147.1
153.8
133.7
129.0
127.7
131.1
125.9
136.3
170.5
133.3
127.3
129.8
130.8
128.2
134.1
161.7
120.6
CH
CH
CH
CH
C
8.32, dd (8.0, 1.5)
3,4a,17
4,4a,17,17a
1,4a
7.65, ddd (8.0, 7.2, 1.5)
7.93, ddd (7.6, 7.2, 1.5)
7.8, dd (7.6, 1.5)
7.67, ddd (8.0, 7.2, 1.1)
7.95, ddd (8.3, 7.2, 1.5)
7.81, dd (8.3, 1.1)
2,3,4a,17,17a
2,3,17a
C
C
C
C
CH
CH
CH
CH
C
C
C
CH
CH
CH
C
7.63, dd (7.6, 1.6)
5a,8,9a
5b,9
6,9a
CH
CH
CH
CH
C
7.60, dd (7.6, 1.5)
5a,8,9a
5b,9
6,9a
7.40, ddd (8.0, 7.6, 1.1)
7.47, ddd (8.0, 7.6, 1.6)
7.18, dd (8.0, 1.1)
7.39, ddd (8.0, 7.6, 1.1)
7.47, ddd (8.0, 7.6, 1.5)
7.20, dd (8.0, 1.1)
9
5a,5b,7
5a,5b,7
9a
11
11a
12
13
14
15
15a
17
17a
C
C
6.90, dd (7.6, 1.3)
7.29, dd (8.3, 7.6)
6.92, dd (8.3, 1.3)
11, 11a
11,11a,12,15,15a
12,15,15a
CH
CH
CH
CH
C
7.48−7.50, m
7.48−7.50, m
7.48−7.50, m
7.33, m
11,11a,14
11a,14
12
13
C
C
C
C
C
Extensive fractionation of the active organic extract led to the
isolation of eight new natural products (1−8), together with
the known compounds amauromine (9),16,17 malbranchea-
mide (10),3 and the epimeric mixture of arugosin N (11) and
its C-8 prenyl derivative, 1,6,10-trihydroxy-8-methyl-2-(3-
methyl-2-butenyl)dibenz[b,e]oxepin-11(6H)-one (12)18 (Sup-
porting Information, S1−S51).
indicated an index of hydrogen deficiency (IHD) of 17. The
IR spectrum revealed absorptions for amide and aromatic
groups (2927, 1684, 1591, and 1566 cm−1). Analysis of the 1H
and 13C NMR data (Table 1 and Figures S1 and S2) indicated
the presence of 18 aromatic carbons (11 protonated and seven
nonprotonated, one of which was oxygenated), two amide
carbonyls, and one imine group. Based on COSY spectra
(Figure S5), the 11 protons were part of two 1,2-disubstituted
and one 1,2,3-trisubstituted benzene ring. HMBC correlations
Compound 1 was isolated as colorless crystals. Its molecular
formula, C21H13N3O3, established from the HRESIMS,
B
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Figure 1. Selected key HMBC (→) correlations of substructures a and b and NOE (↔) correlation between H-9 and H-12 of 1.
Table 2. NMR Data of Compounds 3−5
a
b c
,
a c
,
3
4
5
d
HMBC
1H→13C
HMBC
HMBC
δH, mult. (J in
δH, mult. (J in
δH, mult. (J in
d
c
position
δC
type
Hz)
δC
Hz)
1H→13C
δC
Hz)
1H→13C
1
2
3
4
4a
5
165.0
109.1
164.7
108.2
135.0
120.8
C
CH
C
CH
C
CH
164.8
109.8 6.90, d (2.5)
163.3
109.8 7.47, d (2.5)
135.2
121.4 7.62, dq (1.7, 0.9) 7,8a,9,10,11
165.0
109.9 6.91, d (2.5)
163.3
110.1 7.48, d (2.5)
135.4
121.6 7.64, dq (1.7, 0.7) 7,8a,10,11
6.97, d (2.5)
7.55, d (2.5)
1,4
1,4,9a
3,4a,10
1,4,9a
3,4a,10
3,4a,10
7.67, dq (1.7,
0.7)
7,8a,9,11
6
7
148.8
124.1
C
CH
148.7
148.9
7.13, dq (1.7,
0.7)
5,8,8a,9,11
124.6 7.08, dq (1.7, 0.9) 5,8,8a,11
124.8 7.10, dq (1.7, 0.7) 5,8,8a,9,11
8
8a
9
9a
10
10a
11
1′
2′
161.8
113.4
190.1
110.8
182.4
132.9
21.7
C
C
C
C
C
C
162.6
113.6
190.9
111.2
181.8
133.1
22.2
98.3
71.2
162.8
113.8
191.2
111.5
182.0
133.3
22.3
98.3
71.0
CH3 2.48, s
CH
CH
2.45, s
6.07, d (4.5)
5.20, dd (7.0, 4.5) 4′,2′-COCH3
2.46, s
101.8
74.7
5.88, d (4.5)
4.38, dd (6.9,
4.5)
3,3′,4′
4′
3,3′,4′
6.04, d (4.5)
3,3′,4′
5.18, dd (7.0, 4.5) 4′,2′-COCH3
3′
70.5
CH
CH
4.27, m
1′,5′
70.0
5.42, m
1′,5′,3′-
70.0
5.36, m
1′,5′,3′-
COCH3
COCH3
4′
5′
83.9
62.7
4.28, m
2′,5′
3′,4′
84.2
62.0
4.32, m
3.89, dd (12.2,
3.2)
2′,5′
3′,4′
81.4
63.4
4.45, m
4.34, dd (12.3,
3.2)
2′,5′
3′,5′-COCH3
CH2 3.90, m
3.81, m
3′,4′
3.85, dd (12.2,
3.2)
3′,4′
4.26, dd (12.3,
3.2)
3′,5′-COCH3
2′-COCH3
3′-COCH3
5′-COCH3
2′-
169.9
20.5
170.0
20.9
COCH3
2′-
COCH3
3′-
COCH3
3′-
COCH3
5′-
COCH3
2.16, s
2.20, s
2′-COCH3
3′-COCH3
2.15, s
2.19, s
2.13, s
170.6
20.8
170.5
21.0
170.6
20.6
5′-
COCH3
1-OH
9-OH
12.29, s
12.09, s
2,9a
7,8a
12.23, s
12.05, s
2,9a
7,8a
12.26, s
12.07, s
2,9a
7,8a
a
b
c
d
1H (400 MHz) and 13C (100 MHz). 1H (700 MHz) and 13C (175 MHz). CDCl3. CDCl3 + drops of CD3OD.
