α-Glucosidase Inhibitors from Salvia circinata

Article abstract of DOI:10.1021/acs.jnatprod.7b00155

A dried infusion prepared from the aerial parts of Salvia circinata did not provoke acute toxicity in mice (LD₅₀ > 5 g/kg). This infusion showed poor hypoglycemic and antihyperglycemic effects (100-570 mg/kg) when tested in normal and hyperglycemic mice using acute and oral glucose tolerance tests, respectively. However, this infusion possessed antihyperglycemic action in vivo during an oral sucrose tolerance test (31.6-316 mg/kg), suggesting the presence of α-glucosidase inhibitors in S. circinata. Fractionation of a nonpolar extract of the aerial parts of the plant yielded a new biflavone (1) and four new neoclerodane diterpenoid glucosides (2-5) along with the known compounds amarisolide (6), pedalitin (7), apigenin-7-O-β-d-glucoside (8), and the flavone 2-(3,4-dimethoxyphenyl)-5,6-dihydroxy-7-methoxy-4H-chromen-4-one (9). Compounds 1 and 6-9 were active against mammalian α-glucosidases; 6 and 7 were also active against a recombinant α-glucosidase from Ruminococcus obeum and reduced significantly the postprandial peak during an oral sucrose tolerance test in healthy mice, consistent with their α-glucosidase inhibitory activity. Molecular docking and dynamic studies revealed that compounds 6 and 7 might bind to α-glucosidases at the catalytic center of the enzyme.

Full text of DOI:10.1021/acs.jnatprod.7b00155

Article
pubs.acs.org/jnp
α‑Glucosidase Inhibitors from Salvia circinata
†
‡
§
§
Laura Flores-Bocanegra, Martin Gonzal
́
ez-Andrade, Robert Bye, Edelmira Linares,
,†
and Rachel Mata*
†
‡
§
Facultad de Química, Facultad de Medicina, and Instituto de Biología, Universidad Nacional Auton
́
oma de Mex
́
ico, Cuidad de
Mex
́ ́
ico 04510, Mexico
*
S Supporting Information
ABSTRACT: A dried infusion prepared from the aerial parts of Salvia
circinata did not provoke acute toxicity in mice (LD50 > 5 g/kg). This
infusion showed poor hypoglycemic and antihyperglycemic effects
(
100−570 mg/kg) when tested in normal and hyperglycemic mice
using acute and oral glucose tolerance tests, respectively. However, this
infusion possessed antihyperglycemic action in vivo during an oral
sucrose tolerance test (31.6−316 mg/kg), suggesting the presence of
α-glucosidase inhibitors in S. circinata. Fractionation of a nonpolar
extract of the aerial parts of the plant yielded a new biflavone (1) and
four new neoclerodane diterpenoid glucosides (2−5) along with the
known compounds amarisolide (6), pedalitin (7), apigenin-7-O-β-D-
glucoside (8), and the flavone 2-(3,4-dimethoxyphenyl)-5,6-dihydroxy-
7
-methoxy-4H-chromen-4-one (9). Compounds 1 and 6−9 were
active against mammalian α-glucosidases; 6 and 7 were also active
against a recombinant α-glucosidase from Ruminococcus obeum and reduced significantly the postprandial peak during an oral
sucrose tolerance test in healthy mice, consistent with their α-glucosidase inhibitory activity. Molecular docking and dynamic
studies revealed that compounds 6 and 7 might bind to α-glucosidases at the catalytic center of the enzyme.
ccording to the International Diabetes Federation, in 2015
glucosidase inhibitors in vitro against α-amylase from hog
pancreas and α-glucosidase from Saccharomyces cerevisiae.
S. circinata (syn. Salvia amarissima Ortega) is a perennial
13
A
around 415 million people worldwide were suffering from
1
type II diabetes mellitus (T2DM). The high prevalence of this
disease and its negative economic impact on global national
health systems have triggered the search for new therapeutic
alternatives. In particular, in middle- and low-income countries,
diabetic patients require more economic and efficient treat-
ments to ameliorate their impaired glycemic conditions. To
assist with this problem, plants used in traditional medicine
have proven to be valuable sources of phytotherapeutic
preparations and chemical templates for the development of
herb widely distributed in Mexico, mainly in the Central
1
4
Mexican Valley, Oaxaca, and San Luıs
́
Potosı.
́
This species is
used in folk medicine for treating ulcers, helminthiases, and
diabetes. Previous phytochemical studies led to the isolation
of some clerodane diterpenoids such as amarisolide (6),
several seco-clerodane diterpenoids, and the flavone pedalitin
15
16,17
10
16
(7). Some of the compounds obtained were cytotoxic against
human cancer cell lines and had modulatory activity in a breast
2
17,18
new antidiabetic drugs. Thus, as part of our efforts to establish
cancer cell line resistant to vinblastine.
the efficacy of selected Mexican plants for treating diabetes and
3
−5
to discover new α-glucosidase inhibitors,
useful for the
RESULTS AND DISCUSSION
■
development of new antidiabetic therapies, reported herein is
the investigation of Salvia circinata Cav. (Lamiaceae). Previous
studies on other species of this genus, including S. miltiorrhiza
Acute toxicity analysis in animals is the first step during the
assessment of the efficacy of phytopreparations. The
information obtained from an acute toxicity test is useful for
choosing appropriate doses for pharmacological studies and
providing preliminary identification of target organ toxicity. In
this investigation, acute toxicity was studied in mice following
6
7
8
Bunge, S. splendens Sellow ex Schult., S. hypoleuca Benth.,
9
10
11
S. fruticosa Mill., S. syriaca L., S. santolinifolia Boiss.,
12
10
S. moorcraftiana Wall., S. limbata C.A. Mey., S. atropatana
1
0
10
10
Bunge, S. nemorosa L., and S. multicaulis Vahl., have
revealed its potential as sources of hypoglycemic and α-
glucosidase inhibitory agents. In most cases, the active
ingredients have been flavonoids and other phenolic com-
pounds such as salvianolic acids A and B, which ameliorated
hyperglycemia and dyslipidemia in db/db mice through the
19
the Lorke protocol, which gives reproducible results using a
minimum number of animals. Acute administration of a dried
infusion prepared from the aerial parts of S. circinata did not
provoke behavior alterations, macroscopic tissue injury, or
6
AMPK pathway. More recently, some clerodane diterpenoids
Received: February 21, 2017
from S. chamaedryoides Cav. were found to be active as α-
©
XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.7b00155
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Figure 1. Antihyperglycemic action of the infusion (IS) from S. circinata (31.6−316 mg/kg, po) in normoglycemic (A) and NA-STZ (50/130 mg/
kg, ip)-hyperglycemic mice (B) during an oral glucose tolerance test. VEH: vehicle; ACA: acarbose. Each point represents the mean ± SEM 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 post hoc
test for comparison with respect to vehicle control at the same time.
