Neo-Clerodane Diterpenoids from Salvia polystachya Stimulate the Expression of Extracellular Matrix Components in Human Dermal Fibroblasts

Abstract

Eleven neo-clerodane diterpenoids (1–11) including the new analogues 1, 2, and 10, and 3′,5,6,7-tetrahydroxy-4′-methoxyflavone (12) were isolated from the aerial parts of Salvia polystachya. Polystachyne G (1) and 15-epi-polystachyne G (2) were isolated as an epimeric mixture, containing a 5-hydroxyfuran-2(5H)-one unit in the side chain at C-12 of the neo-clerodane framework. Polystachyne H (10) contains a 1(10),2-diene moiety and a tertiary C-4 hydroxy group. The structures of these compounds were established by analysis of their NMR spectroscopic and MS spectrometric data. The absolute configurations of compounds 3, 4, and 10 were determined through single-crystal X-ray diffraction analysis. The antibacterial, antifungal, and phytotoxic activities of the diterpenoids were determined. In addition, the stimulatory effect of the expression of extracellular matrix components of nine of the isolates (1–8 and 11) was assayed. Compounds 1–4, 8, and 11 increased the expression of the genes codifying for type I, type III, and type V collagens and for elastin.

Full text

neo-Clerodane Diterpenoids from Salvia polystachya Stimulate the
Expression of Extracellular Matrix Components in Human Dermal
Fibroblasts
ElihúBautista,*
,†
NaytzéOrtiz-Pastrana,
§
Guillermo Pastor-Palacios,
†
Angé
lica Montoya-Contreras,
‡
Rubé
n A. Toscano,
§
Jesús Morales-Jimé
nez,
†
Luis A. Salazar-Olivo,
‡
and Alfredo Ortega
§
†
CONACYT-CIIDZA and
‡
División de Biología Molecular, Instituto Potosino de Investigación Científica y Tecnológica A. C.,
Camino a la Presa San José2055, Lomas 4a Sección, 78216 San Luis Potosí, Mé
xico
§
Instituto de Química, Universidad Nacional Autónoma de Mé
xico, Circuito Exterior, Ciudad Universitaria, Coyoacá
n 04510, Mé
xico
City, Mé
xico
*
SSupporting Information
ABSTRACT: Eleven neo-clerodane diterpenoids (1−11)
including the new analogues 1,2, and 10, and 3′,5,6,7-
tetrahydroxy-4′-methoxyflavone (12) were isolated from the
aerial parts of Salvia polystachya. Polystachyne G (1) and 15-
epi-polystachyne G (2) were isolated as an epimeric mixture,
containing a 5-hydroxyfuran-2(5H)-one unit in the side chain
at C-12 of the neo-clerodane framework. Polystachyne H (10)
contains a 1(10),2-diene moiety and a tertiary C-4 hydroxy
group. The structures of these compounds were established by
analysis of their NMR spectroscopic and MS spectrometric
data. The absolute configurations of compounds 3,4, and 10
were determined through single-crystal X-ray diffraction
analysis. The antibacterial, antifungal, and phytotoxic activities
of the diterpenoids were determined. In addition, the stimulatory effect of the expression of extracellular matrix components of
nine of the isolates (1−8and 11) was assayed. Compounds 1−4,8, and 11 increased the expression of the genes codifying for
type I, type III, and type V collagens and for elastin.
neo-Clerodane diterpenoids constitute a group of secondary
metabolites of pharmacological interest due to their structural
diversity, lipophilic properties, and capability to interact with
molecular targets, which makes this class of compounds ideal
molecules for their bioprospecting in several pharmacological
models.
1
These compounds have shown anti-inflammatory
2
and neuroprotective activities
3
and have the potential to be
used to treat Alzheimer’s disease,
4
colitis,
5
and obesity.
6
Other
bioactivities of neo-clerodane diterpenoids are their affinity for
the κ-opioid receptor
7
and their cytotoxicity against different
human tumor cell lines.
8
The broad spectrum of pharmaco-
logical activities attributed to neo-clerodane diterpenoids
prompted an investigation into the phytochemical analysis of
the aerial parts of Salvia polystachya.
Salvia polystachya (Lamiaceae, subgenera Calosphace)isa
shrub endemic to Mexico.
9
This plant is known in folk
medicine as “chı
́
a”, and together with 17 other species of the
genera (including S. hispanica), make up the “chı
́
a”complex of
medicinal plants.