(Figure 1 and Figure S4) and NMR chemical shifts (Table 1)
suggested a quinazolinone moiety attached to an aromatic ring
(ring C) through the imine carbon (substructure a), which is
present in a few anthranilic acid alkaloids including
sclerotigenin,19 circumdatin F,20 asperlicins,21,22 and benzo-
malvins.23−25 The protons of ring C (H-6−H-9) showed the
following HMBC correlations (Figure 1): H-6 to C-5a, C-8,
and C-9a; H-7 to C-5b and C-9; H-8 to C-6 and C-9a; and H-
9 to C-7, C-5a, and C-5b. On the other hand, correlations from
H-12 to C-11, C-11a, and C-15; H-13 to C-11, C-12, C-15,
C
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and C-15a; and H-14 to C-12 and C-15, as well as the spin
system H-12−H-1, typical of a 1,2,3-trisubstituted aromatic
ring, supported a 3-hydroxyanthranilic acid residue (sub-
structure b). Thereby, the extra ring required to fulfill the
unsaturation number required by the molecular formula was
identified connecting fragments a and b through C-11 and N-
10 and between C-15a and N-16. A NOESY correlation
between H-9 and H-12 (distance of 4.3 Å) supported these
joining points due to the bending of ring E as observed in the
most stable conformation of compound 1 (Figures 1 and S78).
Altogether, these data suggest that 1 is a tripeptide formed by
two anthranilic acid units and one 3-hydroxyanthranilic acid
unit.
Compound 2 was isolated as colorless crystals. Its molecular
formula was deduced as C21H13N3O2 based on the molecular
ion peak in the HRESIMS (IHD = 17). Detailed analysis of the
1D and 2D NMR data (Table 1), in particular of the HMBC
correlations, indicated that this compound was closely related
to 1: the key differences were observed in ring D, which was
disubstituted in compound 2. Thus, the 1,2,3-trisubstituted
benzene ring observed in 1 (protons H-12 to H-14) was
replaced by a 1,2-disubstituted aromatic system in 2 (protons
H-12 to H-15) (Table 1). Furthermore, a difference of 16 Da
and the diamagnetically shifted signal of C-15 from δC 152.6 in
1 to δC 128.2 in 2 suggested that 2 lacks the phenolic group at
C-15. Thus, compound 2 was proposed as the 15-deoxy
derivative of 1.
coelution with an authentic sample. The negative optical
rotation sign of the monosaccharide was consistent with D-
ribose.
Based on its NMR data (Table 2 and Figures S15−S35),
compounds 4 and 5 showed the same aglycone as 3; however
their ribofuranosyl unit was di- and triacetylated, respectively.
Thus, the 13C and HSQC spectra 4 revealed the presence of
two additional carbonyl groups at δC 169.9 (C-6′) and δC
170.6 (C-8′) and two methyl groups at δC 20.5 (C-7′) and δC
20.8 (C-9′). The HMBC connectivities of H-2′ to C-4′ and 2′-
COCH3, 2′-COCH3 to 2′-COCH3, H-3′ to C-1′, C-5′, and 3′-
COCH3, and 3′-COCH3 to 3′-COCH3 (Table 2) confirmed
the position of the acetyl groups at C-2′ and C-3′. The NMR
spectra of 5 showed signals for methyl (δC 20.6, 4′-COCH3)
and a third carbonyl (δC 170.6, 4′-COCH3) group, and the
HMBC correlations from H-5′ to C-4′ and 4′-COCH3 and
from 4′-COCH3 to 4′-COCH3 indicated that this acyl unit was
at C-5′ of the carbohydrate moiety. The D-monosaccharide
configuration of 4 and 5 was assumed on biogenetic grounds.
Compound 6 was isolated as orange glassy solid. Its
molecular formula was determined by HRESIMS as
C22H20O9 (IHD = 13). The NMR data (Table 3 and Figures
1
Table 3. H (700 MHz) and 13C (175 MHz) NMR Data of
Compound 6 in CDCl3
1
position
δC
type
δH, mult. (J in Hz)
HMBC H→13C
1
2
3
4
4a
5
6
7
8
162.9
124.7
148.4
120.6
132.4
122.0
136.0
122.4
158.1
121.5
189.0
114.9
182.4
135.5
22.1
C
CH
C
CH
C
CH
CH
CH
C
C
C
C
C
In order to provide further evidence for the NMR structural
1
assignment of 1 and 2, H and 13C chemical shifts were
7.14, dq (1.8, 0.9)
7.65, dq (1.8, 0.9)
1,4,9a,11
calculated and compared with the experimental data. Basically,
the protocol involves a conformational search using molecular
mechanics, geometry optimization using density functional
theory (DFT) with M06-2X/6-31+G(d), and chemical shift
calculations using the GIAO method with the B3LYP/6-
311+G(2d,p) level of theory.26,27 Comparisons of computed
and experimental NMR chemical shifts of 1 and 2 gave a mean
2,9a,10,11
8.08, dd (7.7, 1.2)
7.78, dd (8.0, 7.7)
7.70, dd (8.0, 1.2)
6,7,10
5,8,10a
5,6,8,8a
1
8a
9
absolute error (MAE) lower than 0.11 ppm for H and lower
than 2.0 ppm for 13C (Tables S3 and S4).
