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molecules
Article
Flavonoids and Terpenoids with PTP-1B Inhibitory
Properties from the Infusion of
Salvia amarissima Ortega
Eric Salinas-Arellano 1, Araceli Pérez-Vásquez 1, Isabel Rivero-Cruz 1, Rafael Torres-Colin 2,
Martín González-Andrade 3, Manuel Rangel-Grimaldo 1and Rachel Mata 1,*
1Facultad de Química, Universidad Nacional Autónoma de México, Mexico City 04510, Mexico;
ersalinass@hotmail.com (E.S.-A.); perezva@unam.mx (A.P.-V.); riveroic@unam.mx (I.R.-C.);
manuel_erg_p9@hotmail.com (M.R.-G.)
2Instituto de Biología, Universidad Nacional Autónoma de México, Mexico City 04510, Mexico;
rafael.torres@ib.unam.mx
3Facultad de Medicina, Universidad Nacional Autónoma de México, Mexico City 04510, Mexico;
martin@bq.unam.mx
*Correspondence: rachel@unam.mx; Tel.: +52-55-56225289
Academic Editor: Raffaele Capasso
Received: 17 June 2020; Accepted: 30 July 2020; Published: 1 August 2020
Abstract:
An infusion prepared from the aerial parts of Salvia amarissima Ortega inhibited the enzyme
protein tyrosine phosphatase 1B (PTP-1B) (IC
50
~88 and 33
µ
g/mL, respectively). Phytochemical
analysis of the infusion yielded amarisolide (
1
), 5,6,4
0
-trihydroxy-7,3
0
-dimethoxyflavone (
2
),
6-hydroxyluteolin (
3
), rutin (
4
), rosmarinic acid (
5
), isoquercitrin (
6
), pedalitin (
7
) and a new
neo-clerodane type diterpenoid glucoside, named amarisolide G (
8a
,
b
). Compound
8a
,
b
is a new
natural product, and
2
–
6
are reported for the first time for the species. All compounds were tested for
their inhibitory activity against PTP-1B; their IC
50
values ranged from 62.0 to 514.2
µ
M. The activity
was compared to that of ursolic acid (IC
50
=29.14
µ
M). The most active compound was pedalitin
(
7
). Docking analysis predicted that compound
7
has higher affinity for the allosteric site of the
enzyme. Gas chromatography coupled to mass spectrometry analyses of the essential oils prepared
from dried and fresh materials revealed that germacrene D (
15
) and
β
-selinene (
16
), followed by
β
-caryophyllene (
13
) and spathulenol (
17
) were their major components. An ultra-high performance
liquid chromatography coupled to mass spectrometry method was developed and validated to
quantify amarisolide (1) in the ethyl acetate soluble fraction of the infusion of S. amarissima.
Keywords: Salvia amarissima; PTP-1B activity; amarisolide G; diabetes
1. Introduction
Type 2 diabetes mellitus is a metabolic disease characterized by chronic hyperglycemia due to
insulin resistance, or the relative absence of the hormone. The prevalence of the disease is continuously
increasing, with approximately 463 million people living with diabetes nowadays. Mexico is one of the
countries more affected by type 2 diabetes mellitus, with more than 12 million cases. The population
sector more affected is the indigenous people owing to variations in its traditional way of life and
the effects of industrial developments [
1
]. Mexican population employs more than 300 plant species
to treat the symptoms of diabetes; in some cases, the patients combine allopathic therapies with the
botanical remedies [
2
]. These plants are an essential part of the country’s alternative medical care,
and the best testimony of their efficacy is their persistence in Mexican markets and other places for
crude or fresh drug selling. Therefore, it is crucial to analyze these plants to establish their composition,
Molecules 2020,25, 3530; doi:10.3390/molecules25153530 www.mdpi.com/journal/molecules
Molecules 2020,25, 3530 2 of 18
security, efficacy, and to develop a suitable methodology for quality control of the crude drug following
good practice guidelines.
Quality control of herbal drugs is the base for their efficacy and safety. Quality control
of herbal drugs aims to define their identity, purity and content of active principles or marker
compounds. The chemical composition of plants, and hence of their therapeutic preparations, is
variable, so standardization is necessary to guarantee comparable therapeutic effects. To prove the
constant composition of herbal preparations, there are appropriate standard analytical methods to
establish relevant criteria for uniformity. Standard analytical techniques include, among others,
high-performance liquid chromatography. For many years, the World Health Organization (WHO) has
encouraged all its country members to elaborate pharmacopeic monographs providing comprehensive
scientific information on the quality of their most commonly used medicinal plants. Following WHO
guidelines, Mexico has developed the Mexican Herbal Pharmacopeia, which contained monographs
with definitions, analytical techniques for identity and composition, as well as storage regulations of
the most widely used Mexican Herbal drugs [2,3].
Like other pharmaceutical products, herbal drugs should fulfill the basic requirements of being
efficacious and safe. To establish herbal drugs’ efficacy and safety is necessary to perform preclinical
and clinical assays, including those of the healer or medical doctor in rural communities who apply
locally produced herbal preparations. When assessing the efficacy of the plants, it is essential to
study both the traditional preparations and their components; this is because sometimes the efficacy
is attained with the combinations of compounds in the preparations, which may be acting through
synergy, network pharmacology or by targeting several nonrelated proteins involved in the pathology
of a disease. Altogether, these studies can lead to the development of standardized phytomedicines of
good quality and discover good drug candidates or molecules useful for lead optimization or even
fragment-based drug discovery [3].
Among the species highly valued in Mexico for treating diabetes is Salvia amarissima Ortega (syn.
Salvia circinata Cav.) belonging to the mint family. It is a perennial aromatic shrub native to Mexico,
listed as medicinal in the catalog of plants from the Royal Botanical Expeditions to New Spain. Like
many other New World Salvia species, S. amarissima is melittophilous (bee-pollinated). A tea brewed
from dried aerial parts of the plant is useful in Mexican folk medicine for treating diabetes, ulcers and
helminthiases [
4
,
5
]. The species is commonly regarded as “insulina” (insulin), referring to its efficacy
to improve the diabetic condition [
5
]. Previous phytochemical studies allowed the isolation of some
neo-clerodane diterpenoids, including amarisolide (1) [
6
], a few seco-clerodane diterpenoids [
6
–
10
] and
some flavonoids [6,8]. The traditional preparation of the plant collected in Puebla, Mexico, as well as
amarisolide (1), and some of the flavonoids showed inhibitory activity against mammal
α
-glucosidases
in vitro
and
in vivo
. The preparation and compounds were hypoglycemic and reduced the postprandial
peak significantly during an oral sucrose tolerance test in healthy mice [
8
]. Some of the seco-clerodane
diterpenoids were cytotoxic against a few human cancer cell lines, had modulatory activity in a
breast cancer cell line resistant to vinblastine and exhibited antiprotozoal action [
7
,
9
–
11
]. Furthermore,
the traditional preparation lack toxicity when tested according to the Lorke criteria. [
8
]. More recently,
the antinociceptive properties of an aqueous extract of the plant, 1and 7were demonstrated [12].
Based on the above considerations, this investigation aimed: (i) to determine the effect of the
traditional preparation (infusion) of the plant collected in Oaxaca and their components on the activity
of the protein tyrosine phosphatase (PTP-1B) in order to assess a new molecular target, and get a
better insight in the
in vivo
hypoglycemic effect previously demonstrated [
8
]. This target was chosen,
considering that this enzyme acts as a negative regulator of insulin and leptin dependent signal cascades
holding therapeutic utility in type 2 diabetes mellitus and obesity [
13
]. (ii) To analyze the chemical
composition of the essential oil. (iii) To set up an appropriate procedure using Ultra-High-Performance
Liquid Chromatography (UHPLC) to quantify one active or marker compound of the plant’s infusion.
The chemical composition of the plant’s essential oil and the UHPLC procedure will allow developing
Molecules 2020,25, 3530 3 of 18
a pharmacopeic monograph of S. amarissima, as they represent valuable identity and composition
tests, respectively.
2. Results and Discussion
Scheme 1summarizes the whole work.