10
The aerial parts are used as a purgative and
diuretic and to treat dysentery. A previous bioguided study led
to the isolation of the diterpenoid linearolactone as the active
principle responsible for the antiamoebic and antigiardial effects
of the acetone-soluble extract of S. polystachya leaves.
11
In
addition, other neo-clerodane diterpenoids have been isolated
from the aerial parts including polystachynes A−F, salvifaricin,
and dehydrokerlin.
12
Herein, the isolation and structural
elucidation of three new diterpenoids, as well as their
antimicrobial and phytotoxic properties, and their capability
to modulate the expression of extracellular matrix components
in human dermal fibroblasts are discussed.
■RESULTS AND DISCUSSION
The phytochemical reinvestigation of an acetone-soluble extract
of the leaves of S. polystachya afforded 11 neo-clerodane
diterpenoids (1−11) and 3′,5,6,7-tetrahydroxy-4′-methoxyfla-
vone (12).
13
Three of the diterpenoids, 1,2, and 10, are new
and are accompanied by the known diterpenoids salvifilines A
(3) and C (5), polystachynes A (7), B (8), and E (9), and
linearolactone (11).
Polystachyne G (1) and 15-epi-polystachyne G (2) were
isolated as colorless crystals and occurred as a 1:1 epimeric
mixture. Its molecular formula, C20H22O8, was deduced from its
protonated molecular ion at m/z391.13979 [M + H]+by
Received: July 13, 2017
Published: November 14, 2017
Article
pubs.acs.org/jnp
© 2017 American Chemical Society and
American Society of Pharmacognosy 3003 DOI: 10.1021/acs.jnatprod.7b00591
J. Nat. Prod. 2017, 80, 3003−3009
Cite This:
J. Nat. Prod.
2017, 80, 3003-3009

HRMS (DART-TOF+) and 13C NMR data, indicating 10
indices of hydrogen deficiency. The IR spectrum of 1/2showed
absorption bands at 3362 and 1767 (broad) cm−1, indicating
the presence of hydroxy and γ-lactone groups. The presence of
the γ-lactone moiety was confirmed by the signals observed in
the 1H and 13C NMR spectra (δC174.2, C-18; 79.7, C-19; δH
4.82, dd, J= 7.6, 2.4 Hz, H-19β; 4.02, dd, J= 8.0 Hz, H-19α).
In addition, the 13C NMR and DEPT spectra displayed 20 pairs
of signals; these signals corresponded to a pair of methyls, four
pairs of methylenes, 10 pairs of methines, and five pairs of
nonprotonated carbons (Table 1). The above information and
analysis of their 1D and 2D NMR data showed that 1/2possess
aneo-clerodane framework.
14
In addition, comparison of these
NMR data with those of diterpenoids derived from S.
polystachya revealed that 1/2have structures closely similar
to polystachyne B (8),
12a
differing only in the C-12 side chain
moieties. The 1H NMR signals (Table 1)atδH7.03 (d, J= 6.5
Hz) and 6.14 (dd, J= 8.0, 6.5 Hz) correlated in the COSY
spectrum and showed HMBC correlations with a signal at δC
169.7/169.6 (C-16). The above data suggested the presence of
a 5-hydroxyfuran-2(5H)-one unit at C-12 of the neo-clerodane
framework, and these signals were assigned to H-14 (δC145.2/
144.6, C-14) and H-15 (δC97.1, C-15), respectively. This
assignment was supported by the HMBC correlations of H-14
and H-15 with C-13 (δC137.9/137.6) and of H-12 (δH5.08,
dd, J= 8.0, 7.6 Hz; δC75.2, C-12) with C-14 and C-16. In the
1H NMR spectrum of 1/2, the signals at δH5.33/5.31 (s) and
4.51 (d, J= 4.0 Hz) were assigned to the acetalic protons H-20
(δC110.6/110.4, C-20) and the oxymethine H-7 (δC87.8, C-
7), respectively. This assignment was based on the HMBC
correlations of H-20 with C-8 (δC38.1), C-9 (δC59.0), and C-
10 (δC46.2/46.1) and of H-7 with C-5 (δC43.0), C-9, and C-
17 (δC14.0). Additional signals at δH3.30 (dd, J= 5.6, 3.6 Hz)
and 3.11 (br dd, J= 6.0, 3.2 Hz) were assigned to the protons
of the oxirane moiety, H-2 (δC50.4, C-2) and H-1(δC55.1, C-
1), respectively. These spectroscopic data established the
structure of compounds 1/2as depicted.