9a
10
10a
11
1′
2′
3′
4′
5′
6′
7′
1-OH
There is no precedent in nature of tripeptides like 1 and 2.
However, it was reported that heating of anthranilic acid with
phosphorus pentoxide in refluxing xylene28 or polyphosphoric
acid in an argon current29 furnished compound 2. Its UV
profile (λmax 280 and 306 nm)28 was similar to those of 1 and 2
(λmax 280, 306, and 320 nm); however, there are insufficient
NMR data available for comparison purposes and we could not
duplicate the synthesis of 2.
Compounds 3−5 were isolated as dark orange glassy solids;
their molecular formulas were determined as C20H18O9,
C24H22O11, and C26H24O12 by HRESIMS (IHD = 12, 14,
and 15, respectively). Their NMR spectra (Table 2, Figures
S15−S35) were similar to those of 3-O-(α-D-ribofuranosyl)-
questin (13)30 previously isolated from the endophytic fungus
Eurotium rubrum. In the case of 3, the signals due to a methoxy
group (δH 3.97, s, OMe-12) in 13 were replaced by resonances
for a chelated hydroxy group (δH 12.29, s, OH-1), which was
also supported by the difference of 14 Da in their molecular
weight. In addition, the 1H NMR spectrum of 3 showed signals
for a second chelated phenolic hydroxy proton at δH 12.09
(OH-9) and for an α-D-ribofuranosyl unit (Table 2) as in
compound 13. Finally, the HMBC correlation of the anomeric
proton at δH 5.88 (d, J = 4.5 Hz, H-1′) with the signal at δC
164.7 supported the O-α-glycosidic linkage to C-3. Acid
hydrolysis of 3 afforded ribose, which was identified by TLC
C
CH3
CH
CH
CH
CH
CH2
C
2.48, s
5.85, d (4.4)
4.56, m
2,3,4
8,3′,4′
4′
102.9
72.0
71.4
85.4
62.6
5.29, dd (7.0, 2.1)
4.38, m
1′,4′,5′,6′
2′
3.91, m
3′
170.8
21.0
CH3
2.22, s
6′
12.84, s
1,2,3,9a
S36−S42) for this compound were nearly identical to those of
known chrysophanein (14) (Table 3).31 However, compound
6 possesses a 3′-acetyl-α-D-ribofuranosyl moiety (δH 5.85, d, J
= 4.4 Hz, H-1′; 4.56, m, H-2′; 5.29, dd, J = 7.0, 2.1 Hz, H-3′;
4.38, m, H-4′; 3.91, m, H-5′; 2.22, s, H3-7′) rather than the β-
D-glucopyranosyl group at C-8. The placement of the
ribofuranosyl moiety at C-8 through an O-glycosidic linkage
was based on the HMBC correlations of the α-anomeric
proton H-1′ with C-8 (δC 158.1). On the other hand, HMBC
connectivities from H-3′ and H-7′ to C-6′ indicated the
position of the acetyl groups at C-3′ (Table 3). Therefore,
D
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1
Table 5. H (700 MHz) and 13C (175 MHz) NMR Data of
compound 6 was named chrysophanol O-3′-acetyl-α-D-
ribofuranoside.
Compound 8 in CDCl3
Compound 7 was isolated as a yellow solid. Its molecular
formula was deduced as C19H18O5, based on its molecular ion
peak in the HRESIMS (IHD = 11). The 1D and 2D NMR data
(Table 4 and Figures S43−S49) were consistent with those of
HMBC
position
δC
type
δH, mult. (J in Hz)
1H→13C
1
2
3
4
4a
5
154.5
126.4
128.4
147.0
124.4
76.4
C
C
CH
C
C
7.15, s
1,2,2′,4
1
Table 4. H (700 MHz) and 13C (175 MHz) NMR Data of
Compound 7 in CDCl3
1
HMBC H→13C
CH
4.96, dd (10.5, 4.9)
4a,7,9
position
δC
type
δH, mult. (J in Hz)
6
26.5
CH2 2.51, dddd (13.3, 10.5,
4.9,1.0)
4a,5,7,8
1
2
3
4
4a
5
6
7
8
8a
9
9a
10a
11
1′
2′
3′
4′
5′
159.9
110.7
137.7
119.0
153.0
134.1
135.4
111.7
153.1
105.8
185.9
107.6
142.3
16.7
C
CH
CH
C
C
C
C
CH
C
C
C
6.73, d (8.4)
7.48, d (8.4)
1,4,9,9a
1, 1′,4a,9a
2.21, m
4a,5,7
5,6,8
5,6,8
7
35.5
CH2 2.89, ddd (17.5, 12.6, 4.2)
2.62, dddd (17.5, 12.6, 4.2,
1.0)
8
8a
9
1′
2′
3′
4′
4a′
5′
6′
203.2
115.2
55.5
154.5
126.4
128.4
147.0
124.4
76.4
C
C
6.64, s
5,8,8a,9,10a
CH3 3.58, s
C
C
5
CH
C
7.15, s
1′,2,2′,4′
C
C
C
CH3
CH2
CH
C
CH3
CH3
2.41, s
3.53, dd (6.8, 1.2)
5.34, m
6,7
CH
4.96, dd (10.5, 4.9)
4a′,7′,9′
4a′,5′,7′,8′
4a′,5′,7′
5′,6′,8′
28.5
3,3′,4,4′,4a,5′
4,4′,5′
26.5
CH2 2.51, ddd (13.3, 10.5, 4.9, 1.0)
121.9
133.5
17.9
2.21, m
7′
35.5
CH2 2.89, ddd (17.5, 12.6, 4.2)
1.83, s
1.81, s