Molecules 2020, 25, x 3 of 18
will allow developing a pharmacopeic monograph of S. amarissima, as they represent valuable
identity and composition tests, respectively.
2. Results and Discussion
Scheme 1 summarizes the whole work.
Scheme 1. Workflow diagram of this study. * Efficacy studies. ** Quality control analyses. ***
Discovery of a lead molecule.
2.1. Chemical Constituents of the Aqueous Extract
An aqueous extract (AE) from S. amarissima inhibited the PTP-1B activity significantly, with an
IC
50
value of 88.6 ± 5.4 μg/mL. Workup of AE by solvent partitioning and repeated chromatography
afforded eight compounds (Figure 1), namely amarisolide (1), 5,6,4′-trihydroxy-7,3′-
dimethoxyflavone (2), 6-hydroxyluteolin (3), rutin (4), rosmarinic acid (5), isoquercitrin (6), pedalitin
(7) and a neo-clerodane type diterpenoid glycoside, named amarisolide G (8a,b). Compound 8a,b, is
a new natural product characterized by conventional spectroscopic and spectrometric techniques.
The known compounds were identified by comparing their spectroscopic data with those previously
described (Figures S1-S11, Supplementary Material) [8,14,15]. Compounds 2–6 are reported for the
first time for this species. In addition, thin layer chromatographic (TLC) analysis revealed that these
compounds were present in the AE prepared from the fresh material.
Scheme 1.
Workflow diagram of this study. * Efficacy studies. ** Quality control analyses. *** Discovery
of a lead molecule.
2.1. Chemical Constituents of the Aqueous Extract
An aqueous extract (AE) from S. amarissima inhibited the PTP-1B activity significantly, with an
IC
50
value of 88.6
±
5.4
µ
g/mL. Workup of AE by solvent partitioning and repeated chromatography
afforded eight compounds (Figure 1), namely amarisolide (
1
), 5,6,4
0
-trihydroxy-7,3
0
-dimethoxyflavone
(
2
), 6-hydroxyluteolin (
3
), rutin (
4
), rosmarinic acid (
5
), isoquercitrin (
6
), pedalitin (
7
) and a neo-clerodane
type diterpenoid glycoside, named amarisolide G (
8a
,
b
). Compound
8a
,
b
, is a new natural product
characterized by conventional spectroscopic and spectrometric techniques. The known compounds
were identified by comparing their spectroscopic data with those previously described (Figures S1–S11,
Supplementary Material) [
8
,
14
,
15
]. Compounds
2
–
6
are reported for the first time for this species.
In addition, thin layer chromatographic (TLC) analysis revealed that these compounds were present in
the AE prepared from the fresh material.
Product
8a
,
b
was obtained as a white solid optically active. DART-HRMS (Direct Analysis
In Real Time-High Resolution Mass Spectrometry) established its molecular formula as C
26
H
36
O
11
.
The IR spectrum included bands for hydroxyl (3365 cm
−1
) and
α
,
β
-unsaturated-
γ
-lactone (1749 cm
−1
)
functionalities (Figure S12, Supplementary Material) [
8
]. The NMR spectra of
8a
,
b
(Table 1; Figures
S13–S18, Supplementary Material) were closely similar to those of amarisolide D (
8c
), a neo-clerodane
type of diterpenoid with an
α
,
β
-unsaturated-
γ
-lactone at C-4/C-5, a five-membered keto-
γ
-lactol
methyl ether and a
β
-D-glucopyranosyloxy moiety at C-2 [
8
]. Thus, in compound
8a
,
b
the signals
for the five-membered keto-
γ
-lactol methyl ether were replaced by those of a keto-
γ
-lactol moiety.
Molecules 2020,25, 3530 4 of 18
Accordingly, the NMR spectra showed signals at
δH
5.90, 6.05 (brs, H-14)/
δC
99.4, 99.9 (C-14),
δH
5.96 (brs, H-16)/
δC
116.1 (C-16),
δC
171.4 (C-13) and
δC
171.9 (C-15) (Table 1). Since the resonances
for H-14 appeared as two separate signals, the intensity of each corresponding to one-half proton,
compound
8a
,
b
is a 1:1 mixture of C-14 epimers. The doubling of C-14 signal in the
13
C-NMR spectra
provided further evidenced (Table 1). The
13
C-NMR chemical shifts (Table 1) of C-11-C-16 and the
key HMBC (Heteronuclear Multiple Bond Correlation) correlations of H-12 and H-14 established
the point of attachment of the ethyl fragment (C-11-C-12) to the keto
−γ
-lactol ring. The most
relevant HMBC correlations were H-12a (
δH
1.72) with C-14 (
δC
99.4 and 99.9); H-12b (
δH
1.56) with
C-14 (
δC
99.4 and 99.9); and H-14 (
δH
5.90 and 6.05) with C-12 (
δC
33.8). The NOESY (Nuclear
Overhauser Effect Spectroscopy) interactions revealed that the relative configuration at the stereogenic
centers of
8a
,
b
was identical to that of amarisolides A–D [
8
]. The electronic circular dichroism
spectrum of
8a
,
b
showed negative Cotton effects at ~212 and ~250 nm due to the electronic transitions
π→π
* and n
→π
*, respectively, of the
α
,
β
-unsaturated-
γ
-lactone. The latter data indicated that
the absolute configuration at the stereogenic centers C-2, C-5, C-8, C-9 and C-10 of compound
8a
,
b
was S,S,R,Rand R, respectively. The D configuration of the
β
-glucopyranosyloxy moiety
was established as previously described [
8
]. On the basis of these evidences, compound
8a
,
b
was characterized as (2S,5S,8R,9R,10R,14R,S)-2-(O-
β
-d-glucopyranosyl)-neo-clerodan-14-hydroxy-3,
13-diene-14,15;18,19-diolide (8a,b) and was designated with the trivial name of amarisolide G.
Molecules 2020, 25, x 4 of 18
Figure 1. Structures of compounds (1–8a,b) from S. amarissima.
Product 8a,b was obtained as a white solid optically active. DART-HRMS (Direct Analysis In
Real Time-High Resolution Mass Spectrometry) established its molecular formula as C26H36O11. The
IR spectrum included bands for hydroxyl (3365 cm−1) and α,β-unsaturated-γ-lactone (1749 cm−1)
functionalities (Figure S12, Supplementary Material) [8]. The NMR spectra of 8a,b (Table 1; Figures
S13−S18, Supplementary Material) were closely similar to those of amarisolide D (8c), a neo-clerodane
type of diterpenoid with an α,β-unsaturated-γ-lactone at C-4/C-5, a five-membered keto-γ-lactol
methyl ether and a β-D-glucopyranosyloxy moiety at C-2 [8]. Thus, in compound 8a,b the signals for
the five-membered keto-γ-lactol methyl ether were replaced by those of a keto-γ-lactol moiety.
Accordingly, the NMR spectra showed signals at δH 5.90, 6.05 (brs, H-14)/δC 99.4, 99.9 (C-14), δH 5.96
(brs, H-16)/δC 116.1 (C-16), δC 171.4 (C-13) and δC 171.9 (C-15) (Table 1). Since the resonances for H-14
appeared as two separate signals, the intensity of each corresponding to one-half proton, compound
8a,b is a 1:1 mixture of C-14 epimers. The doubling of C-14 signal in the 13C-NMR spectra provided
further evidenced (Table 1). The 13C-NMR chemical shifts (Table 1) of C-11-C-16 and the key HMBC
(Heteronuclear Multiple Bond Correlation) correlations of H-12 and H-14 established the point of
attachment of the ethyl fragment (C-11-C-12) to the keto−γ-lactol ring. The most relevant HMBC
correlations were H-12a (δH 1.72) with C-14 (δC 99.4 and 99.9); H-12b (δH 1.56) with C-14 (δC 99.4 and
99.9); and H-14 (δH 5.90 and 6.05) with C-12 (δC 33.8). The NOESY (Nuclear Overhauser Effect
Spectroscopy) interactions revealed that the relative configuration at the stereogenic centers of 8a,b
was identical to that of amarisolides A−D [8]. The electronic circular dichroism spectrum of 8a,b
showed negative Cotton effects at ~212 and ~250 nm due to the electronic transitions π→π* and n→
π*, respectively, of the α,β-unsaturated-γ-lactone. The latter data indicated that the absolute
configuration at the stereogenic centers C-2, C-5, C-8, C-9 and C-10 of compound 8a,b was S, S, R, R
and R, respectively. The D configuration of the β-glucopyranosyloxy moiety was established as
previously described [8]. On the basis of these evidences, compound 8a,b was characterized as
(2S,5S,8R,9R,10R,14R,S)-2-(O-β-D-glucopyranosyl)-neo-clerodan-14-hydroxy-3, 13-diene-14,15;18,19-
diolide (8a,b) and was designated with the trivial name of amarisolide G.