A previous phytochemical study of neo-clerodane diterpe-
noids from S. filipes reported the isolation and structural
elucidation of salvifilines A−D; salvifilines A (3) and B as well
as salvifilines C (5) and D are C-4 epimers. In the present work,
only the C-15 epimeric mixtures of 1/2and 3/4and the C-16
epimeric mixture of 5/6were isolated. Comparison of the 1D
and 2D NMR data of 1/2with those of salvifiline A (3)
indicated that 1/2differed from 3/4by the presence of a 1,2-
oxirane moiety.
15
A single-crystal X-ray diffraction study of
salvifiline A/15-epi-salvifiline A (3/4) permitted the assignment
of their (4S,5S,7R,8S,9S,10R,12R,15R/15S,20R) absolute con-
figurations [Flack parameter = 0.05(7)]
16
and confirmed their
epimeric relationship (Figure 1). In addition, the concomitant
occurrence of 1/2with 3/4and the analysis of their NOESY
spectra established the relative configuration of 1/2. The cross-
peaks of H-19αwith H-10 and H-4 suggested a cis-fused decalin
ring and a trans-fused 18,19-γ-lactone moiety (Figure 2). The
orientation of the oxirane ring was established as βby the
correlations of H-1 and H-2 with H-10 and H-3α. The NOE
correlation of H-19βand H-10 with H-20 determined an α-
disposition of the oxymethine. The orientation of the 5-
hydroxyfuran-2(5H)-one unit also was determined as α, based
on the correlations of H-12 with H-11βand H3-17. In addition,
Table 1. 1H (400 MHz) and 13C (100 MHz) NMR Data of
Compounds 1/2 and 10 (in CDCl3−DMSO-d6, 9:1)
1/2
a
,
b
10
c
,
d
position δHmult. (Jin Hz) δC
δHmult. (Jin
Hz) δC
1 3.11 br dd (6.0, 3.2) 55.1, CH 6.33 d (6.0) 118.8, CH
2 3.30 dd (5.6, 3.6) 50.4, CH 6.37 dd (9.0,
6.0)
126.8, CH
3α2.31 m
b
18.9, CH25.51 d (9.0) 120.5, CH
3β1.82 dd (16.0, 16.0)
4 2.31 m
b
45.7, CH 74.7, C
5 43.0, C 47.2, C
6α1.71 dd (13.6, 4.4) 30.5, CH21.24 br d
(14.5)
21.8, CH2
6β1.49 br d (14.0) 1.64 br dd
(14.5, 12.5)
7α87.8, CH 2.14 m 18.2, CH2
7β4.51 d (4.0) 2.24 dd
(14.0, 3.0)
8 2.95 dq (6.8, 4.0) 38.1, CH 2.67 m 46.8, CH
9 59.0, C 37.3, C
10 2.37 br s 46.2/46.1, CH 142.2, C
11α2.04 dd (13.6, 8.0)/
2.00 dd (13.6, 8.0)
34.6/34.4, CH22.03 dd
(14.5, 2.5)
39.4, CH2
11β2.74 dd (13.2, 7.6)/
2.71 dd (13.2, 7.6)
2.63 dd
(14.5, 12.5)
12 5.08 dd (8.0, 7.6) 75.2, CH 5.36 dd
(12.5, 2.0)
70.3, CH
13 137.9/137.6, C 125.1, C
14 7.03 d (6.5) 145.2/144.6, CH 6.62 br s 108.9, CH
15 6.14 d (6.5)/6.10 d
(6.8)
97.1, CH 7.68 br s 143.8, CH
16 169.7/169.6, C 7.75 br s 140.4, CH
17 1.27 d (6.8) 14.0, CH3171.5, C
18 174.2, C 177.2, C
19α4.02 d (8.0) 79.7, CH23.39 dd (8.5,
1.0)
72.7, CH2
19β4.82 dd (7.6, 2.4) 4.58 d (9.0)
20 5.33 s/5.31 s 110.6/110.4, CH 1.18 s 31.9, CH3
a
OH-15 signal at δH7.66 (t, 4.8).
b
Overlapped.
c
In DMSO-d6at 500
MHz (1H) and 125 MHz (13C).
d
OH-4 signal at δH5.94 (s).
Journal of Natural Products Article
DOI: 10.1021/acs.jnatprod.7b00591
J. Nat. Prod. 2017, 80, 3003−3009
3004

a single-crystal X-ray crystallographic study confirmed the
structures and the relative configuration (Figure 3) proposed
for 1/2based on the analysis of their NMR data.