11.85, s
5.15, s
11.03, s
2′,3′,5′
2′,3′,4′
1,2,9a
5,6
2.62, ddd (17.5, 12.6, 4.2, 1.0)
5′,6′,8′
25.6
8′
8a′
9′
1-OH
4-OH
1′-OH
4′-OH
203.2
115.1
55.5
C
C
1-OH
5-OH
8-OH
CH3 3.58, s
12.55, s
7.88, s
12.54, s
7.86, s
5′
1,2,8a
3,4
1′,2′,8a′
3′,4′
7,8,8a
the 1,4,8-trihydroxy-3-methylxanthone ravenelin (15),32 except
for the following key changes: the presence of signals for an
isoprene unit (δC/δH C-1′/H-1′, 28.5/3.53; C-2′/H-2′, 121.9/
5.34; C-3′, 133.5; C-4′/H3-4′, 17.9/1.83; and C-5′/H3-5′,
25.6/1.81) and two aromatic protons (δC/δH C-2/H-2, 110.7/
6.73, and C-3/H-3, 137.7/7.48) as a pair of ortho-coupled
doublets. The position of the isoprene unit at C-4 (δC 119.0)
was confirmed by the key HMBC correlations from H-1′ to C-
3, C-4, and C-4a (δC 153.0) and from H-2′ to C-4. Moreover,
the HMBC correlation from 1-OH to C-1, C-2, and C-9a
supported this proposal. On the basis of these considerations,
compound 7 was given the trivial name 4-isoprenylravenelin.
Compound 8 (yellow solid) was isolated as a racemic
mixture since it was optically inactive, and the CD showed no
Cotton effect. The molecular formula was established as
C22H22O8 according to its HRESIMS peak (IHD = 12).
respectively; and the nonchelated phenolic OH-4 and OH-4′
to C-3 and C-4, and C-3′ and C-4′, respectively, confirmed
that the two identical units were linked throughout C-2−C-2′.
The conformation of the cyclohexenone ring was determined
as a half-chair form on the basis of the NOESY correlation
between H-5 and H-5′ with one of the methylene protons of
H-7 and H-7′, respectively; the J values of H-7 and H-7′
(Table 5) suggested a β- and α-quasi-axial orientation with
respect to the cyclohexenone ring. Interestingly, the chelated
and nonchelated phenolic OH groups were not equivalent,
probably due to a rotational barrier or the presence of
atropoisomerism. Previously, related monomers of 8 were
isolated as enantiomeric mixtures from Juglans mandshurica
Maxim. var. sieboldiana Makino (Juglandaceae).33 In that work,
both enantiomers were separated by chiral HPLC, and CD and
OR values were obtained. However, the amount of 8 obtained
in this work (0.8 mg) precluded separation by this means.
All compounds were evaluated for their inhibitory effects
against both α-glucosidase and PTP-1B. Compounds 1−3, 9,
and 10 showed inhibitory activity against yeast α-glucosidase
(αGHY), with IC50 values of 116.8 7.2, 144.5 4.9, 261.3
Detailed analysis of the H and 13C NMR data (Table 5 and
1
Figures S50−S56) indicated that this compound could be a
homodimer related to 5,8-dihydroxy-4-methoxy-α-tetralone.33
The HMBC correlations (Table 5) from H-3 and H-3′ to C-1,
C-2, C-2′, and C-4, and C-1′, C-2, C-2′, and C-4′, respectively;
from H3-9 and H3-9′ to C-5 and C-5′, respectively; from H-5
and H-5′ to C-4a, C-7, and C-9, and C-4a′, C-7′, and C-9′,
respectively; from H-6 and H-6′ to C-4a, C-5, C-7, and C-8,
and C-4a′, C-5′, C-7′, and C-8′, respectively; and from H-7
and H-7′ to C-5, C-6, and C-8, and C-5′, C-6′, and C-8′,
respectively, supported this affirmation. Furthermore, the
HMBC connectivities between the chelated phenolic OH-1
and OH-1′ to C-1, C-2, and C-8a, and C-1′, C-2′, and C-8a′,
7.8, 57.4
0.2, and 71.3
0.7 μM, respectively (positive
control acarbose, IC50 = 585.8 0.1 μM). The most active
compounds, 9 and 10, were also evaluated against intestinal rat
α-glucosidase enzymes. The calculated IC50 values were 742.6
7.3 and 458.7
6.6 μM, respectively, vs acarbose (IC50
E
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Journal of Natural Products
Article
Figure 2. Putative binding mode for 1 (cyan sticks), 3 (yellow sticks), 9 (orange sticks), 10 (blue sticks), acarbose (purple sticks), and ursolic acid
(red sticks) with (A) αGHY (skyblue cartoon, pdb code 3A4A) and (B) PTP-1B (green cartoon, pdb code 1SUG).