Figure 1. Structures of compounds (1–8a,b) from S. amarissima.
2.2. Chemical Constituents of the Essential Oil
Dried and fresh plant materials were analyzed to assess any change during the drying process.
Since it is an aromatic plant, the chemical profile of the essential oil is valuable as an identity test.
The essential oil content of distilled aerial parts was 0.02% and 0.03% for fresh and dried material,
respectively. In each case, eight major compounds were identified, representing 99.96 and 99.97%
of the composition of the essential oil, respectively. As observed in Table 2and Figures S19 and
S20 (Supplementary Material), the major components in both samples were germacrene D (
15
) and
Molecules 2020,25, 3530 5 of 18
β
-selinene (
16
), followed by
β
-caryophyllene (
13
) and spathulenol (
17
). The only mutually exclusive
constituents were 3-methoxy-p-cymene (
9
) present in the dried material, and
δ
-elemene (
11
) found
only in the fresh plant. These differences are not due to seasonal changes because the plant material
was from the same batch.
Table 1. 1H and 13C NMR spectroscopic data for compound 8a,bin DMSO-d6.
Position δHa(J/Hz) δCb
1α: 1.35 dd (13.6, 3.2) 26.3
β: 1.81 brd (13.7)
2 4.44–4.49 m 70.0
3 6.66 d (6.4) 131.1
4 143.0
5 45.4
6α: 1.72 td (12.7,3.3) 33.8
β: 1.22–1.26 m
7α: 1.53–1.58 m
β: 1.65−1.70 m 27.6
8 1.65–1.70 m 36.2
9 37.8
10 2.21 d (15.1) 40.5
11 1.59–1.64 m 27.6
12 α: 1.71–1.74 m 33.8
β: 1.53–1.64 m
13 171.4
14 α: 5.90 brs
β: 6.05 brs 99.4
99.9
15 171.9
16 5.96 brs 116.1
17 0.80 d (6.5) 15.9
18 169.2
19a 4.39 d (8.2) 70.9
19b 4.02 d (8.2)
20 0.57 s 17.7
104.31 d (7.7) 102.8
202.96–3.05 m 74.1
303.13–3.16 m 77.2
402.96–3.05 m 70.7
503.13–3.16 m 77.4
60α: 3.43–3.47 m
β: 3.63–3.69 m 61.6
14-OH 7.75 brs
20-OH 4.92 brs
30-OH 4.92 brs
40-OH 4.92 brs
60-OH 3.33 s
Assignments based on the analysis of the HSQC (Heteronuclear single quantum coherence spectroscopy) and
HMBC experiments; Jvalues (Hz) in parentheses. a700 MHz. b175 MHz.
It is worth mentioning that the oils from other Salvia species analyzed also contains sesquiterpenes.
In this context, Salvia ceratophylla, S. aethiopis L., S. palaestina Bentham and S. xanthocheila Boiss. ex
Benth., are rich sources of germacrene D (
15
) [
16
];
β
-caryophyllene (
13
) is the major component of
S. nemorosa L., S. verticillata L., S. virgata Ortega and S. hydrangea DC. ex Benth. Finally, germacrene B
was the major compound of S. syriaca L. [17,18].
Molecules 2020,25, 3530 6 of 18
Table 2. GC-MS identified compounds from the essential oils of S. amarissima.
Compound CAS Number Peak Area (%)
RI[a] Sa-Batch 1 Sa-Batch 2
3-Methoxy-p-cymene (9) 1076-56-8 1219 4.40
(E)-Pinocarvyl acetate (10) 1686-15-3 1313 5.98 2.10
δ-Elemene (11) 20307-84-0 1329 1.99
α-Bourbonene (12) 5208-58-2 1378 4.24 4.11
β-Caryophyllene (13) 87-44-5 1413 15.05 21.27
α-Caryophyllene (14) 1139-30-6 1447 7.68 4.21
Germacrene D (15) 37839-63-7 1476 25.09 24.26
β-Selinene (16) 17066-67-0 1491 28.35 30.16
Spathulenol (17) 6750-60-3 1576 11.59 9.45
Total peak area (%) 99.97 99.96
[a] RIretention indices relative to series of n-alkanes (C8-C20) on a DB-5 column.
2.3. Evaluation of Compounds 1–8a,b on the PTP-1B Inhibitory Activity
One of the major causes of type 2 diabetes mellitus is insulin resistance, which occurs when
the hormone cannot activate signaling pathways in major metabolic tissues (muscles, fat and liver).
Insulin resistance involves several inhibitory molecules that interfere with tyrosine phosphorylation
of the insulin receptor. Among those, PTP-1B is a promising therapeutic target since it inactivates
crucial signaling effectors in the insulin- and leptin-signaling cascades by dephosphorylating their
tyrosine residues [
13
]. Therefore, natural products
1
–
8a,b
were tested against PTP-1B (Table 3). Among
the flavonoids tested, the most active component was pedalitin (
7
) with an IC
50
of 62.0
±
4.1
µ
M
(19.0
µ
g/mL), whereas of the diterpenoids was amarisolide (
1
) (279.9
±
26.0
µ
M; 137.9
µ
g/mL). In both
cases, the inhibitory effect was concentration-dependent. The inhibitory activity against PTP-1B
reported for compounds
4
and
6
correlated well with that found in this work [
19
,
20
]. On the other hand,
rosmarinic acid (
5
) is an ingredient of an active extract of Rosmarinus officinalis against PTP-1B [
21
].
However, in this study we report for the first time the effect of the pure
5
. The activity of the isolates
1
–
8a,b
was compared to that of ursolic acid (
UA
, IC
50
=28.1
±
1.2
µ
M), which in other studies displayed
lower IC50 values (~3.1 µM) [22].
Table 3.
Protein tyrosine phosphatase 1B (PTP-1B) inhibitory activity of compounds (
1
–
8a
,
b
) from
S. amarissima expressed as IC50.
µg/mL ±SD µM±SD
1137.9 ±12.8 279.9 ±26.0
262.5 ±2.3 189.4 ±7.0
324.2 ±0.7 80.1 ±2.2
4197.3 ±10.1 323.4 ±16.6
549.4 ±6.7 137.1 ±18.7
6120.7 ±14.7 259.9 ±31.7
719.6 ±1.3 62.0 ±4.1
8a,b 269.7 ±3.6 514.2 ±6.8
UA [a] - 28.1 ±1.2
[a] Positive control. Values are representative of three independent determinations.
The results of the PTP-1B are significant. They suggest that the traditional preparation
of S. amarissima, with hypoglycemic and antihyperglycemic properties demonstrated
in vivo
[
8
]
(i.e., the overall action), contains compounds such as
1
–
8a
,
b
that might weakly target different proteins
(i.e., PTP-1B and others) within the same signaling network thus shutting insulin signaling cascade
process by network pharmacology. It is also possible that compounds
1
–
6
and
8a
,
b
, with weaker activity
than compound
7
, altogether put forth a biochemical effect by synergism (i.e., a synergy between weakly
Molecules 2020,25, 3530 7 of 18
active compounds against PTP-1B). Finally, molecules like compounds
1
,
4
,
5
and
7
can exert their
action binding different targets such as PTP-1B and
α
-glycosidases, among others (polypharmacology).
The fact that rutin (
4
) [
23
] and rosmarinic acid (
5
) [
21
] are multitarget antidiabetic compounds, and
compounds 1and 7inhibited α-glycosidases in vivo [8] strengthen any of these possibilities.