Chart 1
weight loss during a 14-day observation period, and the
estimated LD was higher than 5 g/kg. Therefore, according to
the Lorke criteria, the traditional preparation of S. circinata
proved to be devoid of acute toxic effects for mice (Table S1,
Supporting Information).
1), the infusion (31.6, 100, and 316 mg/kg) significantly
reduced blood glucose when compared with a vehicle-treated
group, with the effect observed comparable to that of acarbose,
a commercially available drug used as positive control. The
good activity observed during the oral sucrose tolerance test
and the poor effect during the oral glucose tolerance test
strongly suggested that the antihyperglycemic effect of the
S. circinata infusion could be due to the presence of α-
glucosidase inhibitors, which may be able to prevent
postprandial hypersecretion of insulin and reactive hypoglyce-
50
Next, the hypoglycemic potential of the S. circinata infusion
was assessed in both normal and nicotinamide−streptozotocin
(
NA 50 mg/kg-STZ 130 mg/kg)-treated mice using half-log
interval doses (100−570 mg/kg), chosen according to a
20
standard protocol of allometric scaling. The NA-STZ model
is used for evaluating potential antidiabetic drugs since it
portrays a similar biochemical blood profile and pathogenesis to
22
mia.
In order to isolate the active principles, a nonpolar extract
from the aerial parts of S. circinata was prepared. Extensive
chromatography of this extract yielded new compounds 1−5,
21
T2DM in humans. Oral administration of the infusion to
normal and NA-STZ mice showed a moderate decrease of
blood glucose level at the doses tested (Figure S1, Supporting
Information). This effect was less than that produced by
glibenclamide (GLI), an antidiabetic drug.
16
16 23
24
along with the known substances 6, 7, 8, and 9, which
were identified by comparison with physical and spectroscopic
data previously reported.
The antihyperglycemic effect of the S. circinata infusion was
Compound 1 was isolated as a yellow, amorphous powder.
initially established throughout an oral glucose tolerance test
Its molecular formula, C H O , was calculated from the
34
26 13
(
Figure S2, Supporting Information). In this, the infusion did
HRESIMS. The IR spectrum displayed characteristic signals for
−
1
not induce a significant drop in the postprandial peak after
glucose challenge in normal and hyperglycemic mice (100−570
mg/kg). Finally, during an oral sucrose tolerance test (Figure
hydroxy and carbonyl groups (3252 and 1663 cm ,
respectively). The chemical shifts of the paired signals in the
NMR spectrum (Table 1, Figures S3 and S4, Supporting
B
DOI: 10.1021/acs.jnatprod.7b00155
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Table 1. H (500 MHz) and 13C (125 MHz) NMR
1
this evidence, compound 1 was characterized as 6,6″,3‴-
25,26
Spectroscopic Data for Compound 1 in CD OD
trihydroxy-7,3′,7″-O-trimethylloniflavone.
3
Compounds 2−5 were isolated as optically active, colorless,
glassy solids. Their molecular formulas were deduced by
HRESIMS as C H O , C H O , C H O , and
position
δC
type
δH, multiplicity (J in Hz)
2
166.7,
104.3,
184.1,
107.0,
147.6,
156.2,
131.8,
92.4,
C
2
4
36
9
26 36 10
27 38 11
3
4
4
5
6
7
8
8
1
2
3
4
5
6
2
3
4
4
5
6
7
8
8
1
2
3
4
5
6
CH
C
6.53, s
C H O , respectively. The IR spectra of all compounds
27
38 11
included bands in the range 3396 to 3385 and from 1763 to
a
C
−1
1
749 cm , consistent with the presence of hydroxy and α,β-
C
unsaturated-γ-lactone groups in the molecules. The IR
C
−1
spectrum of 2 showed also a band at 1705 cm attributable
C
to a carbonyl group. The NMR spectra of 2−5 (Tables 2 and 3,
Figures S12−S39, Supporting Information) showed strong
similarities to those of amarisolide (6), a neo-clerodane type of
diterpenoid possessing a tricyclic skeleton with the six-
membered rings trans-fused and an ethylfuran moiety. Also in
common, clerodanes 2−6 exhibited signals for a β-glucopyr-
anosyloxy moiety at C-2 and an α,β-unsaturated-γ-lactone at C-
CH
C
6.71, s
a
152.3,
131.9,
111.0,
149.9,
152.5,
117.2,
122.2,
165.5,
104.5,
184.2,
107.1,
147.6,
156.3,
131.9,
92.3,
′
′
′
′
′
′
C
CH
C
7.39, d (2.0)
C
CH
CH
C
6.86, d (8.3)
4
/C-5. In order to demonstrated the D configuration of the β-
7.42, dd (8.4, 2.1)
glucopyranosyloxy moiety, compounds 2−6 were hydrolyzed
″
″
″
a″
″
″
″
″
a″
‴
‴
‴
‴
‴
‴
with β-glucosidase; in all cases, the aqueous-soluble fraction
CH
C
6.49, s
20
showed identical optical rotation ([α]
+30) and R (co-
D
f
TLC) to those of an authentic sample of β-D-glucose.