spectra (HRMS) were acquired with a JEOL AccuTOF-DART
JMS-T100LC (JEOL Ltd., Tokyo, Japan) spectrometer in positive
mode. Flash chromatography was accomplished on a CombiFlash Rf+
Lumen system (Teledyne Technologies, Inc., Lincoln, NE, USA)
using RediSep Rf gold silica gel columns (Teledyne) and eluting with
a gradient of n-hexane, CHCl3, and MeOH. Analytical and preparative
HPLC separations were conducted on Gemini C18 columns (5 μm,
110 Å, 250 × 4.6 mm i.d. and 5 μm, 110 Å, 250 × 21.2 mm i.d.,
respectively; Phenomenex, Torrance, CA, USA) in a Waters HPLC
system (Waters, Milford, MA, USA) equipped with a 2535 quaternary
pump, a 2707 autosampler, and the 2998 PDA and 2424 ELSD
detectors. Data management and acquisition were performed with the
Empower 3 software (Waters). Column chromatography (CC) was
carried out on silica gel 60 (70−230 mesh, Merck, Darmstadt,
Germany) or Sephadex LH-20 (GE Healthcare, Chicago, IL, USA).
Thin-layer chromatographic (TLC) analyses were performed on silica
gel 60 F254 plates (Merck), and visualization of the plates was carried
out using a (NH4)4Ce(SO4)4 (10%) solution in H2SO4. Reagent-
grade n-hexane, CHCl3, CH2Cl2, and MeOH, HPLC-grade MeCN,
and H2O were purchased from J.T. Baker (Avantor Performance
Materials, Center Valley, PA, USA).
Fungal Strain and Identification. The fungal strain Malbranchea
circinata Sigler & Carmichael (type strain ATCC 34526)39 was
obtained from the American Type Culture Collection (Manassas, VA,
USA). In order to place this fungus into a phylogenetic context and
since the type strain was deposited over 40 years ago in the ATCC, we
sequenced ATCC 34526 for the ITS rDNA (ITS1, 5.8S and ITS2), as
well as the partial 28S rDNA, using primer combinations ITS1F and
ITS440,41 and LROR and LR642,43 using methods outlined previously
(Supporting Information).44 The sequence data are deposited in
GenBank with accession numbers ITS: MN627784, MN627785;
LSU: MN627782, MN627783.
151.1 6.1 μM). The effect of 10 was corroborated in vivo
using a sucrose tolerance in normoglucemic mice at doses of
3.2 and 10.0 mg/kg. As can be seen in Figure S60, 10 at the
dose of 10 mg/kg decreases the postprandial peak in a similar
way to that of acarbose (positive control). In order to predict
the binding manner of the inhibitors with αGHY, docking
analyses were carried out using the crystallized structure of
αGHY (pdb code 3A4A).34,35 The docking protocol was
validated reproducing the binding mode of acarbose at the
catalytic domain,36 and the results predicted that 1−3, 9, and
10 could bind to the catalytic domain (Figure 2 and Figures
S61−S65).
Likewise, 1−10 showed moderate inhibitory activity against
PTP-1B (Table S5). The most active compounds were 7−10,
with IC50 values of 13.9 1.3, 10.9 0.6, 15.3 0.4, and 14.5
1.5 μM, respectively (positive control ursolic acid, IC50
=
27.8 0.1 μM). In the same fashion, docking analyses of 7−10
using the crystallized structure of PTP-1B (pdb code 1SUG),37
validated with ursolic acid,38 indicated that these compounds
bind into the catalytic site (Figure 2 and Figures S66−S74).
In summary, M. circinata is a new source of α-glucosidase
and PTP-1B inhibitors that could be useful for the develop-
ment of antidiabetic drugs. Specifically, malbrancheamide (10)
possesses vasodilating and antidiabetic effects and holds
promise for the design of novel therapies for metabolic
syndrome, a cluster of related conditions that increases the risk
for developing cardiovascular disease and T2DM.
EXPERIMENTAL SECTION
■
Fermentation, Extraction, and Isolation. M. circinata ATCC
34526 was cultivated on potato dextrose agar plates. Seed cultures of
the fungus were prepared using agar plugs (0.5 cm3), which were
inoculated in potato-dextrose broth (BD, Franklin Lakes, NJ, USA)
medium and incubated at room temperature for 10 days at 100 rpm.
Next, M. circinata was grown in six 2 L Fernbach flasks containing rice
medium (200 g and 400 mL of H2O each). After incubation for 41
days, the cultures were extracted exhaustively with 700 mL of 1:1
CHCl3−MeOH. The mixture was shaken for 3 h in a reciprocating
General Experimental Procedures. IR spectra were recorded
using a Bruker Tensor 27 FT-IR spectrophotometer (Bruker Corp.,
Billerica, MA, USA). Optical rotations were recorded at the sodium
D-line wavelength using a PerkinElmer model 343 polarimeter at 20
°C (PerkinElmer, Waltham, MA, USA). NMR spectra were recorded
on a Bruker AVANCE III HD with TCI CryoProbe 700 H−C
spectrometer at 700 MHz (1H) or 175 MHz (13C), using
tetramethylsilane as an internal standard. High-resolution mass
F
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Journal of Natural Products
Article
shaker and filtered; then equal volumes of H2O and CHCl3 were
added to the filtrate to a total volume of 1 L. After shaking, the
mixture was transferred into a separatory funnel and the organic layer
was drawn off and evaporated to dryness; the resulting residue was
partitioned in a separatory funnel between 180 mL of 1:1 MeOH−
MeCN and 180 mL of n-hexane, and then the bottom layer was
collected and evaporated to dryness. The defatted extract (2.86 g) was
dissolved in a mixture of CHCl3−MeOH, adsorbed onto a minimal
amount of Celite, and fractionated via flash chromatography on a 70 g
silica gel column, using a gradient solvent system of n-hexane−
CHCl3−MeOH at a flow rate of 40 mL min−1 and 35.0 column
volumes (CVs) over 109.4 min. Fractions were collected every 23 mL
and pooled according to UV and ELSD profiles to obtain 14 fractions
(F1−F14).