2.4. Docking Study
To predict the preferred binding orientation of compounds
1, UA
and
7
into PTP-1B
,
we
performed a docking analysis. Compound
1
was not among the most active compounds but
included for comparative purposes. These substances
1
,
7
and
UA
were docked with the co-crystallized
structure of PTP-1B at the catalytic (PDB ID: 1G7F) and allosteric (PDB ID: 1T49) sites. The results in
Figure 2and Table 4indicate that all ligands bind at the same site as the co-crystallized ligands 892
(3-(3,5-dibromo-4-hydroxy-benzoyl)-2-ethyl-benzofuran-6-sulfonic acid (4-sulfamoyl-phenyl)-amide)
and INZ (2-{4-[(2s)-2-[({[(1s)-1-carboxy-2-phenylethyl]amino}carbonyl)amino]-3-oxo-3-(pentylamino)
propyl]phenoxy}malonic acid) [
24
,
25
]. The estimated energy binding is different for each compound.
Compound
1
has a higher affinity for the catalytic site, which is more and conserved site, while
compound
7
and
UA
targeted its more hydrophobic and less conserved allosteric site. The RMSD
values for ligands
1
and
7
are in the range of 2 to 3 Å, which indicates that the ligands do not precisely
overlap but maintain the correct orientation [
26
,
27
]. The amino acids interacting with compounds
1
and
7
, as well as
AU
are similar to those previously reported for other inhibitors (Table 4; Figures S30
and S31 of the Supplementary Material) [
24
,
25
]. At the allosteric site, compound
7
has hydrophobic
interactions with Ala189, Leu192, Phe280 and Phe196 while
AU
with Leu192, Phe280 and Phe196.
Therefore, pedalitin (
7
) behaves as
UA
regarding its higher affinity for the allosteric site. For
UA
, the
key structural feature is the pentacyclic core displaying a nonpolar characteristic, which interacts with
nonpolar residues in the allosteric site [
28
]. For compound
7
, apparently beside the tricyclic structure,
the lack of oxygen at C-3 of the flavonoid core seems to be essential. It will be necessary to pursue a
kinetic analysis to determine if experimentally compound
7
is an allosteric inhibitor of PTP-1B. It is
essential to mention, that the less-conserved PTP-1B allosteric site is an ideal target for a compound to
inhibit PTP-1B activity because the problems associated with inhibition at catalytic site will disappear.
Thus, this study may provide an important lead among flavones for the development of novel PTP-1B
allosteric inhibitors.
Molecules 2020, 25, x 8 of 18
1
K
i
values were calculated using the equation ΔG = RT lnK
i
[26]
2
Residues interacting 4 Å of the
compound;
3
RMSD were calculated from the co-crystallized ligands for UA; and for 1 and 7 from
UA. BE, binding energy.
Figure 2. Structural models of the binding sites of the PTP-1B ligand complexes. In cyan cartoons
(PTP-1B protein), red sticks (1), green sticks (7) purple sticks (UA) at the catalytic (A) and allosteric
sites (B). Images created with Pymol.
2.5. Molecular Dynamics of PTP-1B-Compounds 1, 7 and UA Complexes
Molecular dynamics (MD) studies of the complexes PTP-1B-compounds 1, 7 and UA were
carried out to evaluate the stability of the docked complexes illustrate in Figure 3. Table 5 shows the
theoretical parameters calculated from 100 ns of MD. All PTP-1B-compound complexes show
negative ∆G (affinity parameter) consistent with their stability. Compound 1 has a ∆G similar to UA
at the catalytic site, while compound 7 has a lower ∆G than AU at the same site. Figures 3 and 4 show
the structural models, RMSD and Root Mean Square Fluctuations (RMSF) of the molecular
trajectories for compounds 1 and 7, respectively. The RMSD of the complexes is lower with respect
to PTP-1B in both the catalytic and allosteric MDs, which indicates the conformational stability of the
complexes. In the RMSF analysis, it is observed an area between residues 27–50 (corresponding to a
loop), which is stabilized with the ligands. The data obtained with the MDs are complementary and
conscious with the docking data and experimental results.
Table 5. Calculations of the theoretical energy components from the trajectories of molecular
dynamics.
Complex. ∆E
vdw
∆E
ele
∆G
GB
∆G
NP
∆G
solv
∆G
bind
PTP-1B-UA
1
−21.88 ± 0.44 −20.02 ± 1.08 27.60 ± 1.63 −41.91 ± 1.26 25.39 ± 1.04 −16.51 ± 0.36
PTP-1B-UA
2
−28.67 ± 0.23 −1.21 ± 0.46 10.89 ± 0.42 −27.78 ± 0.48 7.73 ± 4.21 −20.05 ± 0.21
PTP-1B-1 −36.22 ± 0.47 −18.53 ± 0.77 41.62 ± 0.55 −54.76 ± 1.00 36.84 ± 0.53 −17.92 ± 0.61
PTP-1B-7 −35.95 ± 0.45 −4.27 ± 0.31 12.90 ± 0.33 −40.22 ± 0.55 9.06 ± 0.33 −31.16 ± 0.46
1
Catalytic site;
2
Allosteric site; ∆E
vdw
, contributions by van der Waals interactions; ∆E
ele
, electrostatic
energy; ∆G
GB
, polar solvation energy; ∆G
np
, nonpolar solvation energy; ∆G
solv
, desolvation free energy
(∆G
solv
= ∆G
GB
+ ∆G
nonpol
); ∆G
bind
, binding affinity.
Figure 2.
Structural models of the binding sites of the PTP-1B ligand complexes. In cyan cartoons
(PTP-1B protein), red sticks (
1
), green sticks (
7
) purple sticks (
UA
) at the catalytic (
A
) and allosteric
sites (B). Images created with Pymol.
Molecules 2020,25, 3530 8 of 18
Table 4. Results of the dockings analyses.
Catalytic Site Allosteric Site
Ki
(µM) 1BE
(kcal/mol) RMSD (Å) 3Residues 2Ki
(µM) 1BE
(kcal/mol) RMSD (Å) 3Residues 2
10.55 −8.5 2.89 Tyr46, Asp48, Val49, Phe182, Gly183,
Cys215, Ser216, Ala217, Gly220,
Arg221, Gln262, Thr263 and Gln266 8.39 −6.8 2.15 Phe196, Lys197, Arg199,
Glu200, Gly277, Phe280
and Ile281
79.94 −6.8 2.73 Tyr46, Asp48, Val49, Trp179, Asp181,
Phe182, Gly183, Ala217, Ile219,
Arg221, Gln262 and Gln 266 2.16 −7.7 2.27 Ala189, Leu192, Glu276,
Gly277, Phe280, Phe196,
Lys197 and Glu200
UA 2.56 −7.6 1.35 Phe182, Gly183, Cys215, Ala217,
Gly218, Ile219, Gly220, Arg221,
Gln262, Thr263 and Gln266 0.78 −8.3 1.73 Asn193, Phe196,
Asp263, Phe280, Leu192
and Ile281
1
K
i
values were calculated using the equation
∆
G=RT lnK
i
[
26
]
2
Residues interacting 4 Å of the compound;
3RMSD were calculated from the co-crystallized ligands for UA; and for 1and 7from UA. BE, binding energy.
2.5. Molecular Dynamics of PTP-1B-Compounds 1, 7 and UA Complexes
Molecular dynamics (MD) studies of the complexes PTP-1B-compounds
1
,
7
and
UA
were carried
out to evaluate the stability of the docked complexes illustrate in Figure 3. Table 5shows the theoretical
parameters calculated from 100 ns of MD. All PTP-1B-compound complexes show negative
∆
G (affinity
parameter) consistent with their stability. Compound
1
has a
∆
G similar to
UA
at the catalytic site,
while compound
7
has a lower
∆
G than
AU
at the same site. Figures 3and 4show the structural models,
RMSD and Root Mean Square Fluctuations (RMSF) of the molecular trajectories for compounds
1
and
7
, respectively. The RMSD of the complexes is lower with respect to PTP-1B in both the catalytic and
allosteric MDs, which indicates the conformational stability of the complexes. In the RMSF analysis,
it is observed an area between residues 27–50 (corresponding to a loop), which is stabilized with the
ligands. The data obtained with the MDs are complementary and conscious with the docking data and
experimental results.
