Keywords
Zanthoxylum rhoifolium
- Rutaceae - sesquiterpenes -
α-ionone - DFT/NMR - antibacterial
Introduction
Within the Rutaceae family, the genus Zanthoxylum L. comprises 225 species of trees and shrubs that are native to subtropical and temperate
regions worldwide [1]. Among these, the native South American tree Zanthoxylum rhoifolium Lam., commonly referred to as “Indian ash” and “mamica de cadela”, is part of this
genus. This species is primarily distributed in Brazil in the Minas Gerais and Rio
de Janeiro states along the eastern rainforest of the Atlantic coast; additionally,
it is found in the states of Piauí and Ceará in the northeast region [2]. In traditional medicine practices, the extract resulting from Z. rhoifolium bark boiled in water is used to relieve symptoms associated with malaria [3]. This practice has been observed in the Patamona Indians of Huyana and Bolivia [4]. Several traditional applications for this plant have been
described, focusing on the bark efficiency as a tonic and febrifuge, as well as its
value as a mouth rinse for toothaches. It is also worth noting the external use of
a decoction as an antibacterial agent used for eruptions on childrenʼs legs [5].
In addition to the traditional medicinal uses, numerous studies reported in the literature
have assessed the biological activities of extracts obtained from this plant. The
roots of Z. rhoifolium have demonstrated anti-plasmodic activity, supporting the traditional use of this
plant as an antimalarial agent [6]. Extracts obtained from the stem bark have shown antinociceptive effects [7] and gastroprotective activity [8]. In a TLC bioassay, the crude plant bark extracts exhibited moderate antibacterial
activity against gram-positive bacteria such as Staphylococcus aureus, Staphylococcus epidermidis, and Micrococcus luteus, as well as against three gram-negative bacteria, Klebsiella pneumoniae, Salmonella setubal, and Escherichia coli
[9], while the CHCl3-MeOH extract from the plant bark has demonstrated fungistatic
activity against Botrytis cinerea, Sclerotinia sclerotiorum, Alternaria alternata, Colletotrichum gloeosporioides, and Clonostachys rosea in an earlier preliminary study [10].
Numerous phytochemical investigations detailed in the literature have examined extracts
derived from the bark, leaves, and fruits of the plant, revealing the presence of
different metabolites, including alkaloids, coumarins, lignans, terpenes, and flavonoids
[6], [11], [12]; however, few studies have been reported to date on the separation and chemical
identification of the nonpolar constituents [13], a part from the ones mainly focused on the essential oil composition [9].
In the framework of a project study devoted to the antimicrobial evaluation of plant
and fungi extracts and/or pure secondary metabolites [14], [15], a phytochemical study of apolar extracts of the plant stem bark and leaves was
carried out, leading to the isolation and characterization of 29 compounds, including
three new sesquiterpenes and one new α-ionone. Moreover, the antimicrobial activity of the total extracts against a collection
of gram-positive and gram-negative bacteria (E. coli, Staphylococcus aureus, Klebsiella sp., Streptococcus mutans, Citrobacter sp., Salmonella sp., Bacillus subtilis, Shigella sp., Enterococcus fecalis, Bacillus clausii, Pseudomonas aeruginosa, Acinetobacter baumannii,
Streptococcus epidermidis, and Lysteria monocitogenes) was evaluated.
Results and Discussion
The Z. rhoifolium stem bark and leaves were extracted with solvents of increasing polarity. The petroleum
ether, CHCl3, and CHCl3-MeOH extracts subjected to different column chromatographies yielded 29 compounds,
including three new sesquiterpenes (1 – 3) and one new α-ionone (4) ([Fig. 1]).
Fig. 1 Structures of compounds 1 – 4.
Among the known derivatives, five α-ionones: corchoionoside C [16], 6R,9S-3-oxo-α-ionol-β-d-glucopyranoside [17], breyniaionoside A [18], blumenol C [19], and debiloside C [20]; four coumarins: pimpinellin [21], anisocoumarin H [22], auraptene [23], and acetoxyauraptene [23]; thirteen sesquiterpenes: (−)-1,12-oxaguai-10-(15)-ene [24], pancherione [25], 4α-hydroxy-11-hydroxy-guai-10(14)-ene [26], 1β,6α-dihydroxyeudesm-4(15)-ene [27], spathulenol [28], 7-epi-11-hydroxychabrolidione A [29], holostylactone [29], caryolane-1,9-β-diol [30], (4S,5S,7S,10S)-5,12-dihydroxyeudesma-1-one [31], (4R,5S,7S,10S)-5,12-dihydroxyeudesma-1-one [32], (−)-10-epi-5-β-11-dihydroxyeudesmane [33], (1α,4αH,5α,7αH,10αH)-1,5-epoxyguaian-11-ol [34], and (1β,4αH,5α,7αH,10αH)-1,5-epoxyguaian-11-ol [34]; and three lignans episesamin [35]: piperitol-3,3-dimethylallyl ether [36], and alangilignoside C [37] were characterized by comparison of their NMR and MS data with those reported in
the literature.