Comparison of the NMR data of 2−5 with 6 (Tables 2 and
C
C
3
) revealed that the β-substituted furan ring in 6 was replaced
C
by a methyl ketone group in 2, an α,β-unsaturated-γ-lactone in
, and a five-membered ketolactol methyl ether in 4 and 5.
C
3
CH
C
6.67, s
1
13
Assignments of the H and C NMR data of 2−5 (Tables 2
152.4,
125.5,
114.4,
153.0,
148.7,
113.0,
120.5,
57.4,
1
1
and 3) were supported by 2D NMR ( H− H COSY, HSQC,
and HMBC) experiments. The 13C NMR chemical shifts
observed in the spectra of compounds 4 and 5 for C-13 (4/5 δC
C
CH
C
7.30, d 2.2
1
1
70.0/137.9), C-14 (4/5 δ 104.7/171.2), C-15 (4/5 δ 171.0/
C C
C
02.5), and C-16 (4/5 δ 117.2/143.0) revealed that the ethyl
C
CH
CH
6.96, d (8.6)
7.38, dd (7.3, 2.1)
3.92, s
fragment [C-11 (4/5 δ 27.6/34.7) and C-12 (4/5 δ 33.9/
C
C
1
7.7)] is linked to the five-membered ketolactol methyl ether
OCH -7
CH3
3
27,28 29,30
moiety at the β-
or α-
carbon to the carbonyl group,
OCH -3′
OCH -7″
OCH -3‴
57.0,
CH3
3.90, s
3
respectively. The HMBC correlations for H-12 and H-14 in 4
supported this assignment [H-12a (δH 1.56) → C-14 (δC
57.4,
CH3
CH3
3.92, s
3
56.9,
3.89, s
3
1
5
04.7); H-12b (δ 2.09) → C-14 (δ 104.7); and H-14 (δ
H C H
.91) → C-12 (δ 33.9)]. Likewise, the HMBC correlations for
C
Information) were consistent with a biflavone-type dimeric
H-12 and H-15 in 5 [H-12a (δ
12b (δ 2.09) → C-16 (δ 143); and H-15 (δ
(δ 137.9) and C-14 (δ 171.2)] reinforced the structural
assignment made.
H
2.33) → C-16 (δ
C
143.0); H-
25,26
flavonoid, with a C-4′ → C-4‴ interflavanoid ether linkage.
H
C
H
5.92) → C-13
1
The H NMR spectrum (Table 1 and Figure S3, Supporting
Information) showed two ABX systems attributable to the
protons of ring C of the two flavonoid moieties, which were
C
C
The NOESY interactions (Figure 2) observed in the spectra
of 2−5 revealed that the relative configuration at the
stereogenic centers of all clerodanes were identical to that of
6. Thereafter, the electronic circular dichroism (ECD) spectra
of 2−6 were simulated. The experimental and calculated ECD
spectra (Figures 3 and S40, Supporting Information) were
similar in all cases, indicating that the absolute configuration at
the stereogenic centers C-2, C-5, C-8, C-9, and C-10 was S, S,
R, R, and R, respectively, in 2−6. All spectra showed negative
Cotton effects at ∼208 and ∼250 nm due to the electronic
transitions π → π* and n → π*, respectively, of the α,β-
unsaturated-γ-lactone. It is important to point out that the
absolute configuration of 6 was unequivocally determined by X-
then trisubstituted. Four singlets assigned to H-8 (δ 6.75), H-
H
8
″ (δ 6.69), H-3 (δ 6.58), and H-3″ (δ 6.55) were also
H
H
H
observed; thus, rings A of both monomers were pentasub-
stituted. Finally, signals for four methoxy groups between δH
3
.8 and 3.9 were detected. The cross-peaks observed for H-8
and H-8″ with the methoxy group signals in the NOESY
spectrum indicated that two of the methoxy groups were at C-7
and C-7″. On the other hand, the NOESY correlations from H-
2
′ (δ 7.43) → OCH -3′ (δ 3.96) and H-2‴ (δ 7.33) →
H
3
H
H
OCH -3‴ (δ 3.94) were used to locate the other two methoxy
3
H
groups at C-3′ and C-3‴ (Figure S10, Supporting Information).