Polystachyne H (10) was isolated as colorless needles from
EtOAc−hexanes. Its HRMS (DART-TOF+) spectra showed an
ammonium adduct ion at m/z374.15957 [M + NH4]+,
indicating a molecular formula of C20H24N1O6(calcd for
374.16036). The IR spectrum indicated the presence of OH
(3334 cm−1), γ- and δ-lactone (1775 and 1705 cm−1), and
furan (873 cm−1) functionalities. In the 13C NMR spectrum 20
signals were observed, which were classified by the DEPT
experiment as a methyl, four methylenes, eight methines, and
seven nonprotonated carbons (Table 1). As with compounds 1
and 2, the above data indicated that 10 was also a neo-clerodane
diterpenoid. In the 1H NMR spectrum of 10 (Table 1), the
signals at δH7.75 (br s), 7.68 (br s), and 6.62 (br s) were
assigned, respectively, to H-16 (δC140.4), H-15 (δC143.8),
and H-14 (δC108.9) of the furan moiety. The signals of the
olefinic protons at δH6.37 (1H, dd, J= 9.0, 6.0 Hz), 6.33 (1H,
d, J= 6.0 Hz), and 5.51 (1H, d, J= 9.0 Hz) indicated the
presence of a 1(10),2-diene moiety in the ring A of the neo-
clerodane backbone,
14a
which displayed cross-peaks in the
COSY experiment, and were assigned to H-2 (δC126.8, C-2),
H-1 (δC118.8, C-1), and H-3 (δC120.5, C-3). This was
additionally supported by the HMBC correlations of H-1 with
C-10 (δC142.2) and C-9 (δC37.3) and of H-3 with C-4 (δC
74.7), C-5 (δC47.2), and C-18 (δC177.2). In the 1H NMR
spectrum a signal at δH5.94 (s) did not show correlation in the
HSQC spectrum, suggesting the presence a tertiary hydroxy
group.
16
Comparison of the NMR data of 10 with those of
1,10-dehydrosalviarin
14a
indicated that 10 possessed one more
oxygen atom. The C-4 location of the hydroxy group was
indicated by the HMBC correlations of HO-4 with C-3, C-4, C-
5, and C-18. In addition, the relative configuration of 10 was
established by the analysis of the NOESY spectrum (Figure 4),
taking into account the same criteria as for 1and 2. The NOE
correlations of the HO-4 with H-6βsuggested a β-disposition
of the hydroxy group, and this correlation together with those
of H-19βwith H3-20 indicated the presence of a cis-fused γ-
lactone moiety. The cross-peak of H3-20 with H-8 established
the presence of a cis-fused δ-lactone, and the cross-peaks of H-
14 and H-15 with H-11αsuggested an α-oriented furan ring.
The structure and the (4S,5R,8S,9S,12R) absolute configuration
of 10 was confirmed by a single-crystal X-ray diffraction study
[Figure 5, Flack parameter = 0.01(3)].
Previous research showed that naturally occurring diterpe-
noids modulate the expression of genes codifying for
extracellular matrix proteins in several types of animal cells.
Oridonin, purified from Rabdosia rubescens (Lamiaceae),
markedly decreased expression of the gene codifying for
collagen type I, in hepatic stellate cells.
17
On the contrary,
kirenol, a diterpenoid present in Siegesbeckia orientalis
(Asteraceae), increased the expression of the type I collagen
gene in MC3T3-E1 cells.
18
Genkwadaphnin, a daphnane-type
diterpenoid from the flower buds of Daphne genkwa
(Thymelaeaceae), markedly induced the expression of genes
codifying for type I, type II, and type X collagens in ATDC5
chondroprogenitor cells.
19
In the same manner, tanshinone IIA
Figure 1. ORTEP drawing of compounds 3/4.
Figure 2. Key NOESY correlations for compounds 1/2.
Figure 3. ORTEP drawing of compounds 1/2.
Figure 4. Key NOESY correlations for compound 10.
Figure 5. ORTEP drawing of compound 10.
Journal of Natural Products Article
DOI: 10.1021/acs.jnatprod.7b00591
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stimulates the synthesis and deposition of elastin in cultures of
human cardiac fibroblasts.
20
To initiate the characterization of the biological effects of the
diterpenoids described here, their roles in the expression of
genes codifying for the type I collagen, COL1A1, type III
collagen, COL3A3, type V collagen, COL5A1, and elastin, ELN,
in human dermal fibroblasts (Figures 6 and 7) were evaluated.