Compound 5: dark orange glassy solid; [α]2D01 +209.1 (c 0.09,
CHCl3); FTIR νmax 3560, 1745, 1677, 1223 cm−1; H and 13C NMR
see Table 2; HRESIMS m/z 529.13605 [M + H]+ (calcd for
C26H25O12 529.13460).
Chrysophanol O-3′-acetyl-α-D-ribofuranoside (6): orange glassy
solid; UV (MeOH) λmax (log ε) 268 (4.43), 344 (3.99), 405 (3.38)
nm; FTIR νmax 3450, 1660, 1632, 1582, 1485, 1250, 1226, 1190 cm−1;
1H and 13C NMR see Table 3; HRESIMS m/z 429.11742 [M + H]+
(calcd for C22H21O9 429.11856).
4-Isoprenyl ravenelin (7): dark orange glassy solid; FTIR νmax
3560, 1745, 1677, 1223 cm−1; 1H and 13C NMR see Table 4;
HRESIMS m/z 327.12308 [M + H]+ (calcd for C19H19O5
327.12325).
Compound 8: yellow glassy solid; [α]2D0 0 (c 0.08, CHCl3); UV
(MeOH) λmax (log ε) 368 (3.24), 262 (3.59), 228 (3.79), 207 (3.84)
F1 (121.4 mg) was subjected to silica gel CC eluting with n-
hexane−CH2Cl2 (75:25 → 0:100) to afford eight fractions (F1I−
F1VIII). From fraction F1II (eluted with 75:25 n-hexane−CH2Cl2),
13.9 mg of 6 was obtained. Fraction F1V (eluted with 65:35 n-
hexane−CH2Cl2) yielded 11.8 mg of the tautomeric mixture of 11
and 12. F2 (248.6 mg) was first subjected to flash chromatography on
a 12 g silica gel column using a gradient solvent system of n-hexane−
CHCl3−MeOH at a flow rate 30 mL min−1 and 54.0 CVs over 30.2
min, to yield seven fractions (F2I−F2VII). F2IV (146.6 mg) was further
purified by preparative RP-HPLC using as mobile phase 65:35
MeCN−H2O [0.1% formic acid (FA)] and increasing linearly to
100% MeCN over 15 min, at a flow rate of 21.24 mL min−1. This
procedure afforded 15.7 mg of 9 (tR = 8.2 min). F3 (159.1 mg) was
purified by silica gel CC with a gradient of n-hexane−CH2Cl2−
MeOH (40:60 → 0:100 → 85:15) to afford six fractions (F3I− F3VI).
F3III eluted with 25:75 n-hexane−CH2Cl2 afforded 23.7 mg of 5. F5
(86.9 mg) was purified by silica gel CC with a gradient of n-hexane−
CH2Cl2−MeOH (30:70 → 0:100 → 70:30) to afford four fractions
(F5I− F5IV). F5II eluted with 20:80 n-hexane−CH2Cl2 afforded 3.7
mg of 4. F7 (265.7 mg) was fractionated by flash chromatography on
a 12 g silica gel column eluted with a gradient of n-hexane−CHCl3−
MeOH at a 30 mL min−1 flow rate and 60.3 CVs over 30.2 min, to
yield three major fractions (F7I−F7III). F7II (179.4 mg) was purified
by preparative RP-HPLC using a gradient of 75:25 MeCN−H2O
(0.1% FA) and increasing linearly to 100% MeCN in 15 min, at a flow
rate of 21.24 mL min−1, to obtain 15.9 mg of 10 (tR = 5.3 min) and
2.0 mg of 2 (tR = 6.5 min). F8 (215.7 mg) was subject to silica gel CC
with a gradient of n-hexane−CH2Cl2−MeOH (20:80 → 0:100 →
70:30). This process yielded 10 fractions (F8I−F8X). F8VI (51.7 mg)
was further fractionated via Sephadex LH-20 eluted with 3:7
acetone−MeOH to afford six fractions (F8VI‑A−F8VI‑F). F8VI‑B (7.1
mg) and F8VI‑D (5.9 mg) were further purified by preparative TLC
using 98:2 CHCl3−MeOH and 97:3:0.1 CHCl3−MeOH−FA as
mobile phases, to obtain 1.2 mg of 8 and 1.6 mg of 7, respectively.
Finally, F9 (72.3 mg) was purified by preparative RP-HPLC using a
gradient of 70:30 MeCN−H2O (0.1% FA) and increasing linearly to
MeCN in 12 min, at a flow rate of 21.24 mL min−1, to obtain 1.2 mg
of 1 (tR = 5.2 min) and 3.4 mg of 3 (tR = 6.8 min).
1
nm; FTIR νmax 3560, 1745, 1677, 1223 cm−1; H and 13C NMR see
Table 5; HRESIMS m/z 415.13951 [M + H]+ (calcd for C22H23O8
415.13929).
Acid Hydrolysis of Compound 3. A 2 mg amount of compound
3 dissolved in 1 mL of water was refluxed with 1 mL of HCl (1 M) for
1 h. The reaction mixture was diluted with H2O (2 mL) and extracted
twice with EtOAc (4 mL). The H2O phase was concentrated to
dryness to yield α-D-ribofuranoside as detected by TLC with an
authentic sample. [α]2D0 −10 (c 0.09, H2O).