The long-range HMBC correlations from H-5′ (δ 6.90) → C-
H
31
1
′ (δ 131.9) and C-3′ (δ 149.9) and H-5‴ (δ 7.03) → C-1‴
ray analysis; since the optical rotation of 6 isolated in this
C
C
H
16,31
(δ 125.5) and C-3‴ (δ 153.0) as well as those for H-2′ (δ
work was identical to that previously reported,
the strategy
C
C
H
7
7
.39) and H-6′ (δ 7.42) → C-4′ (δ 152.5), and H-2‴ (δ
employed to establish the configuration at the streogenic
centers at the tricyclic skeleton with the six-membered rings
trans-fused of all diterpenes appears to be sound. In the case of
compounds 4 and 5, the ECD were calculated for the two
possible epimers at C-14 or C-15, respectively. The results
H
C
H
.30) and H-6‴ (δ 7.38) → C-4‴ (δ 148.7), corroborated
H
C
these assignments and established the interlinkage bond as C-
1
3
4
′−O−C-4‴. The C NMR spectrum was fully assigned by
means of the HSQC and HMBC experiments. On the basis of
C
DOI: 10.1021/acs.jnatprod.7b00155
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Journal of Natural Products
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1
Table 2. H (500 MHz) NMR Spectroscopic Data for Compounds 2−5 in DMSO-d
6
δ, multiplicity (J, Hz)
position
1
2
3
4
5
1.76,
.28,
ddd (13.8, 12.0, 12.0)
1.80,
1.34,
4.47,
6.63,
1.72,
1.25,
1.24,
1.71,
2.27,
1.54,
ddd (13.8, 12.0, 12.0)
1.76,
1.34,
4.47,
6.67,
1.67,
1.25,
1.54,
1.67,
2.25,
1.54,
ddd (13.8, 12.0, 12.0)
1.81,
1.34,
4.46,
6.66,
1.71,
1.25,
1.60,
1.54,
2.24,
1.53,
1.44,
2.33,
2.09,
ddd (13.8, 12.0, 12.0)
a
a
a
a
1
m
m
m
m
a
a
a
a
2
3
6
4.43,
6.63,
1.69,
m
m
m
m
d (6.4)
d (6.4)
d (6.3)
d (6.3)
m
m
m
m
1.18,
m
m
m
m
7
8
1.52,
1.53,
2.12,
1.44,
m
m
m
m
m
m
m
m
1
1
0
1
d (12.9)
m
d (12.6)
d (12.9)
m
d (12.9)
m
m
m
m
m
m
m
m
1
.43,
12
2.61,
m
m
s
2.37,
2.06,
1.56,
2.09,
5.91,
m
2.32,
m
1
1
1
1
1
4
5
6
7
9
2.05,
d (0.8)
4.79,
7.45,
0.80,
4.00,
4.38,
0.54,
4.31,
2.94,
3.11,
2.97,
3.11,
3.60,
3.44,
m
5.92,
7.13,
0.79,
4.02,
4.39,
0.56,
4.30,
2.93,
3.10,
2.95,
3.10,
3.64,
3.33,
brs
t (1.6)
d (6.6)
d (8.3)
d (8.2)
s
6.04,
0.80,
4.02,
4.38,
0.56,
4.30,
2.97,
3.11,
2.98,
3.11,
3.67,
3.34,
3.45,
t (0.7)
d (6.4)
d (8.3)
d (8.2)
s
brs
0.74,
3.97,
d (6.6)
d (8.1)
d (8.1)
s
d (6.5)
d (8.3)
d (8.2)
s
4.53,
20
1′
2′
3′
4′
5′
6′
0.51,
4.29,
2.94,
3.10,
2.96,
3.10,
3.65,
d (7.7)
dd (8.0)
dd (8.0)
dd (8.0)
m
d (7.7)
dd (8.0)
dd (8.0)
dd (8.0)
m
d (7.7)
dd (8.0)
dd (8.0)
dd (8.0)
m
d (7.7)
dd (8.0)
dd (8.0)
dd (8.0)
m
dd (11.3, 2.5)
dd (11.0, 2.5)
dd (11.6, 2.5)
dd (11.6, 2.0)
a
a
a
a
3.34,
m
m
m
m
OCH3
OCH3
s
3.45,
s
a
Signal overlapped.
revealed that the ECD calculated for the β-epimer at C-15 of 5
was identical to the experimental spectrum; thus, the absolute
configuration at the chiral center C-15 in 5 was established as S.
In the case of compound 4 the configuration at C-14 could not
be established, since the calculated ECD spectra for the two
epimers at C-14 did not match exactly with the experimental
spectrum. On the basis of this evidence, compounds 2−5 were
characterized as (2S,5S,8R,9R,10R)-2-(O-β-D-glucopyranosyl)-
n e o c l e r o d a - 3 - e n - 9 - o x o b u t y l - 1 8 , 1 9 - o l i d e ( 2 ) ,
from Ruminococcus obeum, a bacterium found in the human
32
intestine that is involved in carbohydrate metabolism. This
protein is a structural homologue to human intestinal N-
maltase-glucoamylase (2QMJ.pdb, 85% coverage) with a highly
conserved catalytic domain (Figures S41 and S42, Supporting
Information). This enzyme is phylogenetically closer to human
N-maltase-glucoamylase than those of rat small intestinal
enzymes; therefore, it is possible to make better inferences
regarding the potential in vivo effects in humans of any
compound assayed with this enzyme. The maltase-glucoamy-
lase enzyme from R. obeum shows substrate preference for
α(1→6) over α(1→4) glycosidic linkages and produces glucose
(
2S,5S,8R,9R,10R)-2-(O-β-D-glucopyranosyl)neocleroda-3,13-
diene-14,15;18,19-diolide (3), (2S,5S,8R,9R,10R)-2-(O-β-D-
glucopyranosyl)neocleroda-14-methoxy-3,13-diene-
32
1
5,14;18,19-diolide (4), and (2S,5S,8R,9R,10R,15S)-2-(O-β-D-
glucopyranosyl)neocleroda-15-methoxy-3,13-diene-
4,15;18,19-diolide (5), which were given the trivial names
from isomaltose as well as maltose. The results of the assays
showed that 6, 7, and acarbose inhibited the activity of the pure
enzyme with IC50 values of 400 ± 19.0, 60 ± 3.0, and 1030 ±
14.0 μM, respectively.
1
amarisolides B−E (2−5).
Compounds 1−9 were tested in vitro against mammalian α-
glucosidase. The more active compounds were the flavonoids,
and, in particular, compound 1 showed an IC50 value of 39 ±
Compounds 6 and 7 were also evaluated in vivo in an oral
sucrose tolerant test in healthy mice (Figure 4); acarbose was
the positive control. As expected, oral administration of 6 and 7
reduced significantly the postprandial peak in a dose-dependent
manner. In both cases, the effect was comparable to that of the
positive control, thus revealing their antihyperglycemic
potential. These results were consistent with the α-glucosidase
inhibition activity demonstrated in vitro for these compounds.