As shown in Figure 6A, compounds 5/6 and 11 did not affect
the transcription of COL1A1, while 1/2 induced only moderate
increases in the expression of this gene. Compounds 3/4,7,
and 8induced COL1A1 transcription by 1.6-fold (7at 29.2 μM
and 8at 0.28 μM) or 1.8-fold (3/4 at 0.27 μM) with respect to
MB control. The effect of 3/4 on COL1A1 expression was
higher than that observed for the positive control, ascorbic acid
(AA) at 100 μM.
Genes codifying for other proteins of dermal extracellular
matrix were also clearly induced by the diterpenoids isolated
from S. polystachya (Figures 6 and 7). Compounds 1/2 and 5/6
induced expression of the COL3A3 gene between 1.5- and 2.2-
fold, while 8stimulated the expression of this gene by 2.1- and
2.5-fold at the assayed concentrations, and 11 2.5-fold at 0.29
μM and 3.4-fold at 2.93 μM(Figure 6B). The expression of
COL5A1, the gene codifying for type V collagen, was strongly
induced by 11 at 2.93 μM (2.5-fold), by 8at 0.28 μM (2.2-
fold), by 1/2 at 25.6 μM (1.9-fold), and by 5/6 at 26.7 μM
(1.7-fold) (Figure 7A).
In the same manner, 1/2 stimulated the expression of the
elastin gene by 1.7- and 2.4-fold with respect to the MB control,
exhibiting an effect comparable to the AA control (Figure 7B).
Moreover, elastin gene expression was induced between 2.7-
and 3.2-fold by 8and between 2.1- and 3.4-fold by 11. All the
remaining compounds discretely induced the expression of this
gene, and none inhibited the expression of the elastin gene
(Figure 7B). Compounds 1−4,8, and 11 were biologically
evaluated in an antimicrobial assay, and compounds 1−6for
their phytotoxicity; however, none of them showed detectable
activity.
■EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were
measured on a PerkinElmer 343 polarimeter. UV spectra were
recorded on a Shimadzu UV 160U spectrophotometer. IR spectra
were obtained on a Bruker Tensor 27 spectrometer. NMR
experiments were performed on a Bruker Avance III 400 MHz or
on a Varian Unity Plus 500. Chemical shifts were relative to
tetramethylsilane, and Jvalues are given in Hz. X-ray crystallographic
data were obtained on a Bruker D8 Venture κ-geometry diffractometer
with Cu Kαradiation (λ= 1.541 78 Å). HRDARTMS data were
recorded on a JEOL AccuTOF JMS-T100LC mass spectrometer.
Figure 6. Effect of compounds 1/2,3/4,5/6,7,8, and 11 on the expression of extracellular matrix proteins in human dermal fibroblasts. The
expression of genes codifying for collagen type I (COL1A1) and collagen type III (COL3A3) was analyzed by RT-PCR in total mRNA obtained from
proliferative normal human dermal fibroblasts exposed for 48 h to described treatments. Expression of GAPDH was used as a housekeeping gene.
Results are presented as mean ±SD. Statistical significance of treatments with respect to the control was determined by one-way ANOVA. P< 0.05
was considered significant (*).
Journal of Natural Products Article
DOI: 10.1021/acs.jnatprod.7b00591
J. Nat. Prod. 2017, 80, 3003−3009
3006

Column chromatography (CC) assisted with vacuum was performed
on silica gel 60 (Merck G), unless otherwise stated. Silica gel 230−400
mesh (Macherey−Nagel) was used for flash chromatography. TLC
was carried out on precoated Macherey−Nagel Sil G/UV254 plates of
0.25 thickness, and spots were visualized by UV light at 254 nm and
then spraying with 3% CeSO4in 2 N H2SO4, followed by heating.
Plant Material. The leaves and flowers of S. polystachya were
collected in Huitzilac, Morelos, Mé
xico, in September 2011. A voucher
specimen was deposited (MEXU-573762) at the National Herbarium,
Instituto de Biologí
a, Universidad Nacional Autónoma de Mé
xico.