Compound 1 possessed a molecular formula of C15H26O3 and three indices of hydrogen deficiencies, as determined by the HRESIMS spectrum
(m/z 277.1766 [M + Na]+). Analysis of the 1D NMR data ([Table 1]) showed the presence of four methyl groups at δ
H 1.04 (3H, d, J = 6.8 Hz), 1.06 (3H, d, J = 6.5 Hz), and 1.18 (6H, s), one hydroxymethine (δ
H 4.13, br d, J = 6.0 Hz), four methylenes, three methines, and three oxygenated quaternary carbons,
suggesting a tricyclic skeleton of 1. A comparison of 1H and 13C NMR data, obtained with the analysis of HSQC and HMBC spectra ([Fig. 2]), with those of related guaiane sesquiterpenes suggested close similarities [34], [38]. A COSY experiment registered for 1, revealed the following
connectivities: H-2–Me-15 (through H2-3 and H-4) and H2-6–Me-14, supporting the presence of a tertiary hydroxyl function at C-11. Signals at
δ
C 73.0 and 74.7 indicated the presence of an epoxy ring. HMBC correlations between
H2-3 and C-4; H2-6 and C-1, C-4, C-5, C-8, C-11; Me-12 and Me-13 and C-11; Me-14 and C-1 and C-10
suggested that the hydroxymethine function was placed at C-2, with the epoxy ring
between C-1 and C-5. The determination of the relative configuration of C-1, C-2,
C-4, C-5, C-7, and C-10 of 1 was performed by applying the quantum mechanical (QM) methods combined with NMR spectroscopy
(QM/NMR) approach. This methodology, discovered and optimized by our group [39], is based on the computation of NMR properties (13C/1H NMR chemical shift) at the density functional theory (DFT) level and the subsequent
comparison of the experimental
and predicted 13C and 1H NMR chemical shifts using statistical parameters [39]. The latter step is fundamental for suggesting the proper prediction of the stereochemical
assignment of organic molecules. First, using Monte Carlo molecular mechanics (MCMM),
low-mode conformational sampling (LMCS), and molecular dynamics (MD) simulations,
a thorough empirical conformational search related to all the possible investigated
stereoisomers of 1 was conducted. Specifically, five stereocenters were undefined, since C-1 and C-5
were considered as a single stereocenter for the presence of the epoxide. Therefore,
among the possible 32 stereoisomers, 16 diastereoisomers (1a-1p, vide infra) were considered for the QM/NMR predictions. The MM conformer ensembles were further
subjected to geometry and energy optimization processes at the DFT at the MPW1PW91/6 – 31 g(d)
level of theory. Finally, additional visual
inspection was performed on the optimized conformers to eliminate any additional redundant
ones. The Boltzmann distribution of the conformers for each stereoisomer derived at
the same level of theory was then taken into consideration for predicting the 13C and 1H NMR chemical shifts for 1a-1p at the MPW1PW91/6 – 31 g(d,p) level. The integrated equation formalism model (IEFPCM)
for modelling methanol as a solvent was used for all DFT calculations (see Material
and Methods). The computed and experimental values were then compared using the mean
absolute error (MAE) values (see Computational Details, Material and Methods). The
relative configuration of 1 was thus elucidated based on the MAE values for the 13C and 1H NMR chemical shifts (13C MAE = 1.67 ppm, 1H MAE = 0.10 ppm for 1b) (Tables 1S and 2S, Supporting Information). Specifically, the relative configuration was
determined as follows: 1R*, 2R*, 4R*, 5S*, 7S*, 10R*. Additionally, we used the DP4+ method [40] to corroborate our findings further. Again, the stereoisomer 1b showed the highest DP4+ probability (100%), corroborating the relative configuration
1R*, 2R*, 4R*, 5S*, 7S*, 10R*. Thus, 1 was elucidated as (1R*,2R*,4R*,5S*,7S*,10R*)-epoxy-guaian-2,11-diol.
Table 1 1H and 13C NMR data of compounds 1 and 2
a.