In order to complete the inhibition studies, docking and
molecular dynamics studies of the R. obeum α-glucosidase
complexes were performed with acarbose, 6, and 7 (Figures 5
and 6). According to the results obtained, the three products
0.06 μM, which was 2.5 times more active than acarbose (IC50
=
100 ± 0.3 μM), used as positive control. Flavonoids 7−9
exhibited IC50 values of 810 ± 39, 200 ± 12, and 1800 ± 140
μM, respectively. Regarding the diterpenoids, only compound 6
was active, with an IC value of 500 ± 32.3 μM. In the case of
5
0
compounds 2−5, the IC values were higher than 10 000 μM
5
0
(
Table S2, Supporting Information). The major compounds of
the plant, 6 and 7, and acarbose were also tested against a
recombinant α-glucosidase with maltase-glucoamylase activity
D
DOI: 10.1021/acs.jnatprod.7b00155
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Table 3. 13C (125 MHz) NMR Spectroscopic Data for Compounds 2−5 in DMSO-d6
δ, type
position
2
3
4
5
1
2
3
4
5
6
7
8
9
25.7,
69.2,
130.6,
142.8,
45.1,
33.6,
27.2,
35.8,
36.8,
39.3,
30.3,
36.0,
208.7,
30.0,
CH2
CH
CH
C
25.7,
69.0,
130.7
142.6,
45.1,
33.3,
27.2,
35.8,
37.4,
39.2,
34.9,
17.8,
132.8,
174.2,
70.4,
146.4,
15.4,
168.8,
70.6,
17.2,
101.9,
73.6,
76.7,
70.0,
76.9,
61.1,
CH2
CH
CH
C
26.8,
69.1,
131.0,
143.2,
45.6,
33.7,
26.2,
35.9,
37.7,
39.5,
27.6,
33.9,
170.0,
104.7,
171.0,
117.2,
15.9,
169.2,
70.9,
17.6,
102.0,
74.0,
77.2,
70.7,
77.4,
61.8,
56.5,
CH2
CH
CH
C
25.7,
69.2,
130.8,
142.6,
45.1,
33.3,
27.2,
35.7,
37.4,
39.2,
34.7,
17.7,
137.9,
171.2,
102.5,
143.0,
15.5,
168.8,
70.6,
17.3,
102.8,
73.5,
76.7,
70.8,
77.0,
61.2,
CH2
CH
CH
C
C
C
C
C
CH2
CH2
CH
C
CH2
CH2
CH
C
CH2
CH2
CH
C
CH2
CH2
CH
C
1
1
1
1
1
1
1
1
1
1
2
1
2
3
4
5
6
0
1
2
3
4
5
6
7
8
9
0
′
CH
CH2
CH2
C
CH
CH2
CH2
C
CH
CH2
CH2
C
CH
CH2
CH2
C
CH3
C
CH
C
C
CH
CH
CH3
C
CH
CH
CH3
C
CH
CH3
C
15.5,
168.7,
70.5,
17.3,
101.9,
73.9,
76.8,
70.3,
77.0,
61.2,
CH3
C
CH2
CH3
CH
CH
CH
CH
CH
CH2
CH2
CH3
CH
CH
CH
CH
CH
CH2
CH2
CH3
CH
CH
CH
CH
CH
CH2
OCH3
CH2
CH3
CH
CH
CH
CH
CH
CH2
′
′
′
′
′
OCH3
OCH3
56.1,
OCH3
bind to the catalytic site of the enzyme. On the other hand, the
molecular dynamics studies (Figure 6) indicated that the total
energy of the complexes remained stable throughout the
analysis, revealing that the binding of the compounds was
stable. The study was conducted for 20 ns considering that the
system was stable during this period of time (Figure S44,
Supporting Information). The most stable complex was
compound 7 since the calculated ΔG was −31.26 ± 2.9,
while the ΔG values for acarbose and 6 were −23.12 ± 4.6 and
clerodane diterpenoid against α-glucosidase and of the
occurrence of biflavones in the genus Salvia.
EXPERIMENTAL SECTION
■
General Experimental Procedures. Melting points were
determined on a Fisher-Johns apparatus and are uncorrected. IR,
UV, and ECD spectra were recorded using a PerkinElmer 400 FT-IR, a
Shimadzu U160, and a JASCO J720 instrument, respectively. Optical
rotations were recorded at the sodium D-line wavelength using a
PerkinElmer model 343 polarimeter at 20 °C. 1D and 2D NMR
spectra were recorded in CD OD or DMSO-d solution on a Bruker
−
20.03 ± 3.4, respectively. These results are in agreement with
the inhibition data (IC ) obtained experimentally. Animated
5
0
3
6
1
images of the trajectories of molecular dynamics may be found
in the Supporting Information (Figure S43).
Avance III HD spectrometer at either 500 MHz ( H) or 125 MHz
13
1
13
(
C) or a Varian VNRMS at 400 MHz ( H) or 100 MHz ( C), using
tetramethylsilane as an internal standard. HRESIMS were obtained
using a Thermo LTQ Orbitrap XL mass spectrometer. Preparative
HPLC was carried out with a Waters instrument (Milford, MA, USA)
equipped with a 2535 pump and a 2998 photodiode array detector,
using an XBridge C18 packed column (21.1 × 250 mm) and different
gradient systems of MeCN and 0.1% aqueous formic acid, at a flow
rate of 17.0 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 (GE
Healthcare, Little Chalfont, Buckinghamshire, UK) and/or silica gel 60
In summary, the aerial parts of S. circinata possess an
antihyperglycemic action in vivo in an oral sucrose tolerance
test, supporting the medicinal use of S. circinata for treating
diabetes in Mexican folk medicine. This plant produces several
flavonoid and clerodane diterpene glycoside constituents with
α-glucosidase inhibitory effects in vitro, and this target seems to
be involved in their in vivo antihyperglycemic action. The
maltase-glucoamylase enzyme from R. obeum demonstrated to
be a good tool to assess α-glucosidase inhibitory activity in
vitro. Molecular docking and dynamic studies revealed that
compounds 6 and 7 might bind to α-glucosidase at the catalytic
center of the enzyme. As hypothesized by Bisio et al., the
enzymatic inhibitory capacity of species of Salvia is due not
only to their content of phenolic compounds but also of other
secondary metabolites such as the clerodane-type diterpenes,
which are characteristic metabolites of this genus. To our
knowledge, this is the first report of the in vivo action of a neo-
(0.063−0.200). Each Sephadex LH-20 column was 60 cm long with 4
13
cm internal diameter. Thin-layer chromatographic analyses were
performed on silica gel 60 F254 plates (Merck, Kenilworth, NJ, USA),
and visualization of the plates was carried out using a Ce (SO )
4 3
2
(
10%) solution in H SO .