Extraction and Isolation. The powdered plant material (4.25 kg)
was defatted with hexanes (16 L) and extracted by percolation with
Me2CO (12 L) to obtain a dried extract (247 g), which was dissolved
in a mixture of MeOH−H2O (4:1, 1.5 L) at 50 °C and partitioned
with hexanes (3 ×1 L). The MeOH was evaporated under reduced
pressure, water (0.5 L) was added, and the mixture was partitioned
again with EtOAc (4 ×0.3 L) to give 68 g of residue. The EtOAc
fraction was subjected to silica gel 60 G CC (10.5 ×12.0 cm, 500 mL)
using hexanes−EtOAc mixtures as eluents. The 57 fractions obtained
were analyzed by TLC and grouped as follows: fraction A (frs. 12−17,
7.0 g, eluted with hexanes−EtOAc, 7:3), fraction B (frs. 18−21, 4.81 g,
eluted with hexanes−EtOAc, 7:3), fraction C (frs. 22−24, 4.40 g,
eluted with hexanes−EtOAc 3:2), and fraction D (frs. 25−38, 27.0 g,
eluted with hexanes−EtOAc, 1:1). Fraction A was subjected to several
silica gel CC eluted with hexanes−EtOAc in increasing polarity to give
259 mg of 7, which was purified by crystallization with acetone−
hexanes. Fr. B was subjected to flash CC (4.0 ×9.0 cm, 100 mL) using
mixtures of CHCl3−acetone. The fractions were analyzed by TLC in
the following manner: Fr. B1 (frs. 1−16, eluted with CHCl3−acetone,
95:5), Fr. B2 (frs. 17−23, eluted with CHCl3−acetone, 90:10), and Fr.
B3 (frs. 24−31, eluted with CHCl3−acetone, 80:20). From Fr. B2
compounds 3/4(387 mg) were obtained and purified by
crystallization from EtOAc. The mother liquors were subjected to
silica gel CC (1.7 ×8.5 cm, 20 mL), eluted with a mixture of hexanes−
EtOAc (1:1), to yield from frs. 8−9 compounds 5/6(25.8 mg) and
from frs. 16−18 compounds 3/4(39.1 mg). Fr. B3 was resubjected to
silica gel CC (1.7 ×9.0 cm, 15 mL), eluted with a mixture of hexanes−
CHCl3−MeOH, 45:50:5, to give compounds 1/2(25.3 mg). Fr. C was
purified by silica gel CC (4.5 ×8.0 cm, 100 mL) using mixtures of
hexanes−EtOAc in increasing polarity to give 7(100 mg). Compound
8(6.5 g) from fr. D was obtained by multiple crystallizations from
acetone−hexanes. The mother liquors were subjected to several silica
gel CC using as eluents mixtures of hexanes−EtOAc, CHCl3−acetone,
and CH2Cl2−EtOAc to afford compounds 11 (1.2 g), 9(7.1 mg), and
10 (6.0 mg).
Polystachyne G (1)/15-epi-Polystachyne G (2): colorless crystals,
mp 240−242 °C (EtOAc); [α]25D+2 (c0.1, Me2CO); UV (MeOH)
λmax (log ε) 205 (2.62); IR (KBr) νmax 1767, 873 cm−1;1H and 13C
NMR (CDCl3−DMSO-d6) see Table 1; HRMS (DART-TOF+) m/z
391.13979 [M + H]+(calcd for C20H23O8, 391.13929). X-ray
crystallographic analysis of polystachyne G (1)/15-epi-polystachyne
G(2): formula C20H22O8, MW = 390.38, monoclinic, space group P21,
Figure 7. Effect of compounds 1/2,3/4,5/6,7,8, and 11 on the expression of extracellular matrix proteins in human dermal fibroblasts. The
expression of genes codifying for collagen type V (COL5A1) and elastin (ELN) was analyzed by RT-PCR in total mRNA obtained from proliferative
normal human dermal fibroblasts exposed for 48 h to described treatments. Expression of GAPDH was used as a housekeeping gene. Results are
presented as means ±SD. Statistical significance of treatments with respect to the control was determined by one-way ANOVA. P< 0.05 was
considered significant (*).
Journal of Natural Products Article
DOI: 10.1021/acs.jnatprod.7b00591
J. Nat. Prod. 2017, 80, 3003−3009
3007

unit cell dimensions a= 10.9433(12) Å, b= 7.8994(9) Å, c=
11.2708(17) Å, α=90°,β= 118.237(7)°,γ=90°,V= 858.36(19) Å3,
Z=2,Dc= 1.510 g/cm3,F(000) = 412. A total of 3289 unique
reflections were collected, with 1891 reflections greater than I≥2σ(I)
(Rint = 0.2394). The structure was solved by direct methods and
refined by full-matrix least-squares on F2, with anisotropic displace-
ment parameters for non-hydrogen atoms at final Rindices [I>
2σ(I)], R1= 0.1321, wR2= 0.3358; Rindices (all data), R1= 0.1919,
wR2= 0.3676. Flack parameter = 0.5(4). Crystallographic data have
been deposited in the Cambridge Crystallographic Data Centre
(deposition number: CCDC 1561999) and can be obtained free of
charge via http://www.ccdc.ac.uk./data_request/cif.