Computational Section. Minimum energy structures for the
different structures were built with Spartan’10 software (Wave-
function Inc., Irvine, CA, USA). Conformational analysis was
performed with the Monte Carlo search protocol as implemented in
the same software under the MMFF94 molecular mechanics force
field. The two most stable conformers were submitted to the Gaussian
09 program (Gaussian Inc., Wallingford, CT, USA)45 calculation for
their geometry optimization performed using the M06-2X/6-31+G-
(d,p) level of theory. The resulting NMR shielding tensors were
computed with the gauge-independent atomic orbital (GIAO)
method and the polarizable continuum model using the integral
equation formalism variant (IEFPCM) as the SCRF method using the
DFT method at the B3LYP/6-311+G(2d,p) level of theory.
Assay for α-Glucosidase Inhibitors. The fungal extract,
fractions, compounds, and acarbose (positive control) were dissolved
in MeOH or phosphate buffer solution (PBS, 100 mM, pH 7).
Aliquots of 0−10 μL of testing materials (triplicated) were incubated
for 10 min with 20 μL of enzyme (Sigma-Aldrich, St. Louis, MO,
USA) stock solution (0.4 units/mL) in PBS. After incubation, 10 μL
of substrate (pNPG 5 mM) was added and incubated a further 20 min
at 37 °C, and the absorbances were determined. For the extract and
fractions, the inhibitory activity was determined as percentage in
comparison to the blank (PBS) according to the following equation:
i
y
A415t
A415c
j
z
j
z
z
z
z
j
%αGHY = 1 −
× 100%
j
j
k
{
where % αGHY is the percentage of inhibition, A415t is the corrected
absorbance of the extract, fractions, or compound under testing
(A415 end − A415 initial), and A415c is the absorbance of the blank
(A415 end blank − A415 initial blank). The IC50 was calculated by regression
analysis, using the following equation:
Compound 1: white crystalline solid; mp 287 °C; HPLC-UV
[(MeCN in H2O + 0.1% FA)] λmax 280, 306, 320 nm; FTIR νmax
2927, 1684, 1591 1566 cm−1; 1H and 13C NMR see Table 1;
HRESIMS m/z 356.10324 [M + H]+ (calcd for C21H14N3O3
356.10352).
Compound 2: white solid; mp 282 °C; HPLC-UV [(MeCN in
H2O + 0.1% FA)] λmax 280, 306, 320 nm; FTIR νmax 2927, 1684, 1591
1566 cm−1; 1H and 13C NMR see Table 1; HRESIMS m/z 340.10942
[M + H]+ (calcd for C21H14N3O2 340.10860).
Compound 3: orange glassy solid; UV (MeOH) λmax (log ε) 268
(3.66), 225 (3.91) nm; FTIR νmax 3330, 2954, 2921, 1628, 1593,
1545 cm−1; 1H and 13C NMR see Table 2; HRESIMS m/z 403.10315
[M + H]+ (calcd for C20H19O9 403.10291).
A100
%Inhbition =
s
I
1 +
(IC )
50
where A100 is the maximum inhibition, I is the inhibitor concentration,
IC50 is the concentration required to inhibit activity of the enzyme by
50%, and s is the cooperative degree.
Expression and Purification of the Enzyme hPTP-1B.
Recombinant hPTP-1B was expressed from the PTPN1 gene (protein
tyrosine phosphatase nonreceptor type 1 from Homo sapiens, gene ID:
5770). The gene was optimized for overexpression in E. coli and
subcloned into the pET28 vector to obtain the pET28-PTPN1 system
by GenScript (Piscataway, NJ, USA). The overexpression system was
transformed into E. coli BL21 (DE3) cells with karamycin resistance,
Compound 4: orange glassy solid; UV (MeOH) λmax (log ε) 270
1
(3.06), 223 (3.22) nm; FTIR νmax 3564, 1741, 1630, 1215 cm−1; H
and 13C NMR see Table 2; HRESIMS m/z 487.12352 [M + H]+
(calcd for C24H23O11 487.12404).
G
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Journal of Natural Products
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inducing expression with 1 mM of IPTG for 8 h and purifying the
enzyme using a His Trap excel column from General Electric. The
overexpression and purification process was followed by PAGE-SDS,
obtaining a yield of about 90 mg per liter with a purity of 98%.46
Assay for PTP-1B inhibitors. The fungal extract, fractions,
compounds, and positive control were dissolved in DMSO, MeOH, or
Tris buffer solution (Tris, 20 mM, pH 7). Aliquots of 0−10 μL of
testing materials (triplicated) were incubated for 5 min with 20 μL of
enzyme stock solution (22 nm) in Tris. After incubation, 10 μL of
substrate (pNPP 5 mM) was added and incubated a further 15 min at
25 °C, and the absorbances were determined. For the extract and
fractions, the inhibitory activity was determined as percentage in
comparison to the blank (Tris) according to the following equation:
macromolecule was set as the center of the box. The default
parameters of exhaustiveness and number of modes were not altered.