2 4
Plant Material. The aerial parts of S. circinata were collected in
June 2014 in Huauchinango Puebla, Mexico. The plant was identified
by authors R.B. and E.L. A voucher specimen (R. Bye and E. Linares
E
DOI: 10.1021/acs.jnatprod.7b00155
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Journal of Natural Products
Article
Figure 2. Key NOESY correlations of compounds 2−5.
(
10:90), precipitated 3.87 g of 6 as a white solid, mp 206−208 °C.
Fraction OE5 (6.3 g), eluted with hexane−AcOEt (40:60), was
subjected to CC on Sephadex LH-20 eluting with CH OH to afford six
3
secondary fractions (OE 1−OE 6). From fraction OE 6 (423.5 mg)
5
5
5
precipitated 7 (368.0 mg) as a yellow solid, mp 300−302 °C. Fraction
OE 3 (91.0 mg) was also subjected to CC on Sephadex LH-20 eluting
5
with CH OH−acetone (1:1) to afford 1 (10.3 mg), mp 223−225 °C,
3
and 9 (30.6 mg), mp 212−214 °C, both as yellow solids. Fraction OE6
(
20 mg) was purified on a Sephadex LH-20 column (CH OH) to yield
3
8
(15.5 mg), mp 230−232 °C. Fraction OE7 (25 g) was separated on
a silica gel column eluting with hexane−EtAcO (20:80 → 0:100) and
then with EtAcO−CH OH (100:0 → 80:20) to afford four secondary
3
fractions (OE 1−OE 4). Fractions OE 1 (53.0 mg) and OE 2 (75.3
7
7
7
7
Figure 3. Comparison of theoretical and experimental ECD spectra of
mg) were purified by reversed-phase HPLC (XBridge C , 21.1 × 250
18
2.
mm, 5 μm) using as mobile phase 30:70 MeCN−H O (acidified with
2
0
.1% formic acid) and increasing linearly to 50% over 15 min at a flow
rate of 17.0 mL/min. These processes yielded 25.0 mg of 3 (t 14.3
R
3
7855) has been deposited at the National Herbarium (MEXU),
min) and 12.5 mg of 5 (t 17.0 min) from fraction OE 1 and 21.0 mg
R
7
UNAM, Mexico City.
of 4 (t 16.2 min) and 17.0 mg of 2 (t 11.8 min) from fraction OE 2.
R
R
7
Extraction and Isolation. Two hundred grams of dried and
ground plant material was extracted with 5.5 L of boiling water for 20
min and was filtered and dried in vacuo to yield 19 g of aqueous
extract. A nonpolar extract was prepared from 820 g of plant material
by exhaustive maceration (8 days, three times) with 8 L of a solvent
mixture of CH Cl −CH OH (1:1). Thereafter, the extract was filtered
Compound 1: yellow powder; mp 223−225 °C; UV (MeOH) λ
max
(
log ε) 271 (3.8), 330 (3.2) nm; IR (FTIR) ν 3252, 2925, 1663
max
−
1
1
13
cm ; H and C NMR in Table 1; HRESIMS m/z 643.1430 [M +
H] (calcd for C H O [M + H] , 643.1452).
+
+
34 26 13
2
0
Compound 2: glassy solid; [α]
+54.73 (1 mg/mL, CH OH);
D
3
2
2
3
UV (MeOH) λmax (log ε) 206 (0.412); ECD (CH OH, c 0.1 mM, 0.1
and dried in vacuo to yield 200 g of extract. A 150 g quantity of this
extract was subjected to CC on silica gel (5000 g), eluting with
3
cm); Δε −8.94 (210 nm), −8.98 (250 nm); IR (FTIR) νmax 3378,
−
1
1
13
hexane−EtAcO (100:0 → 0:100) and then with EtAcO−CH OH
1763, 1705 cm ; H and C NMR in Table 2 and Table 3;
3
+
+
(
(
100:0 → 80:20). This procedure yielded eight primary fractions
OE1−OE8). From fraction OE6, eluted with hexane−AcOEt
HRESIMS m/z 469.2403 [M + H] (calcd for C24
H
36
O
9
[M + H] ,
469.2438).
F
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Journal of Natural Products
Article
Figure 4. Antihyperglycemic action of 6 (A, 3.1−31.6 mg/kg, po) and 7 (B, 1.0−10.0 mg/kg, po) in normoglycemic mice during an OSTT. VEH:
vehicle; ACA: acarbose. Each point represents the mean ± SEM for 6 mice in each group. **p < 0.01 and ***p < 0.001 represent significantly
different two-way ANOVA followed by Bonferroni’s post hoc test for comparison with respect to vehicle control at the same time.
Figure 5. 3D Structural model of complexes of α-glucosidase with acarbose (A), 6 (B), and 7 (C). Binding sites and amino acid involved in the
binding of acarbose (D), 6 (E), and 7 (F).
Compound 3: glassy solid; [α]20 +43.05 (1 mg/mL, CH OH);
Enzymatic Hydrolysis. Compounds 2−6 (5 mg each) were
dissolved in phosphate buffer solution (5 mL, 100 mM at pH 7), and
enzyme β-glucosidase (10 g, Sigma-Aldrich, St. Louis, MO, USA) was
added to the solution and kept at 40 °C for 15 days, in each case. The
D
3
UV (MeOH) λmax (log ε) 207 (0.514); ECD (CH OH, c 0.1 mM, 0.1
3
cm); Δε −6.92 (208 nm), −7.29 (250 nm); IR (FTIR) ν at 3385,
max
−
1
1
13
1
5
749 cm ; H and C NMR in Table 2 and Table 3; HRESIMS m/z
09.2352 [M + H] (calcd for C H O [M + H] , 509.2387).