Position
|
1
|
2
|
δ
H
|
δ
C
|
δ
H
|
δ
C
|
aSpectra were recorded in CD3OD at 600 (1H) and 150 MHz (13C); J values are in parentheses and reported in Hz; chemical shifts are given in ppm; assignments
were confirmed by COSY, 1D-TOCSY, HSQC, and HMBC experiments; boverlapped signal
|
1
|
|
75.6
|
|
158.9
|
2a
|
4.13 br d (6.0)
|
73.0
|
5.73 br s
|
125.1
|
2b
|
|
|
|
|
3a
|
1.62 dd (13.0, 8.0)
|
38.0
|
2.27 ddd (11.2, 9.0, 3.5)
|
38.8
|
3b
|
1.21 m
|
|
2.01b
|
|
4
|
2.28 m
|
36.2
|
2.38 m
|
41.5
|
5
|
|
73.4
|
2.29 m
|
48.8
|
6a
|
2.21 d (15.0)
|
26.0
|
2.01b
|
31.6
|
6b
|
1.41 m
|
|
0.92 m
|
|
7
|
1.73b
|
46.0
|
1.35 m
|
54.6
|
8a
|
1.74b
|
29.0
|
1.83b
|
25.2
|
8b
|
1.33b
|
|
1.25 m
|
|
9a
|
1.51 m
|
28.0
|
1.85b
|
43.0
|
9b
|
1.32b
|
|
1.66 br dd (14.0, 11.0)
|
|
10
|
2.44 m
|
29.0
|
|
75.0
|
11
|
|
74.7
|
|
73.8
|
12
|
1.18 s
|
27.0
|
1.18 s
|
27.4
|
13
|
1.18 s
|
27.0
|
1.15 s
|
26.3
|
14
|
1.06 d (6.5)
|
18.0
|
1.27 s
|
32.0
|
15
|
1.04 d (6.8)
|
13.5
|
1.07 d (6.5)
|
15.7
|
Fig. 2 Main HMBC correlations of compounds 1 – 4.
The HRESIMS of compound 2 showed a protonated molecular ion at m/z 239.2017 [M + H]+, consistent with the molecular formula C15H26O2 and three indices of hydrogen deficiencies. Its 1D and 2D NMR ([Table 1]) features indicated the presence of a guaiane-type sesquiterpene and similarities
with compound 1. The 13C NMR chemical shift assignments were derived from the analysis of HSQC and HMBC experiments
([Fig. 2]). COSY and HSQC experiments were able to establish the spin system in the molecule
starting from the sp2
proton a δ
H 5.73 at C-2. Two quaternary hydroxyl functions were also evident from the HMBC spectra
at δ
C 73.8 and 75.0 that were located at positions 10 and 11 from the respective correlations
between Me-14–C-1, Me-14–C-9, Me-14–C-10, H2-9–C-10,
Me-12–C-7, Me-12–C-11, and Me-13–C-7, Me-13–C-11. Keys HMBC correlations were also observed among H2-3, H2-6, H2-9, and C-1, confirming the presence of a double bond between C-1/C-2. The relative
configuration was obtained through the QM/NMR approach, as described for compound
1. After evaluating the obtained MAE, the isomer 2c featured the lowest one (13C MAE = 1.12 ppm, 1H MAE = 0.10 ppm) (Tables 3S and 4S, Supporting Information), with the relative configuration of 4R*, 5R*, 7S*, and 10S*. Thus, compound 2 was identified as (4R*,5R*,7S*)-guaia-1,2-en-10,11-diol, also confirmed after applying the DP4+ approach, in which
2c showed the highest DP4+ probability (100%).
In the HRESIMS spectrum, compound 3 showed a sodiated molecular ion at m/z 277.1768 [M + Na]+, indicating a molecular formula of C15H26O3, with three indices of hydrogen deficiencies. The 1H NMR spectrum ([Table 2]) indicated three methyl groups as two singlet signals (δ
H 1.05 3H, 1.12 6H), one methyl group as a doublet signal (δ
H 1.05, J = 6.5 Hz) and four methylenes, three methines, and one hydroxymethine (δ
H 3.82, br dd, J = 2.5, 2.5 Hz). From the COSY experiment, the spin system H2-2–H2-9 was observed. The 13C NMR data suggested an eudesmane skeleton for 3 since one of the three hydrogen deficiencies was consumed by one keto group (δ
C 220.0) [32], [38]. An oxygenated tertiary carbon
(δ
C 74.0) and a quaternary carbon (δ
C 53.4) were also evidenced. The HMBC experiment ([Fig. 2]) was helpful in establishing the substituent locations; thus, the keto group was
placed at C-1 from the H-3–C-1, H-4–C-1, H2-6–C-1, H2-9–C-1, and Me-14–C-1 correlations. The hydroxy groups were located at C-3 and C-11
from the respective correlations Me-15–C-3 and H2-6–C-11. Unfortunately, despite various attempts to derive the stereochemical configuration
of compound 3, the obtained data lacked sufficient confidence to elucidate and suggest the stereogenic
centers of this molecule properly. For this reason, only the novel 2D chemical scaffold
was reported. Thus, we suggest compound 3 as 3,11-dihydroxyeudesma-1-one.