Molecules 2020, 25, x 9 of 18
Figure 3. Molecular dynamics at the catalytic site of PTP-1B for compound 1. A) Structural models,
B) RMSD and C) Root Mean Square Fluctuations (RMSF) from molecular trajectories. AU = UA.
Figure 3.
Molecular dynamics at the catalytic site of PTP-1B for compound
1
. (
A
) Structural models,
(B) RMSD and (C) Root Mean Square Fluctuations (RMSF) from molecular trajectories. AU =UA.
Molecules 2020,25, 3530 9 of 18
Table 5.
Calculations of the theoretical energy components from the trajectories of molecular dynamics.
Complex. ∆Evdw ∆Eele ∆GGB ∆GNP ∆Gsolv ∆Gbind
PTP-1B-UA 1−
21.88
±
0.44
−
20.02
±
1.08
27.60 ±1.63 −
41.91
±
1.26
25.39 ±1.04 −
16.51
±
0.36
PTP-1B-UA 2−
28.67
±
0.23
−1.21 ±0.46 10.89 ±0.42 −
27.78
±
0.48
7.73 ±4.21 −
20.05
±
0.21
PTP-1B-1−
36.22
±
0.47
−
18.53
±
0.77
41.62 ±0.55 −
54.76
±
1.00
36.84 ±0.53 −
17.92
±
0.61
PTP-1B-7−
35.95
±
0.45
−4.27 ±0.31 12.90 ±0.33 −
40.22
±
0.55
9.06 ±0.33 −
31.16
±
0.46
1
Catalytic site;
2
Allosteric site;
∆
E
vdw
, contributions by van der Waals interactions;
∆
E
ele
, electrostatic energy;
∆
G
GB
, polar solvation energy;
∆
G
np
, nonpolar solvation energy;
∆
G
solv
, desolvation free energy (
∆
G
solv
=
∆
G
GB
+
∆Gnonpol); ∆Gbind , binding affinity.
Molecules 2020, 25, x 10 of 18
Figure 4. Molecular dynamics at the allosteric site of PTP-1B for compound 7. A) Structural models,
B) RMSD and C) RMSF from molecular trajectories.
2.6. Drug Likeness for Compounds 1, 7 and UA
According to the SwissTargetPrediction (http://www.swisstargetprediction.ch/index.php), and
Molinspiration (http://www.molinspiration.com/cgi-bin/properties) databases which predict the
most probable targets of small bioactive molecules, compounds 1, 7 and UA 1, 7 could target any
protein with percentages of 26.7, 40 and 40 %, respectively. SwissTargetPrediction predicted PTP-1B
inhibition for these compounds with probabilities of 0, 0.1266 and 0.95, respectively. According to
these predictions, UA should have been 7.5 times more active than compound 7; however,
experimentally, UA was only 2.2 times more than compound 7 (Figures S31–S34).
Next, using the Osiris Property Explorer server (http://che minformatics.ch/propertyExplorer),
relevant properties for compounds 1, 7 and UA were calculated and summarized in Table 6. These
properties indicate whether a molecule is a potential drug. The logP value is a measure of a
compound’s hydrophilicity. Low hydrophilicity and, therefore, high logP values cause poor
absorption. For a compound being well absorbed, its logP value must not be greater than 5.0; logP
values between 1.35 and 1.8 indicate perfect oral and intestinal absorption. Thus, compounds 1 and
7 could have proper absorption, but not UA.
The aqueous solubility of a compound influences its absorption and distribution characteristics;
a low solubility goes along with inadequate absorption. More than 80% of the drugs on the market
have a logS value higher than −4. Compounds 1 and UA present −3.48 and −6.11, respectively. The
drug-likeness parameter is a complex balance of various molecular properties and structural features
Figure 4.
Molecular dynamics at the allosteric site of PTP-1B for compound
7
. (
A
) Structural models,
(B) RMSD and (C) RMSF from molecular trajectories.
2.6. Drug Likeness for Compounds 1,7and UA
According to the SwissTargetPrediction (http://www.swisstargetprediction.ch/index.php),
and Molinspiration (http://www.molinspiration.com/cgi-bin/properties) databases which predict
the most probable targets of small bioactive molecules, compounds
1
,
7
and
UA 1
,
7
could target any
protein with percentages of 26.7, 40 and 40 %, respectively. SwissTargetPrediction predicted PTP-1B
inhibition for these compounds with probabilities of 0, 0.1266 and 0.95, respectively. According to these
predictions,
UA
should have been 7.5 times more active than compound
7
; however, experimentally,
UA was only 2.2 times more than compound 7(Figures S31–S34).
Next, using the Osiris Property Explorer server (http://cheminformatics.ch/propertyExplorer),
relevant properties for compounds
1
,
7
and
UA
were calculated and summarized in Table 6.
Molecules 2020,25, 3530 10 of 18
These properties indicate whether a molecule is a potential drug. The logP value is a measure
of a compound’s hydrophilicity. Low hydrophilicity and, therefore, high logP values cause poor
absorption.
For a compound
being well absorbed, its logP value must not be greater than 5.0; logP
values between 1.35 and 1.8 indicate perfect oral and intestinal absorption. Thus, compounds
1
and
7
could have proper absorption, but not UA.
Table 6. Physicochemical properties of compounds 1,7and UA.
1 7 UA
LogP 1.01 1.92 6
Solubility (LogS) −3.48 −2.58 −6.11
Molecular weight 492.56 316.26 456.71
Druglikeness −2.96 1.8 −3.66
H bond acceptor 9 7 3
H bond donor 4 4 2
Nb stereocenters 10 0 10
Nb rotable bonds 6 2 1
Drug score 0.40 0.52 0.26
The data was calculated using the OSIRIS Property Explorer server (http://www.cheminfo.org/Chemistry/
Cheminformatics/Property_explorer/index.html).
The aqueous solubility of a compound influences its absorption and distribution characteristics;
a low solubility goes along with inadequate absorption. More than 80% of the drugs on the market
have a logS value higher than
−
4. Compounds
1
and
UA
present
−
3.48 and
−
6.11, respectively.
The drug-likeness parameter is a complex balance of various molecular properties and structural
features that determine whether a molecule is similar to the known drugs. These properties, mainly
hydrophobicity, electronic distribution, hydrogen bonding characteristics, molecule size and flexibility,
and of course presence of various pharmacophoric features influence the behavior of molecule in a
living organism, including bioavailability, transport properties, affinity to proteins, reactivity, toxicity,
metabolic stability and many others. A positive value indicates that a molecule contains predominantly
fragments, which are frequently present in commercial drugs [
29
]; compound
7
has a drug-likeness
value of 1.8. The H bond acceptor and H bond donor’s parameters indicate a molecule’s ability to
interact to a greater or lesser degree with a protein; compounds
1
and
7
present a higher number of
possible hydrogen bridges than
UA
. Finally, the drug-score is an indicator that qualifies the potential
of a compound for being a drug based on all the calculated parameters; compound
7
has the best
drug-score (0.52), which is in harmony with the experimental and theoretical data.
2.7. Development and Validation of an UHPLC-MS Method for Quantifying 1
Initial assessments about the complexity of samples of AE were based on visual comparisons
of their chromatographic profiles. The ethyl acetate soluble fraction of AE yielded the best
profile. Chromatographic separation was performed on Acquity UHPLC
®
BEH Shield C
18
column
(
2.1 ×100 mm, 1.7 µm
) applying a binary gradient elution of water (0.1% formic acid) and MeCN.