Polystachyne H (10): colorless crystals, mp 234−236 °C (acetone−
hexanes); [α]25D−26 (c0.2, Me2CO); UV (MeOH) λmax (log ε) 205
(3.76), 268 (3.53); IR (KBr) νmax 3334, 1775, 1705, 873 cm−1;1H and
13C NMR (CDCl3−DMSO-d6) see Table 1; HRMS (DART-TOF+)
m/z374.15957 [M + NH4]+(calcd for C20H24N1O6, 374.16036). X-
ray crystallographic analysis of polystachyne H (10): formula
C20H20O6, MW = 356.36, orthorhombic, space group P212121, unit
cell dimensions a= 7.7431(7) Å, b= 14.6879(14) Å, c= 14.9959(14)
Å, V= 1705.5(3) Å3,Z=4,Dc= 1.388 g/cm3,F(000) = 752. A total of
3479 unique reflections were collected, with 3435 reflections greater
than I≥2σ(I)(Rint = 0.0261). The structure was solved by direct
methods and refined by full-matrix least-squares on F2,with
anisotropic displacement parameters for non-hydrogen atoms at final
Rindices [I>2σ(I)], R1= 0.0284, wR2= 0.0750; Rindices (all data),
R1= 0.0287, wR2= 0.0753. Flack parameter = 0.01(3). Crystallo-
graphic data have been deposited in the Cambridge Crystallographic
Data Centre (deposition number: CCDC 1562001) and can be
obtained free of charge via http://www.ccdc.ac.uk./data_request/cif.
X-ray Crystallographic Analysis of Salvifiline A (3)/15-epi-
salvifiline A (4): moiety formula C20H22O7, MW = 374.38, monoclinic,
space group P21, unit cell dimensions a= 10.9006(6) Å, b= 7.8767(5)
Å, c= 11.1971(6) Å, α=90°,β= 118.648(2)°,γ=90°,V= 843.70(9)
Å3,Z=2,Dc= 1.474 g/cm3,F(000) = 396. A total of 3395 unique
reflections were collected, with 3260 reflections greater than I≥2σ(I)
(Rint = 0.0514). The structure was solved by direct methods and
refined by full-matrix least-squares on F2, with anisotropic displace-
ment parameters for non-hydrogen atoms at final Rindices [I>
2σ(I)], R1= 0.0303, wR2= 0.0724; Rindices (all data), R1= 0.0321,
wR2= 0.0737. Flack parameter = 0.05(7). Crystallographic data have
been deposited in the Cambridge Crystallographic Data Centre
(deposition number: CCDC 1562000) and can be obtained free of
charge via http://www.ccdc.ac.uk./data_request/cif.
Expression of the Extracellular Matrix Components. Cell
Culture. Normal human dermal fibroblasts (HDFs) were derived from
biopsies of patients subjeted to plastic surgery procedures who gave
their informed consent for the use of discarded tissues. Collagenase-
dissagregated HDFs were cultured in L15 Leibovitz medium added
with 10% fetal bovine serum (HyClone, Logan UT) and antibiotics
(penicillin 80 U/mL, streptomycin 80 μg/mL) (basal medium; BM)
under a conventional humidified atmosphere at 37 °C. HDFs at 70%
confluence were refed with BM added with 0.26−0.29, 2.56−2.93, or
25.6−29.3 μM of compounds 1/2,3/4,5/6,7,8,or11. Control
cultures were maintained in BM or BM added with 100 μM AA.