Table 2 1H and 13C NMR data of compounds 3 and 4
a.
Position
|
3
|
4
|
δ
H
|
δ
C
|
δ
H
|
δ
C
|
aSpectra were recorded in CD3OD at 600 (1H) and 150 MHz (13C) for 3 and at 400 (1H) and 100 MHz (13C) for 4; J values are in parentheses and reported in Hz; chemical shifts are given in ppm; assignments
were confirmed by COSY, 1D-TOCSY, HSQC, and HMBC experiments; boverlapped signal
|
1
|
|
220.0
|
|
39.3
|
2a
|
2.34 dd (15.0, 3.0)
|
33.6
|
2.43 d (13.0)
|
56.2
|
2b
|
1.60b
|
|
2.03 d (13.0)
|
|
3a
|
3.82 br dd (2.5, 2.5)
|
78.3
|
|
213.9
|
4a
|
2.43 m
|
32.3
|
2.32 ddd (13.0, 4.5, 2.3)
|
49.8
|
4b
|
|
|
2.16 br t (13.0)
|
|
5
|
2.47 m
|
56.3
|
1.94 m
|
34.4
|
6
|
1.79 br dd (9.0, 8.0)
|
22.0
|
2.01 dd (13.0, 2.0)
|
58.1
|
7
|
1.61b
|
44.0
|
5.65 br d (15.3, 9.0)
|
135.9
|
8a
|
1.62b
|
22.8
|
5.55 br d (15.3, 7.7)
|
131.3
|
8b
|
1.45 m
|
|
|
|
9a
|
2.20 ddd (16.0, 14.0, 3.5)
|
33.0
|
4.41 m
|
79.6
|
9b
|
1.31 m
|
|
|
|
10
|
|
53.4
|
3.64 m
|
66.5
|
11
|
|
74.0
|
0.82 s
|
21.0
|
12
|
1.12 s
|
24.9
|
0.99 s
|
31.8
|
13
|
1.12 s
|
27.3
|
1.01 d (6.0)
|
21.8
|
14
|
1.05 s
|
20.7
|
|
|
15
|
1.05 d (6.5)
|
17.6
|
|
|
Glc 1
|
|
|
4.38 d (7.8)
|
100.7
|
2
|
|
|
3.16 dd (9.0, 7.8)
|
74.9
|
3
|
|
|
3.31 dd (9.0, 9.0)
|
77.8
|
4
|
|
|
3.28 dd (9.0, 9.0)
|
71.4
|
5
|
|
|
3.20 m
|
77.8
|
6a
|
|
|
3.86 dd (12.0, 3.0)
|
62.4
|
6b
|
|
|
3.66 dd (12.0, 5.0)
|
|
Compound 4 (C19H32O8) displayed a sodiated molecular ion at m/z 411.1985 [M + Na]+ and a fragment in the HRESIMS at m/z 249.13 [M + Na – 162]+, consistent with the presence of a hexose moiety in the molecule. Its NMR features
([Table 2]) suggested the presence of an α-ionone glycoside [18] for the signals attributable to two methyl groups as singlets (δ
H 0.82, 0.99), one methyl group as a doublet (δ
H 1.01, d, J = 6.0 Hz), one oxygenated methine (δ
H 4.41, m), one hydroxymethylene (δ
H 3.64, m), two methines, two methylenes, two olefinic protons (δ
H 5.55 br d, J = 15.3, 7.7 Hz, and 5.65 br d, J = 15.3, 9.0 Hz), one keto group (δ
C 213.9), and signals for a β-glucopyranose moiety. The COSY experiment suggested
the spin sequence H2-4–H-5–H-6–H-7–H-8–H-9–H2-10. The HMBC spectrum ([Fig. 2]) led to the identification of the hydroxymethylene C-10, showing cross-peaks between
H2-10–C‐8 and H2-10–C‐9 and the hydroxymethine C-9 thanks to the H-7–C-9 and H‐8–C-9 correlations. Finally,
the relative configuration of compound 4 was obtained through the application of the QM/NMR combined approach, as described
above. Specifically, we firstly performed the computation of the chemical shifts considering
only the aglycone part of compound 4, thus removing the glucopyranose from the structure and replacing it with a simplified
group (-OCH3), considering four diastereoisomers (4a-4d). The obtained MAE highlighted two possible stereoisomers featuring the relative
configuration of 5R*, 6R*, and 9R* (4a) and
5R*, 6R*, and 9S* (4b) (see Table 5S and 6S, Supporting Information), thus suggesting the relative configuration of the aglycone
part as 5R* and 6R*. Therefore, in order to elucidate the relative configuration of C-9, the prediction
of NMR parameters was carried out also considering the entire glycoside, accounting
for 5R*, 6R*, and 9R* (4e) and 5R*, 6R*, and 9S* (4 f). In detail, the glucopyranose moiety included in this structure was considered for
the computation of the relative energy and the final Boltzmann distribution and the
prediction of the chemical shifts, while it was not considered for the computation