The total run time was 10 min. As illustrated in Figure 5compounds
1
,
2
and
4
–
7
are present in the
chromatogram. All compounds were identified by their retention times and m/zvalues corresponding
to [M
−
H]
−
ions. Compounds
1
,
2
and
4
–
7
showed an effective baseline resolution. The pseudo
molecular ions of these compounds appeared at m/z609.54 [M
−
H]
−
(
4
; R
T
1.36 min), 463.46 [
M−H
]
−
(
6
; R
T
1.60 min), 359.23 [M
−
H]
−(5
; R
T
2.46 min), 315.48 [M
−
H]
−
(
7
; R
T
2.82 min), 329.70 [M
−
H]
−
(
2
;
R
T
3.65 min) and 491.23 [M
−
H]
−
(
1
; R
T
4.89 min). The main component of the ethyl acetate fraction
was amarisolide (
1
), then selected as a marker for validation. So far amarisolide (
1
) has been only
isolated from this species, which makes it an excellent marker compound for quality control. It is
worth mentioning that the neo-clerodanes type compounds detected in the infusion were
1
and
8a,b
,
but not the minor diterpenoids we previously isolated from the organic extract of the plant [8].
Molecules 2020,25, 3530 11 of 18
Molecules 2020, 25, x 12 of 18
Figure 5. Representative LC chromatogram of the ethyl acetate soluble fraction prepared from the
infusion of S. amarissima (Detection: 270 nm). Peak identification: 4: R
T
1.36 min; 6: R
T
1.60 min; 5: R
T
2.46 min; 7: R
T
2.82 min; 2: R
T
3.65 min; 1: R
T
4.89 min.
The linearity of the method was tested by recovery assay; the linear regression equation were
found to be y = 5636.35x − 4337.55 (UV detection) and y = 5252.80x + 64101.76 (ESI-MS detection). The
recovery ranges for the standard were expressed as the concentration detected as a percentage of the
expected concentration and were found in the ranges of 100.7–101.7 % for UV detection and 83.7–95.7
% for ESI-MS detection. The reproducibility and repeatability of the analytical method were
evaluated in terms of the intermediate precision by analyzing 6 replicates of the stock solution (50
μg/mL) in two different days. The relative SD (RSD; n = 6) was calculated for each sample evaluated.
The results indicated that their chromatographic patterns were similar showing the presence of
amarisolide (1). The CV values for accuracy were less than 0.11%. Subsequently, compound 1 was
quantified, and the mean concentration calculated was 116 mg/g in dry matter.
3. Materials and Methods
3.1. General 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 Perkin
Elmer model 343 polarimeter at 20 °C (Perkin Elmer, MA, USA). NMR spectra were registered on a
Bruker AVANCE III HD with TCI CryoProbe 700 H-C spectrometer at 700 MHz (
1
H) or 175 MHz
(
13
C), using TMS as an internal standard (Bruker Corp., Billerica, MA, USA). DARTHRMS were
acquired with a JEOL AccuTOF-DART JMS-T100LC (JEOL Ltd., Tokyo, Japan) spectrometer in
positive mode. For GC-MS analyses, an Agilent 6890N series gas chromatograph coupled to a LECO
(Laboratory Equipment Corporation) time-of-flight mass spectrometer detector (MS-TOF; Agilent
Technology, Santa Clara, CA, USA) was used. UHPLC-MS analyses were performed on a Waters
Acquity UHPLC-H® Class system (Waters, Darmstadt, Germany) equipped with a quaternary
pump, sample manager, column oven and photodiode array detector (PDA) interfaced with an SQD2
single mass spectrometer detector with an electrospray ion source. Column chromatography (CC)
was carried out on Sephadex LH-20 (GE Healthcare, IL, USA). Thin layer chromatographic (TLC)
analyses were performed on silica gel 60 F
254
plates (Merck, Darmstadt, Germany), or C
18
-silica gel
matrix plates Analtech plates (Merck, Darmstadt, Germany), visualization of the plates was carried
out using an (NH
4
)
4
Ce(SO
4
)
4
(10%) solution in H
2
SO
4
. Reagent-grade EtOAc, CHCl
3
, CH
2
Cl
2
and
MeOH were purchased from J.T. Baker (Avantor Performance Materials, PA, USA). MeCN, MeOH
and water LC-MS or HPLC grades were purchased from Honeywell Burdick & Jackson (Morristown,
NJ, USA). All other analytical grade solvents and reagents were obtained from various commercial
Figure 5.
Representative LC chromatogram of the ethyl acetate soluble fraction prepared from the
infusion of S. amarissima (Detection: 270 nm). Peak identification:
4
: R
T
1.36 min;
6
: R
T
1.60 min;
5
: R
T
2.46 min; 7: RT2.82 min; 2: RT3.65 min; 1: RT4.89 min.
The analytical method was validated in terms of precision, accuracy, linearity and recovery
according to the Q2 (R1) guideline published by the International Conference on Harmonisation
(ICH) [
30
]. The linearity of the system was tested using a concentration range of
1
between 5
to 100
µ
g/mL and was found to be linear (R
2
=0.9994 (UV) and 0.9921 (Electrospray Ionization
Mass Spectrometry, ESI-MS) in the concentration range used. The CV was less than 0.13% at each
concentration level analyzed. The limit of identification (LOD) and quantification (LOQ) values
were 1.22 and 3.70
µ
g/mL, respectively for UV detection; and 0.60 and 1.82
µ
g/mL, respectively for
ESI-MS detection.
The linearity of the method was tested by recovery assay; the linear regression equation were
found to be y =5636.35x
−
4337.55 (UV detection) and y =5252.80x +64,101.76 (ESI-MS detection).
The recovery ranges for the standard were expressed as the concentration detected as a percentage of the
expected concentration and were found in the ranges of 100.7–101.7 % for UV detection and 83.7–95.7%
for ESI-MS detection. The reproducibility and repeatability of the analytical method were evaluated in
terms of the intermediate precision by analyzing 6 replicates of the stock solution (50
µ
g/mL) in two
different days. The relative SD (RSD; n=6) was calculated for each sample evaluated. The results
indicated that their chromatographic patterns were similar showing the presence of amarisolide (
1
).
The CV values for accuracy were less than 0.11%. Subsequently, compound
1
was quantified, and the
mean concentration calculated was 116 mg/g in dry matter.
3. Materials and Methods
3.1. General 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 Perkin
Elmer model 343 polarimeter at 20
◦
C (Perkin Elmer, MA, USA). NMR spectra were registered
on a Bruker AVANCE III HD with TCI CryoProbe 700 H-C spectrometer at 700 MHz (
1
H) or 175
MHz (
13
C), using TMS as an internal standard (Bruker Corp., Billerica, MA, USA). DARTHRMS
were acquired with a JEOL AccuTOF-DART JMS-T100LC (JEOL Ltd., Tokyo, Japan) spectrometer in
positive mode. For GC-MS analyses, an Agilent 6890N series gas chromatograph coupled to a LECO
(Laboratory Equipment Corporation) time-of-flight mass spectrometer detector (MS-TOF; Agilent
Technology, Santa Clara, CA, USA) was used. UHPLC-MS analyses were performed on a Waters
Molecules 2020,25, 3530 12 of 18
Acquity UHPLC-H
®
Class system (Waters, Darmstadt, Germany) equipped with a quaternary pump,
sample manager, column oven and photodiode array detector (PDA) interfaced with an SQD2 single
mass spectrometer detector with an electrospray ion source. Column chromatography (CC) was carried
out on Sephadex LH-20 (GE Healthcare, IL, USA). Thin layer chromatographic (TLC) analyses were
performed on silica gel 60 F
254
plates (Merck, Darmstadt, Germany), or C
18
-silica gel matrix plates
Analtech plates (Merck, Darmstadt, Germany), visualization of the plates was carried out using an
(NH
4
)
4
Ce(SO
4
)
4
(10%) solution in H
2
SO
4
. Reagent-grade EtOAc, CHCl
3
, CH
2
Cl
2
and MeOH were
purchased from J.T. Baker (Avantor Performance Materials, PA, USA). MeCN, MeOH and water LC-MS
or HPLC grades were purchased from Honeywell Burdick & Jackson (Morristown, NJ, USA). All other
analytical grade solvents and reagents were obtained from various commercial sources. Amarisolide
(
1
) was isolated from the species S. amarissima in the present study. The purity was determined to be
more than 98% by UHPLC-MS.