Next, a refined docking was performed with a smaller box of searching
space (50 × 50 × 50 and 0.375 Å of grid spacing), setting as the
center of the grid box the lower state pose obtained from the blind
docking. The conformational states from the docking simulations
were analyzed using the AutoDockTools program, which also
identified the H-bonds and van der Waals interactions between the
catalytic site of α-glucosidase or PTP1B and the ligand. The predicted
docked complexes (protein−ligand) were those conformations
showing the lowest binding energy. Preparation of the figures was
accomplished with the PyMOL visualization tool (PyMOL Molecular
49,50
̈
Graphics System v1.7.4, Schrodinger, New York, NY, USA
and
51
̈
Maestro (Schrodinger).
i
y
A415t
A415c
j
z
j
z
z
j
%PTP1B = 1 −
× 100%
j
j
z
z
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jnatprod.9b01108.
k
{
■
S
where % PTP1B is the percentage of inhibition, A415t is the corrected
absorbance of the extract, fractions, or compound under testing
(A415 end − A415 initial), and A415c is the absorbance of the blank
(A415 end blank − A415 initial blank). The IC50 was calculated by regression
analysis, using the following equation:
1D and 2D NMR spectra of 1−8 and 1H NMR of 9−12;
phylograms of the most likely tree based on the analyses
of the ITS and LSU regions; docking analyses showing
the interactions of the active compounds with α-
glucosidase and PTP-1B (PDF)
A100
%Inhbition =
s
I
1 +
( )
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%, and s is the cooperative degree.
AUTHOR INFORMATION
Corresponding Author
*Tel: +52-55-5622-5289. E-mail: rachel@unam.mx.
■
Sucrose Tolerance Test of Malbrancheamide (10). ICR male
mice 3−4 weeks old (25−30 g body weight) were purchased from
Envigo-UNAM. Mice were housed in a room with a 12 h light:dark
cycle and controlled for temperature and humidity with free access to
standard laboratory rodent diet (Teklad 2018S, Envigo) and water ad
libitum until the beginning of each experiment. Mice were treated
according to the International Ethical Guidelines for the care and use
of laboratory animals and following the recommendations of the
Mexican Official Norm for Animal Care and Handling (NOM-062-
ORCID
Manuel Rangel-Grimaldo: 0000-0003-1261-3317
Martha L. Macías-Rubalcava: 0000-0001-9650-0920
́
Martin Gonzalez-Andrade: 0000-0002-8910-3035
Huzefa Raja: 0000-0002-0824-9463
Mario Figueroa: 0000-0001-7004-0591
Rachel Mata: 0000-0002-2861-2768
́
ZOO-1999). The Animal Studies Committee of Facultad de Quımica,
UNAM, approved the experimental protocol (FQ/CICUAL/292/
18). After 4 h of food deprivation, mice were divided into five groups
(I−V) of six animals each. The animals of group I were administered
with VEH (saline solution with 0.05% Tween 80) and group II with
reference drug (acarbose 5 mg/kg). Groups III−V were treated orally
with 10 at the doses of 3.1 and 10 mg/kg. Thirty minutes later,
sucrose (3 g/kg) was orally administrated to each animal. Blood
glucose concentrations 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:
Author Contributions
‡Taken in part from the Ph.D. thesis of Manuel Rangel-
Grimaldo.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by grants from CONACyT CB A1-S-
11226 and DGAPA IN 217320. M.R.G. acknowledges the
fellowship from CONACYT (294309) to pursue graduate
studies. This study made use of UNAM’s NMR lab, LURMN
at IQ-UNAM, which is funded by CONACYT-0224747, and
UNAM. The authors also recognize the valuable support of I.
Ä
É
Å
Å
Å
Ñ
Ñ
Ñ
(G − G )
t
i
Å
Å
Ñ
Ñ
%variation of glycemia =
× 100
Å
Å
Å
Ñ
Ñ
Ñ
Gi
Å
Ñ
Å
Ç
Ñ
Ö
where Gi is the basal glycemia and Gt is the different glycemia values
after treatment administration.10,47
́
́
́
Rivero-Cruz and A. Vazquez-Perez from Facultad de Quımica,
́
́
M. I. Velasquez-Lopez from Facultad de Medicina, and B.
Docking Studies. The minimized structures for docking
simulations were prepared using Autodock Tools package v1.5.4
(ADT, http://mgltools.scripps.edu/).48 For metabolites, addition of
Gasteiger charges and number of torsions were set, and nonpolar
hydrogens were merged. The crystallographic structure of α-
glucosidase from yeast and PTP-1B from human was obtained from
the Protein Data Bank (pdb code 3A4A and 1SUG, respectively). For
the receptor polar hydrogens and Kollman charges were added, and
solvation parameters were assigned by default. Molecular docking
studies were achieved with AutoDock v1.1.2.15. First, a blind docking
was performed in order to establish the common site of interaction of
the metabolites with the α-glucosidase and PTP1B. The search space
for this preliminary docking was defined as a box size of 90 × 90 × 90
Å in the x, y, and z dimensions, with a grid spacing of 0.375 Å, and the
́
́
Quiroz Garcıa, M. A. Pena Gonzalez, E. Huerta Salazar, J.
̃
́
́
́
Perez Flores, M. C. Garcıa-Gonzalez, and R. Patin
̃
o Maya from
́
Instituto de Quımica, for recording NMR, IR, UV, and MS
́
́
spectra. We are indebted to Direccion General de Computo y
́
́
́
de Tecnologıas de Informacion y Comunicacion (DGTIC),
UNAM, for the resources to carry out computational
calculations through the Miztli supercomputing system
(LANCAD-UNAM-DGTIC-313).
DEDICATION
■
Dedicated to Dr. Jon Clardy of Harvard Medical School for his
pioneering work on bioactive natural products.
H
DOI: 10.1021/acs.jnatprod.9b01108
J. Nat. Prod. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.jnatprod.9b01108
J. Nat. Prod. XXXX, XXX, XXX−XXX