of the statistical parameters for two reasons: (1) the sugar moiety was already identified
as glucopyranose and (2) to avoid additional errors in the quantitative analysis that
could compromise the identification of the correct stereoisomer. In particular,
these steps were performed to evaluate the influence of the β-glucopyranose moiety on the conformational ensemble and, therefore, on the chemical
shift predictions. Specifically, we manually selected the conformers considering (a)
the chair arrangement of the aglycone part, (b) the arrangement of the alkene at the
C-7 and C-8 position, and (c) the β-glucopyranose moiety orientation (Fig. 26S, Supporting Information), for a total of 12 conformers for each stereoisomer (4e and 4f). Finally, the stereoisomer 4e featured the lowest MAE (13C MAE = 1.27 ppm, 1H MAE = 0.14 ppm) (Tables 7S and 8S, Supporting Information). Therefore, the relative configuration of compound 4 was suggested as 5R*, 6R*, and 9R*, and was confirmed by the obtained DP4+ probability of 99.71%. From all these data,
4 was characterized as
(5R*,6R*,9R*)-10-dihydroxy-megastigman-7-en-3-one 9-O-β-d-glucopyranoside, a new specialized metabolite also for the aglycone portion.
Minimum inhibitory concentrations (MICs) were determined to assess the Z. rhoifolium extracts antimicrobial effect against a collection of gram-positive and gram-negative
bacteria. The MIC of the crude extracts against the bacteria were determined by the
reference protocol of the “Agar and broth dilution methods to determine the MIC of
antimicrobial substances” [41]. The MIC was determined as the lowest extract concentration that inhibited visible
bacterial growth. The results showed no particular antimicrobial activity, except
for the chloroform-methanol bark extract that, at 500 µg/mL, gave a 20% vitality inhibition
on S. mutans and Citrobacter sp.
Material and Methods
General experimental procedures
Optical rotations were measured on an Atago AP-300 digital polarimeter equipped with
a sodium lamp (589 nm) and a 1-dm microcell. NMR data were acquired on a Bruker Ascend-600
NMR spectrometer (Bruker BioSpin GmBH) equipped with a Bruker 5-mm PATXI Probe and
a Bruker DRX-400 spectrometer at 300 K. Data processing was carried out with Topspin
3.2 software. 2D NMR spectra were acquired in methanol-d
4 and standard pulse sequences and phase cycling were used for COSY, HSQC, HMBC, 1D-TOCSY,
and ROESY spectra. HRESIMS data were obtained in the positive ionization mode on a
Q Exactive Plus mass spectrometer, Orbitrap-based FT-MS system, equipped by an electrospray
ionization (ESI) source (Thermo Fischer Scientific Inc.). Column chromatographies
were carried out over silica gel (70 – 220 mesh; Merck) and Sephadex LH-20 (40 – 70 µm;
Pharmacia). RP-HPLC separations were conducted using a Shimadzu LC-8A series pumping
system equipped with a Shimadzu RID-10A refractive
index detector and Shimadzu injector on a C18
µ-Bondapak column (Waters; 30 cm × 7.8 mm, 10 µm, flow rate 2.0 mL/min). TLC separations
were carried out using silica gel 60 F254 (0.20 mm thickness) plates (Merck) and cerium sulphate as a spray reagent.
Plant material
Z. rhoyfolium stem bark and leaves were collected in April 2010 near Merida, Venezuela, and identified
by Eng. Juan Carmona, Universidad de Los Andes, Merida, Venezuela. A voucher specimen
(n. 607) was deposited at Jardin de Plantas Medicinales de la Facultad de Farmacia
y Bioanalisis, Merida, Venezuela.