3.2. Plant Material
Salvia amarissima was collected in Capul
á
lpam de M
é
ndez, Ixtl
á
n de Ju
á
rez, Oaxaca, in January
2019 (Sa-Batch 1 (fresh) and Sa-Batch 2 (air-dried)). A voucher specimen (Number 1502277) was
deposited at the National Herbarium of Mexico (MEXU), Instituto de Biolog
í
a, UNAM. R. Torres-Colin
achieved the botanical identification of the plant. The plant was air-dried and ground to a fine powder
(2 mm) in a Thomas Wiley Model 4 Mill.
3.3. Extracts and Essential Oils Preparation
AE from S. amarissima (dried aerial parts) was prepared with 250 mL of boiling water and 12.5 g
of the crude drug for 30 min. After filtration, the aqueous extract was concentrated in vacuo to obtain
0.1 g of a green residue. This process was repeated as necessary to prepare 10 g of AE. The ethyl acetate
soluble fraction was prepared via partitioning with EtOAc (3
×
250 mL) from the aqueous extract.
The resulting organic phase was dried over anhydrous sodium sulfate and concentrated in vacuo to
yield 130 mg of a brown residue (yield 1.0%).
EOs were prepared from fresh (Sa-batch 1) and air-dried (Sa-batch 2) plant material (200 g in 1.5 L
of distilled water) by hydrodistillation in a modified Clevenger type apparatus for 3 h. In both cases,
the hydrodistilled was extracted with CH
2
Cl
2
(3
×
2 L). The resulting organic phases were dried over
Na
2
SO
4
and concentrated in vacuo to yield an oily yellow residue (0.040 g, yield 0.02% in the case of
the fresh material, and 0.063 g, yield 0.03% for the dried plant). All samples were stored at
−
4
◦
C until
chemical analysis.
3.4. Separation of Active Compounds from the Ethyl Acetate Soluble Fraction
The ethyl acetate soluble fraction (100 mg) was subjected to CC on Sephadex LH-20 (400 g)
using MeOH as eluent; fractions were pooled into 20 secondary fractions (F
1
–F
20
) according to their
TLC profiles. From fraction F
16
(73 mg) crystallized 60 mg of amarisolide (
1
). Preparative RP-TLC
of fraction F
20
(10 mg) yielded 1.2 mg of 5,6,4
0
-trihydroxy-7,3
0
-dimethoxyflavone (
2
) and 4.2 mg of
6-hydroxyluteolin (3).
3.5. Separation of Active Compounds from AE
AE (4.3 g) was fractionated via CC on Sephadex LH-20 using a gradient system of methanol–water
(water 40–100%); this process gave 12 secondary fractions (AE
1
–AE
12
). From fraction AE
9
(35 mg)
crystallized 30 mg of rutin (
4
; m.p. 241–242
◦
C). From fraction AE
11
(15 mg) crystallized 5 mg of
isoquercitrin (
6
). Fraction AE
6
(220 mg) was further purified on a Sephadex CC, eluting with MeOH,
to yield 16 mg of rosmarinic acid (
5
). Preparative TLC on silica gel [ethyl acetate-methanol (85:15)] of
fraction AE
5
(11 mg) afforded 1 mg of pedalitin (
7
). Finally, preparative RP-TLC of AE
6
(10 mg, MeOH)
afforded 4 mg of an epimeric mixture of amarisolide G (8a,b).
Molecules 2020,25, 3530 13 of 18
Amarisolide G (
8a,b
): White solid; m.p. 133–135
◦
C.
[α]20
D
=–149 (c =1 mg/mL, MOH). UV
(MeOH):
λmax
(log
ε
) 206 (0.612) nm. IR (KBr):
νmax
3365, 1749 cm
−1
. ECD (c 0.2 mM, MeOH):
λmax
(
∆ε
) 212 (–5.21), 250 (–4.65) nm.
1
H and
13
C-NMR: see Table 1. HRESIMS: m/z525.2317 [M +H]
+
(calc.
525.2330 for C26H37O11).
3.6. Enzymatic Hydrolysis of 8a,b
Compound
8a
,
b
(2 mg) was mixed with
β
-glucosidase (2 mg, Sigma-Aldrich, MO, USA) in
phosphate buffer solution (2 mL, 100 mM at pH 7); and kept at 40
◦
C for 15 days. Subsequently,
the reaction mixture was extracted with CHCl
3,
and the aqueous phase was concentrated to dryness
and subjected to TLC analysis. d-Glucose was identified by comparison of the retention factor and
optical rotation value with those of the authentic sample.
3.7. UHPLC-MS Analysis and Method Validation
The analytical method (Figure 5) was developed using an Acquity UHPLC
®
BEH Shield C
18
column (2.1
×
100 mm, 1.7
µ
m) at 40
◦
C. The mobile phase consisted of (A) water (0.1% formic acid)
and (B) acetonitrile with a linear gradient elution program: 0–10 min, 20–100% (B); 10–10.5 min, 20%
(B); 10.5–13 min, 20% (B). The flow rate was set to 0.3
µ
L/min, and the sample injection volume was
3.0
µ
L; detection was achieved with a PDA detector at 270 nm. For the identification of compounds,
each sample was analyzed with the electrospray ion source operating in both positive (ESI
+
) and
negative (ESI
−
) ionization modes. The ESI-MS conditions consisted of capillary voltage at 3.0 or 2.5 kV
in positive and negative ion modes, respectively; dry heater temperature 150
◦
C; and nitrogen as the
sheath gas flow. MS spectra were obtained within a mass range of m/z100–1000 using nitrogen as the
collision gas. The MassLynk software (version 4.1) was used to control of the UHPLC-MS system and
for data acquisition and processing.
The method was validated according to the ICH guidelines [
30
]. For linearity, amarisolide (
1
) was
accurately weighed and dissolved in dioxane-methanol (v/v, 1:1) to prepare stock solution at a final
concentration of 1 mg/mL. Six working solutions in the range of 5–100
µ
g/mL for the standard were
prepared from serial dilutions from the stock solution. Each concentration was prepared in sextuplicate.
The linearity was assessed estimating the slope, y-intercept and coefficient of determination (R
2
) using
the least squares method. Limits of detection (LOD) and quantification (LOQ) for the standard were
determined at signal-to-noise (S/N) ratios of 3 and 10, respectively. Recovery experiments were carried
out to evaluate the accuracy, assaying independently three amounts equivalent to 50 (ca. 10
µ
g/mL),
100 (ca. 50
µ
g/mL) or 125% (ca. 75
µ
g/mL). At each level, compound
1
was added simultaneously to
the ethyl acetate soluble fraction (50
µ
g/mL). Each sample was injected twice and analyzed according
to the method previously described. The mean percentage recovery for the standard was found to
be between 98 and 102% by means of Fisher’s F test [
30
]. Finally, the repeatability and inter-day
precision was evaluated by testing six identical samples according to the above described method on
two consecutive days and by two different analysts by triplicate. The relative standard deviation (RSD)
was calculated for each determination as a measure of precision and repeatability.
3.8. GC-MS Analysis of the Essential Oils
For GC-MS analyses, compounds were separated on a DB-5 capillary column (Supelco, Bellefonte,
PA, USA) with the following temperature program: oven temperature was programmed from 40 to
260
◦
C at 4
◦
C/min during 20 min, and finally up to 340
◦
C for 20 min isothermally; injector and MS
transfer line temperatures were set at 200 and 300
◦
C, respectively; Helium was used as the carrier gas
at a constant flow rate of 1 mL/min; split ratio, 1:20. A mixture of the homologous series of n-alkanes
(C
8
–C
20
) in CH
2
Cl
2
was directly injected into the GC under the above temperature program, in order
to calculate the linear retention indices (R
I
). All mass spectra were acquired in EI mode (scan range m/z
40–400, ionization energy 70 eV). The components were identified using retention index (R
I
) of peaks
in the chromatogram [
31
,
32
] and by comparison of their mass spectra with those of standard library
Molecules 2020,25, 3530 14 of 18
data (NIST) of the GC-MS system and literature data or with those of authentic samples available
commercially. All determinations were performed in triplicate.