21
RT-PCR Analysis. Total RNA from HDF maintained for 48 h under
the aforementioned conditions was extracted with TRIzol and
subjected to reverse transcription. cDNA was synthesized from 1 μg
of total RNA using 200 U of reverse transcriptase (M-MLV RT) and
0.5 μg of oligo dT. PCR was performed in a final volume of 15 μL
reaction mix containing 300 ng of the RT reaction mixture, 1.5 mM
MgCl2, 0.2 mM dNTP, 0.2 μM of each primer, and 1.25 U of Taq
DNA polymerase. Thermal cycling over 30 cycles consisted in an
initial denaturation at 95 °C for 4 min, then 95 °C for 30 s, 56.5 °C for
1 min for collagen type I, III, and V and elastin, and 62 °C for 1 min
for GAPDH and then 72 °C for 1 min. It was terminated by a final
extension at 72 °C for 5 min. GAPDH mRNA level was used for
sample standardization. The following oligonucleotides were used as
primers: collagen type I (fw: 5′-CCCCTGGAAAGAATGGAGATG-
3′, rv: 5′-TCCAAACCACTGAAACCTCTG-3′), collagen type III
(fw: 5′-AAGTCAAGGAGAAAGTGGTCG-3′,rv:5′-
CTCGTTCTCCATTCTTACCAGG-3′), collagen type V (fw: 5′-
CGGAACCTTGACGAGAACTAC-3′,rv:5′-TCTCCCTTTTG-
GCCTTTCTC-3′), elastin (fw: 5′-CCTGGCTTCGGATTGTCTC-
3′, rv: 5′-CAAAGGGTTTACATTCTCCACC-3′), and GAPDH (fw:
5′-GAAGGTGGTGAAGCAGGCGT-3′,rv:5′-ATGTGGGC-
CATGAGGTCCACCA-3′). After electrophoresis on 1.5% agarose
gel, each band was quantified by ImageLab software (Bio-Rad).
Antimicrobial Activities. Qualitative antibacterial tests against
Escherichia coli and Bacillus sp. of compounds 1−4,8, and 11 were
carried out in vitro by the agar diffusion method. The medium used
was Bioxon nutrient agar for both bacterial strains (pH 6.8). Bacterial
cells were suspended in saline solution at a density of 108CFU mL−1,
and 0.1 mL was inoculated in nutrient agar plates. Paper filter disks
with 8, 4, 2, 1, and 0.5 μg of each compound were placed onto
inoculated plates. Plates were incubated at 37 °C for 18 h under
aerobic conditions. Filter disks with ampicillin and DMSO were used
as positive and negative controls. All compounds were dissolved in
DMSO. The zone of growth inhibition was measured manually using a
Vernier caliper.
22
The qualitative antifungal tests against Candida
albicans ATCC 10235 and C. glabrata CBS138 were carried out in the
same way as the antibacterial test. However, the medium was Bioxon
potato dextrose agar, the fungal inocula were prepared at a final
concentration of 105CFU mL−1, and the plates were incubated at 37
°C for 24 h under aerobic conditions. Filter disks with amphotericin B
and DMSO were used as positive and negative controls.
23
Phytotoxic Activity. The phytotoxic activity was evaluated using a
germination inhibition bioassay.
24
Lycopersicum esculentum,Capsicum
annuum, and Lactuca sativa seeds were used for the tests. The surface
seeds were sterilized in 2% NaOCl for 10 min, rinsed three times in
sterile distilled water, and air-dried on filter paper. A layer of Whatman
No. 1 filter paper was placed in 90 mm diameter glass Petri dishes.
Compounds 1−6were dissolved in acetone and water containing 0.1%
DMSO in a 1:1 proportion at a concentration of 10 mM.
Corresponding dilutions were made to obtain the assayed concen-
trations (100, 10, and 1 μM). In each dish, 30 seeds were placed
equidistantly, and 2.5 mL of a solution of each compound was added.
The Petri dishes were sealed with Parafilm to prevent solvent
evaporation and incubated for 6 days in the dark inside a growth
chamber at 25 ±2°C. A similar treatment with distilled water served
as a control. Starting from the second day of the experiment,
germinated seeds were counted daily. A seed with 2 mm of radicle was
considered germinated. The percentage of germination in each case
was calculated and compared to the control (pendimethalin), and the
data were analyzed by ANOVA (p< 0.05).
■ASSOCIATED CONTENT
*
SSupporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.7b00591.
NMR spectra for compounds 1,2, and 10 (PDF)
CIF files of 1−4and 10 (CIF)
(CIF)
(CIF)
■AUTHOR INFORMATION
Corresponding Author
*Tel: +52 444 834 2000. Fax: +52 444 834 2010. E-mail:
francisco.bautista@ipicyt.edu.mx (E. Bautista).
ORCID
ElihúBautista: 0000-0002-7050-9182
Notes
The authors declare no competing financial interest.
Journal of Natural Products Article
DOI: 10.1021/acs.jnatprod.7b00591
J. Nat. Prod. 2017, 80, 3003−3009
3008

■ACKNOWLEDGMENTS
We acknowledge the technical assistance of R. Gaviño, H. Rios,
I. Chá
vez, E. Huerta, A. Peña, B. Quiroz, C. Má
rquez, E. Garci ́
a,
L. Marti ́
nez, and R. Patiño. E.B. is grateful to CONACYT for a
Research Fellowship.
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