Extraction and isolation
Dried stem bark (814 g) and leaves (800 g) of Z. rhoyfolium were extracted with solvents of increasing polarity, starting from petroleum ether,
CHCl3, CHCl3-MeOH 9 : 1, and MeOH by exhaustive maceration (3 × 2.0 L) to give 20.0, 15.0, 35.0,
and 50.0 g of the respective residues from the stem bark and 11.5, 22.0, 8.2, and
35.0 g of the respective residues from the leaves. Part of the petroleum ether stem
bark extract (7.0 g) was submitted to silica gel column chromatography (5 × 30 cm),
eluting with n-hexane, followed by increasing concentrations of CHCl3 in n-hexane (between 1 and 100%), continuing with CHCl3, followed by increasing concentrations of MeOH in CHCl3 (between 1 and 50%). There were 300 fractions of 25 mL collected, analyzed by TLC
(silica gel plates, in n-hexane, or mixtures of n-hexane-CHCl3, 9 : 1, 3 : 7 and CHCl3-MeOH, 95 : 5, 9 : 1), and grouped
into ten major fractions, A1-J1 (A1 = 1 – 52, B1 = 53 – 118, C1 = 119 – 148, D1 = 149 – 177, E1 = 178 – 203, F1 = 204 – 221, G1 = 222 – 243, H1 = 244 – 264, I1 = 265 – 290, J1 = 291 – 300). An aliquot (100 mg) of fraction D1 (727.2 mg) was dissolved in 1 mL of MeOH-H2O (7 : 3) and submitted to RP-HPLC with the same solvent (10 injections) to give holostylactone
(1.9 mg, t
R 9 min), (1α,4αH,5α,7αH,10αH)-1,5-epoxyguaian-11-ol (2.3 mg, t
R 17 min), (1β,4αH,5α,7αH,10αH)-1,5-epoxyguaian-11-ol (2.8 mg, t
R 18 min), and piperitol-3,3-dimethylallyl ether (2.2 mg, t
R 25 min). Fraction E1 (85.1 mg) was dissolved in 850 µL of MeOH-H2O (6.5 : 5.5) and purified by RP-HPLC with
the same solvent (8 injections) to yield panchierone (3.2 mg, t
R 14 min). An aliquot (40 mg) of fraction G1 (97.8 mg) was solubilized in 400 µL of MeOH-H2O (3 : 2) and subjected to RP-HPLC with the same solvent (4 injections) to yield (4S,5S,7S,10S)-5,12-dihydroxyeudesma-1-one (2.2 mg, t
R 15 min). An aliquot (50 mg) of fraction H1 (146.2 mg) was solubilized in 500 µL of MeOH-H2O (3 : 2) and subjected to RP-HPLC with the same solvent (5 injections) to give 7-epi-11-hydroxychabrolidione A (2.0 mg, t
R 16 min), (−)-1,12-oxaguai-10-(15)-ene (3.2 mg, t
R 55 min), and 2 (1.5 mg, t
R 58 min). Finally, an aliquot (70 mg) of fraction I1 (143.4 mg) was dissolved in 700 µL of MeOH-H2O (5.5 : 4.5) and chromatographed by RP-HPLC with the same solvent (7 injections)
to yield compounds 3
(2.3 mg, t
R 7 min), (H-5)-5,12-dihydroxyeudesma-1-one (3.0 mg, t
R 10 min), 1 (1.0 mg, t
R 14 min), (−)-10-epi-5-β-11-dihydroxyeudesmane (1.5 mg, t
R 20 min), 4α-hydroxy-11-hydroxy-guai-10(14)-ene (2.4 mg, t
R 22 min), and caryolane-1,9-β-diol (1.8 mg, t
R 30 min).
A side of the CHCl3 leaf extract (7.0 g) was submitted to flash silica gel column chromatography (SNAP
340 g, 7 × 18 cm) by Biotage, eluting with n-hexane, followed by increasing concentrations of CHCl3 in n-hexane (between 1 and 100%), continuing with CHCl3, followed by increasing concentrations of MeOH in CHCl3 (between 1 and 50%). There were 320 fractions of 25 mL collected, analyzed by TLC
(silica gel plates, mixtures of n-hexane-CHCl3, 7 : 3, 1 : 1, CHCl3, and CHCl3-MeOH, 95 : 5), and grouped into five major fractions, A2-E2 (A2 = 1 – 93, B2 = 94 – 188, C2 = 189 – 230, D2 = 231 – 292, E2 = 293 – 320. An aliquot (120 mg) of fraction C2 (537 mg) was solubilized in 1.2 mL of MeOH-H2O (7.5 : 2.5) and purified through RP-HPLC with the same solvent (10 injections) to
yield
pimpinellin (2.7 mg, t
R 8 min), 1β,6α-dihydroxyeudesm-4(15)-ene (2.4 mg, t
R 9 min), anisocoumarin H (2.4 mg, t
R 12 min), episesamin (2.3 mg, t
R 14 min), acetoxyauraptene (3.8 mg, t
R 18 min), spathulenol (1.4 mg, t
R 25 min), and auraptene (1.8 mg, t
R 35 min).