+
+
reaction mixtures were extracted with CHCl , and the aqueous phases
26
36 10
3
20
Compound 4: glassy solid; [α]
+67.90 (1 mg/mL, CH OH);
were compared directly with an authentic D-glucose sample by co-TLC
and optical rotation.
D
3
UV (MeOH) λmax (log ε) 207 (0.631); ECD (CH OH, c 0.4 mM, 0.1
3
cm); Δε −16.4 (208 nm), −16.5 (250 nm); IR (FTIR) ν at 3396,
Electronic Circular Dichroism Spectra. Minimum energy
structures for the stereoisomers neo- and ent- of 2−6 were built with
Spartan’10 software (Wavefunction Inc., Irvine, CA, USA). Conforma-
tional analysis was performed with the Monte Carlo search protocol as
implemented in the same software under the MMFF94 molecular
mechanics force field. The resulting conformers were minimized using
the DFT method at the B3LYP/6-311G+(2d,p) level of theory. The
time-dependent DFT (TDDFT) method at the B3LYP/6-31G+(d)
level of theory was employed for ECD calculations using the same
max
−1
1
13
1
754 cm ; H and C NMR in Table 2 and Table 3; HRESIMS m/z
+
+
5
39.2452 [M + H] (calcd for C H O [M + H] , 539.2492).
27
38 11
20
Compound 5: glassy solid; [α]
+59.21 (1 mg/mL, CH OH);
D
3
ECD (CH OH, c 0.9 mM, 0.1 cm); Δε −15.92 (208 nm), −12.91
3
(
3
251 nm); UV (MeOH) λ (log ε) 207 (0.614); IR (FTIR) ν at
max max
−
1
1
13
396, 1750 cm ; H and C NMR in Table 2 and Table 3;
+
+
HRESIMS m/z 539.2455 [M + H] (calcd for C H O [M + H] ,
27
38 11
539.2492).
G
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Journal of Natural Products
Article
Figure 6. Molecular dynamics trajectory analysis. (A) Total energy of the system as a function of the time of 6, 7, and acarbose (ACA). (B) RMSD
as a function of time.
DFT-minimized conformers. The self-consistent reaction field with a
conductor-like continuum solvent model was used to perform the
ECD calculations of the major conformers in MeOH. The calculated
excitation energy (nm) and rotatory strength (R) in dipole velocity
Molecular Dynamics Simulation. The coordinates of the ligands,
resulting from the docking study, were processed with an antechamber
to generate suitable topologies for the LEaP module from AMBER
3
7,38
12.
Each structure and complex was subjected to the following
(
Rvel) and dipole length (Rlen) forms were simulated into an ECD
protocol: Hydrogens were added using the LEaP module with the
parm99 parameter set, Na counterions were added to neutralize the
+
curve. All calculations were performed employing the Gaussian’09
program package (Gaussian Inc.).
system, and the structures were then solvated in an octahedral box of
explicit TIP3P model water molecules, localizing the box limits at 12 Å
α-Glucosidase Genetic Optimization, Cloning, Expression,
and Purification. The α-glucosidase gene from Ruminococcus obeum
ATCC 29174 was optimized (Figure S41, Supporting Information) for
expression in Escherichia coli BL21(DE3), using the software
OptimumGene from GenScript (Piscataway, NJ, USA). The optimized
gene was cloned into the pUC57 vector and subsequently subcloned
into the expression vector pET-12b Novagen (EMD Chemicals,
Darmstadt, Germany) using the restriction enzyme sites SalI and
BamHI for their correct orientation. E. coli BL21-DE3/pET12b was
grown in 500 mL of LB medium at 37 °C; the expression was induced
by the addition of IPTG (0.5 mM). After 8 h, the cells were harvested
by centrifugation (10 min, 4000g), suspended in 50 mM bis-TRIS
propane at pH 8.0, and lysed by sonication with pulses of 5 min at an
amplitude of 35% three times; cellular debris was removed by
centrifugation (15 min × 15000g). The protein was identified by
3
9,40
from the protein surface.
MD simulations were performed. Frames
were saved at 100 ps intervals for subsequent analysis. The analyses
4
1
were conducted with cpptraj on the trajectory time intervals where
the convergence criteria were met. Binding free energies were
calculated by molecular mechanics/Poisson−Boltzmann surface
4
2,43
area.
Experimental Animals. ICR male mice, aged between 3 and 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
2
018S, Envigo) and water ad libitum until the beginning of each
experiment. All animal experimental protocols were in conformity with
the Mexican Official Norm for Animal Care and Handling (NOM-062-
ZOO-1999) and the 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
32
activity tests and gel electrophoresis.
In Vitro Assay for α-Glucosidase Inhibitors. Acarbose, used as
positive control, was dissolved in phosphate buffer solution (PBS, 100
mM, pH 7), and compounds 1−10 were dissolved in PBS or MeOH−
PBS (1:1). The substrate used was p-nitrophenyl-α-D-glucopyranoside
́
Quimica-UNAM approved the protocols in March 2016: FQ/
CICUAL/134/16 (Lorke assay) and FQ/CICUAL/132/16 (hypo-
glycemic and antihyperglycemic tests).