3.9. Protein Tyrosine Phosphatase 1B Inhibition Assay
The expression and purification of hPTP-1B was performed as previously described [
33
]. Aqueous
extract (AE), ethyl acetate soluble fraction, pure compounds and positive control were dissolved in
DMSO or MeOH or Tris buffer solution (Tris-HCl, 20 mM, pH 7). Aliquots of 0–10
µ
L of testing
materials (triplicated) were incubated for 5 min with 20
µ
L of enzyme stock solution in Tris-HCl
(22 nM). After incubation, 10
µ
L of p-nitrophenylphosphate (pNPP; 5 mM) was added and further
incubated for 15 min at 25
◦
C; then, the absorbance was determined (
λmax
415 nm). For all samples,
the inhibitory activity was determined as a percentage in comparison to the blank (Tris-HCl) according
to the following equation:
% PTP1B = 1−A415t
A415C !×100 (1)
where % PTP-1B is the percentage of inhibition, A
415t
is the corrected absorbance of the extracts, fraction,
or compounds under testing (A
415end −
A
415initial
) and A
415C
is the absorbance of the blank (A
415end blank
−A415initial blank). The IC50 was calculated by regression analysis, using the following equation:
%Inhibition =A100
1+I
IC50 s(2)
where A
100
is the maximum inhibition, Iis the inhibitor concentration, IC
50
is the concentration required
to inhibit the activity of the enzyme by 50% and Sis the cooperative degree.
3.10. Docking Studies
To perform the docking at the catalytic site, the PTP1B-INZ complex corresponding to the PDB
1G7F was used, which has a resolution of 1.8 A. For the allosteric site, the PDB 1T49 (resolution of 1.9)
was used. The two PDBs used were selected considering the resolution and that they had co-crystallized
ligands at the sites of interest. All compounds were built using the HyperChem 8.0 release program
and optimized geometrically using the Gaussian 09 program, revision A.02 (Gaussian Inc., Wallingford,
CT, USA) at DFT B3LYP/3-21G level of theory. The protein and ligands were further prepared using
the utilities implemented by AutoDockTools 1.5.4 (http://mgltools.scripps.edu/). The protein was
adding polar hydrogen atoms, Kollman united-atom partial charges, and to the ligands computing
Gasteiger–Marsilli formalism charges, rotatable groups which were assigned automatically as were the
active torsions. Blind docking was carried out using AutoDock Vina version 2.0 [
34
]. The root mean
square deviation (RMSD) values were obtained by comparing the best pose generated in AutoDock.
The initial parameters used for the active site were a grid box size was 42 Å
×
40 Å
×
40 Å in the x, y
and z dimensions and grid center 9.73, 18.00, 971 to 1G7F.PDB. For the allosteric site docking were
a grid box size was 42 Å
×
40 Å
×
40 Å in the x, y and z dimensions and grid center 9.73, 18.00, 971
used 1T49.pdb. For both sites, the exhaustiveness was 25, and the ten best poses were obtained. The
analysis of the docking was made with PyMol (Maestro, Schrödinger, LLC, New York, NY) [35].
3.11. Molecular Dynamics Simulation
All the structural complexes were verified, cleaned and ordered with the pdb4amber scrip before
starting the preparation in order to generate suitable topologies from the LEaP module of AMBER
19 [
36
,
37
]. Each structure and complex was subjected to the following protocol: hydrogens and
other missing atoms were added using the LEaP module with the leaprc.protein.ff19SB parameter
set; Cl
−
or K
+
counterions were added to neutralize the system; the complexes were then solvated
in an octahedral box of explicit TIP3P model water molecules localizing the box limits at 12 Å from
the protein surface. Molecular dynamic simulations were performed at 1 atm and 315 K, maintained
Molecules 2020,25, 3530 15 of 18
with the Berendsen barostat and thermostat, using periodic boundary conditions and particle mesh
Ewald sums (grid spacing of 1 Å) for treating long-range electrostatic interactions with a 10 Å cutofffor
computing direct interactions. The SHAKE algorithm was used to satisfy bond constraints, allowing
the employment of a two fs time step for the integration of Newton’s equations as recommended
in the Amber package [
36
,
38
]. Amber leaprc.protein.ff19SB force field [
39
] parameters were used
for all residues. All calculations were made using Graphics Processing Units (GPU) accelerated MD
engine in AMBER (pmemd.cuda), a program package that runs entirely on CUDA
®
(Compute Unified
Device Architecture)-enabled GPUs [
40
]. The protocol consisted of performing a minimization of the
initial structure, followed by 50ps heating and pressure equilibration at 315 K and 1.0 atm pressure,
respectively. Finally, the system is equilibrated with 500ps before starting the production of MD.
The production of the MD consisted of 100 ns for each complex. Frames were saved at ten ps intervals
for subsequent analysis. All analyses were done using CPPTRAJ [
41
] part of AMBER19 utilities and
OriginPro 9.1. The calculations of RMSD and Root Mean Square Fluctuations (RMSF) were made,
considering the C, CA and N. The charts were built with OriginPro 2018 SR1, and the trends were
adjusted with the function processing smooth (method lowess span). VMD and PyMOL [
35
] were
used to visualize and create the images from the MD.
3.12. Chemoinformatic Properties of Compounds 1, 7 and AU
The biological and chemoinformatic properties of compounds
1
,
7
and
AU
were explored using
the servers Swiss TargetPrediction (http://www.swisstargetprediction.ch/index.php), Molinspiration
(http://www.molinspiration.com/cgi-bin/properties) and Osiris Property Explorer server (http://che
minformatics.ch/propertyExplorer/) [29,42].
4. Conclusions
The AE of S. amarissima contains rutin (
4
) and rosmarinic acid (
5
), which inhibit intestinal glucose
absorption, promote glucose uptake in muscle cells and suppress insulin-resistance, among other
effects. On the other hand, pedalitin (7), which behave in silico as an allosteric inhibitor of PTP-1B,
could contribute to its overall antidiabetic action via
α
-glucosidase and selective PTP-1B inhibition,
and other mechanisms yet to be determined. The overall action of AE could be attained via network
pharmacology, synergism and or polypharmacology. Altogether, our studies on S. amarissima tend to
support its medicinal use for the treatment of diabetes in Mexican folk medicine. The chromatographic
analyses developed and validated in this study will allow the development of a pharmacopeic
monograph, and generate standardized preparations of this very Mexican plant. The analytical UHPLC
method was suitable for its intended purpose, the quantification of amarisolide (
1
) according to the Q2
(R1) guideline. Overall, the scientific information generated for this plant will contribute to its rational
use in Mexican folk medicine. Like many other New World melittophilous Salvia species, S. amarissima
is a rich source of bioactive compounds.
Supplementary Materials:
The following are available online. NMR, IR and MS spectrum of the isolated or
identified compounds, TIC chromatograms of the essential oils and Residues of interaction to 4 Å of compounds
1,
7and UA at the catalytic and allosteric sites of PTP-1B are available online.
Author Contributions:
This work is part of the PhD thesis of E.S.-A. who performed the experiments; analysis of
spectral data, A.P.-V.; UHPLC-MS method validation, I.R.-C.; docking analysis, M.R.-G.; expression and purification
of hPTP-1B, docking, molecular dynamics and parameters calculations, M.G.-A.; botanical characterization of the
species, R.T.-C.; conception, funding acquisition, structure elucidation, writing and editing, R.M. All authors have
read and agreed to the published version of the manuscript.
Funding:
The research was supported by grants from CONACyT CB A1-S-11226 and DGAPA IN 217320 awarded
to R.M.
Molecules 2020,25, 3530 16 of 18
Acknowledgments:
The authors recognize the valuable support of M.I. Vel
á
squez-L
ó
pez from Facultad de
Medicina; and Georgina Duarte from Facultad de Qu
í
mica. We are indebted to Biol. Itzi Fragoso and Martha
Mart
í
nez Gordillo (Facultad de Ciencias, UNAM) for their valuable help in the identification of the plant material.
We also recognize the support of Direcci
ó
n General de C
ó
mputo y de Tecnolog
í
as de Informaci
ó
n y Comunicaci
ó
n
(DGTIC, UNAM) for the resources to carry out computational calculations through the Miztli supercomputing
system (LANCAD-UNAM-DGTIC-313). E.S.-A. acknowledges the fellowship from CONACyT (289212) to pursue
graduate studies.
Conflicts of Interest: The authors declare no conflict of interest.
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©
2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).