A side of the CHCl3-MeOH leaf extract (2.8 g) was chromatographed over Sephadex LH-20 column chromatography
using MeOH as an eluent (3 × 100 cm; flow rate 0.8 mL/min, collection volume 10 mL),
collecting 54 fractions that were grouped by TLC (silica gel plates, CHCl3-MeOH-H2O 80 : 18 : 2, n-BuOH-CH3COOH-H2O 60 : 15 : 25) into six major collections, A3-F3 (A3 = 1 – 14, B3 = 15 – 27, C3 = 18 – 22, D3 = 23 – 33, E3 = 34 – 45, F3 = 46 – 54). An aliquot (100 mg) of fraction B3 (535.2 mg) was dissolved in 1.0 mL of MeOH-H2O (3.5 : 6.5) and subjected to RP-HPLC with the same solvent (10 injections) to yield
debiloside C (3.0 mg, t
R 9 min), breyniaionoside A (2.4 mg, t
R 12 min), corchoionoside C (1.8 mg, t
R 16 min), compound 4 (4.0 mg,
t
R 74 min), 6R,9S-3-oxo-α-ionol-β-d-glucopyranoside (3.9 mg, t
R 66 min), and blumenol C (2.4 mg, t
R 21 min). An aliquot (80 mg) of fraction C3 (295 mg) was solubilized in 700 µL of MeOH-H2O (3.5 : 6.5) and purified through RP-HPLC with the same solvent (8 injections) to
give alangilignoside C (1.4 mg, t
R 33 min).
Compound 1: amorphous powder; [α]
d
25 + 168 (c 0.1, MeOH); 1H and 13C NMR data, see [Table 1]; HRESIMS m/z 277.1766 [M + Na]+ (calcd. for C15H26O3Na, 277.1780).
Compound 2: amorphous powder; [α]
d
25 + 103(c 0.1, MeOH); 1H and 13C NMR data, see [Table 1]; HRESIMS m/z 239.2017 [M + H]+ (calcd. for C15H27O2, 239.2006).
Compound 3: amorphous powder; [α]
d
25 + 145 (c 0.1, MeOH); 1H and 13C NMR data, see [Table 2]; HRESIMS m/z 277.1768 [M + Na]+, 255.1958 [M + H]+ (calcd. for C15H26O3Na, 277.1780).
Compound 4: amorphous powder; [α]
d
25 − 70 (c 0.1, MeOH); 1H and 13C NMR data, see [Table 2]; HRESIMS m/z 411.1985 [M + Na]+, 249.13 [M + Na – 162]+ (calcd. for C19H32O8Na, 411.1995).
Computational details
Maestro 12.7 (Schrödinger Schrödinger Suite) was used for generating the starting
3D chemical structures of compounds 1, 2, and 4. Optimization of the 3D structures was performed with MacroModel (version 13.1, Schrödinger
Suite, LLC) [42] using the OPLS force field and the Polak-Ribier conjugate gradient (PRCG) algorithm
(maximum derivative less than 0.001 kcal/mol). In particular, for compound 1, showing five stereogenic centers (C-1 and C-5 were considered as unique center,
since an epoxide was present), 16 isomers were considered:
-
1a (1R*, 2R*, 4R*, 5S*, 7R*, 10R*) (9 conformers), 1b (1R*, 2R*, 4R*, 5S*, 7S*, 10R*) (14 conformers), 1c (1R*, 2R*, 4R*, 5S*, 7R*, 10S*) (13 conformers), 1d (1R*, 2R*, 4R*, 5S*, 7S*, 10S*) (8 conformers), 1e (1R*, 2S*, 4R*, 5S*, 7R*, 10R*) (19 conformers), 1f (1R*, 2S*, 4R*, 5S*, 7S*, 10R*) (15 conformers), 1g (1R*, 2S*, 4R*, 5S*, 7R*, 10S*) (9 conformers), 1h (1R*, 2S*, 4R*, 5S*, 7S*, 10S*) (13 conformers), 1i (1R*, 2R*, 4S*, 5S*, 7R*, 10R*) (9 conformers), 1j (1R*, 2R*, 4S*, 5S*, 7S*,
10R*) (11 conformers), 1k (1R*, 2R*, 4S*, 5S*, 7R*, 10S*) (9 conformers), 1l (1R*, 2R*, 4S*, 5S*, 7S*, 10S*) (17 conformers), 1m (1R*, 2S*, 4S*, 5S*, 7R*, 10R*) (10 conformers), 1n (1R*, 2S*, 4S*, 5S*, 7S*, 10R*) (11 conformers), 1o (1R*, 2S*, 4S*, 5S*, 7R*, 10S*) (10 conformers), 1p (1R*, 2S*, 4S*, 5S*, 7S*, 10S*) (14 conformers).