(
20 mM in PBS; Sigma-Aldrich). The mammalian enzyme solution
was prepared with 40 mg of rat intestinal acetone powder in 1 mL of
PBS following the manufacturer’s (Sigma-Aldrich) protocol. The
R. obeum enzyme solution was used at a concentration of 5.2 mg/mL
in bis-TRIS propane. Aliquots of 0−10 μL of compounds 1−9 and
acarbose were incubated with 20 μL of enzyme solution in the case of
the mammalian enzyme; in addition, compounds 6, 7, and acarbose
were incubated with 5 μL of the enzyme solution of R. obeum; in both
cases the incubation period was 10 min at 37 °C. After incubation, the
substrate was added to each preparation (5 μL for mammalian and 10
μL for R. obeum), which were incubated for 30 min and 3 h more at 37
Acute Oral Toxicity in Mice of the Initial Infusion of
S. circinata. The acute oral toxicity of the initial aqueous infusion
of S. circinata was assessed and analyzed according to the Lorke
method. The dose schemes are provided in the Supporting
Information.
1
9
Nicotinamide−Streptozotocin (NA-STZ)-Induced Experi-
mental Hyperglycemia in Mice. Experimental hyperglycemia in
4,21
mice was induced as described earlier,
using a single dose of NA
(50 mg/kg) and STZ (130 mg/kg). One week later mice having blood
glucose ≥ 200 mg/dL were considered hyperglycemic and selected for
the study.
°
C, respectively. Absorbances were determined in a Bio-Rad 680
microplate reader (Hercules, CA, USA) at 405 nm. The inhibitory
activity expressed as IC50 was determined as previously described.
Collection of Blood Samples and Determination of Blood
Glucose Levels. Blood samples were collected from the caudal vein,
and the glucose levels (mg/dL) determined by the enzymatic glucose
2
5,33
Docking Protocol. Docking was conducted using the PDB X-ray
structure of α-glucosidase from R. obeum (3N04.pdb); the structures
of acarbose, 6, and 7 were constructed using HyperChem 8 software
and subsequently minimized using Gaussian 09, revision A.02
4
oxidase method as previously described. The percentage variations of
4
blood glucose levels were calculated following a standard protocol.
Acute Hypoglycemic Assay. The hypoglycemic action of the
dried plant initial infusion (100, 316, and 517 mg/kg) in normal and
NA-STZ mice was performed following the procedures previously
34,35
(
Gaussian Inc., Wallingford, CT, USA).
Electrostatic grid maps,
docking, and docking analyses were performed with AutoDockTools,
36
4
AutoDock, and PyMOL softwares.
reported. The percentage variation of glycemia was calculated as
H
DOI: 10.1021/acs.jnatprod.7b00155
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
previously stated. Areas under the curve were calculated with the
trapezoidal method.
Oral Glucose Tolerance Test (OGTT). The antihyperglycemic
action in an OGTT of the dried plant initial infusion (100, 316, and
(2) Hung, H.; Quian, K.; Morris-Natschke, S.; Hsu, C.; Lee, K.-H.
Nat. Prod. Rep. 2012, 29, 580−606.
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(3) Anaya-Eugenio, G.; Rivero-Cruz, I.; Rivera-Chavez, J.; Mata, R. J.
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Ethnopharmacol. 2014, 155, 416−425.
4) Ovalle-Magallanes, B.; Medina-Campos, O. N.; Pedraza-Chaverri,
J.; Mata, R. Phytochemistry 2014, 110, 111−119.
5) Martínez, A. L.; Madariaga-Mazon, A.; Rivero-Cruz, I.; Bye, R.;
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7) Fatma Abd-Elkader, M.; Mohamed-Soubhi, M.; Siham Mustafa,
5
17 mg/kg) in normal and NA-STZ mice was assayed following the
(
4
methodology previously reported by our research group.
Oral Sucrose Tolerance Test (OSTT). The antihyperglycemic
action in an OSTT of the dried plant initial infusion (31.6, 100, and
(
́
3
16 mg/kg), 6 (3.1, 10, and 31.6 mg/kg), and 7 (1.0, 3.1, and 10 mg/
(
kg) in normal and NA-STZ mice were assayed following the standard
methodology reported elsewhere.
4
9
(
Statistical Analysis. Data were expressed as means ± standard
error. 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, GraphPad Inc., La Jolla, CA,
USA) was used for statistical analysis.
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ASSOCIATED CONTENT
■
*
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Supporting Information
(
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ACS Publications website at DOI: 10.1021/acs.jnat-
prod.7b00155.
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S. cicrcinata in normoglycemic and NA-STZ-hyper-
glycemic mice; antihyperglycemic action of an infusion
from S. cicrcinata in normoglycemic and NA-STZ-
hyperglycemic mice during an oral glucose tolerant
test; one- and two-dimensional NMR spectra of
compounds 1−5; HRESIMS of 1−5; alignment of the
synthetic gene ATCC29174 after optimization; animated
images of the trajectories of molecular dynamics of
complexes acarbose-α-glucosidase, 6-α-glucosidase, and
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AUTHOR INFORMATION
■
*
2
(
Corresponding Author
Phone: +52-555-622-5289. E-mail: rachel@unam.mx.
ORCID
(
Rachel Mata: 0000-0002-2861-2768
Notes
The authors declare no competing financial interest.
(
(
A.; van Vuuren, S. F.; Ferreira, D.; Hamburger, M.; van der
Westhuizen, J. J. Nat. Prod. 2015, 78, 2494−2504.
ACKNOWLEDGMENTS
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This work was supported by grants from CONACyT 219765,
DGAPA IN217516 and IA 204116, and PAIP-UNAM 5000-
(28) Wu, T. H.; Cheng, Y. Y.; Liou, J. R.; Way, T. D.; Chen, C. J.;
Kuo, S. C.; El-Shazly, M.; Chang, F. R.; Wu, Y. H.; Liaw, C. C. RSC
Adv. 2014, 4, 23707−23712.
9
140. We thank R. del Carmen, I. Rivero-Cruz, M. Gutier
Perez-Vasquez, M. I. Velasquez Lopez, and R. I. del Villar for
their valuable technical assistance. We are indebted to
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(DGTIC), UNAM, for the
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y Comunicacion
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5
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