For compound 2, showing four stereogenic centers, 8 isomers were considered:
-
2a (4R*, 5R*, 7R*, 10R*) (17 conformers), 2b (4R*, 5R*, 7R*, 10S*) (15 conformers), 2c (4R*, 5R*, 7S*, 10R*) (14 conformers), 2d (4R*, 5R*, 7S*, 10S*) (14 conformers), 2e (4R*, 5S*, 7R*, 10R*) (16 conformers), 2 f (4R*, 5S*, 7R*, 10S*) (17 conformers), 2g (4R*, 5S*, 7S*, 10R*) (14 conformers), 2h (4R*, 5S*, 7S*, 10S*) (14 conformers).
For compound 4, showing three stereogenic centers considering the aglycone, four isomers of the
aglycone (4a-4d) and two isomers (4e-4f) considering the β-glucopyranose were accounted for:
-
4a (5R*, 7R*, 9R*) (5 conformers), 4b (5R*, 7R*, 9S*) (8 conformers), 4c (5R*, 7S*, 9R*) (9 conformers), 4d (5R*, 7S*, 9S*) (5 conformers), 4e (5R*, 7R*, 9R*) (12 conformers), 4f (5R*, 7R*, 9S*) (12 conformers).
Using the produced 3D structures as input, exhaustive conformational searches were
conducted at the empirical MM level. In particular, LMCS rounds (50 000 steps) and
MCMM rounds (50 000 steps) were carried out. Moreover, MD simulations with a time
step of 2.0 fs, an equilibration period of 0.1 ns, and a simulation duration of 10 ns
were run at 450, 600, 700, and 750 K. A constant methanol dielectric term that mimicked
the solvent presence was taken into account for each of these simulations. For each
isomer, using the “Redundant Conformer Elimination” module of MacroModel (version
13.1, Schrödinger Suite, LLC) [42], the sampled conformers were then minimized (PRCG, maximum derivative less than
0.001 kcal/mol) for each isomer and compared. Specifically, a minimum cutoff of 0.5 Å
RMSD (root-mean-square deviation) was set for saving structures. Afterwards, nonredundant
conformers were considered for QM calculations using Gaussian 09 software [43]. Specifically, the sampled conformers were subjected to a further step of geometry
optimization at the DFT level using the MPW1PW91 functional and the 6–31 G(d) basis set. MeOH solvent effects were reproduced by setting the related integral
equation formalism version of the polarizable continuum model (IEFPCM) [44]. After that, the optimized geometries were additionally visually inspected in order
to remove possible residual redundant conformers. The computation of the 13C and 1H NMR chemical shifts was performed on each ensemble of the considered isomers of
1, 2, and 4, using the MPW1PW91 functional and the 6–31 G(d,p) basis set and MeOH (IEFPCM). For each case study compound, final 13C and 1H NMR sets of data were produced for each investigated isomer, considering the weight
of each conformer on the total Boltzmann distribution considering their
relative energies. The multi-standard approach (MSTD) [45] was employed for the calibrations of predicted 13C/1H chemical shifts and, followed this procedure:
-
chemical shifts for sp2
carbons were computed accounting for benzene as a reference considering the carbonyl
group (i.e., C-3 for compound 4);
-
chemical shifts for sp2
carbons (i.e., C-7 and C-8 of compound 4) and their related protons were computed accounting for allyl alcohol as a reference
for C-7, C-8, C-9 and O-β-d-Glc of compound 4
[45].
Tetramethylsilane (TMS) was used to compute chemical shift data of sp3
carbons and their related protons (Tables 1S–8S, Supporting Information). The comparison of experimental and calculated 13C and 1H NMR chemical shifts was carried through the Δδ parameter (Tables 1S–8S, Supporting Information):
where δ
exp (ppm) and δ
calc (ppm) are the 13C/1H experimental and predicted chemical shifts, respectively. In this way, the MAE values
were computed for all the considered isomers of each case study compound using the
following equation:
namely, the sum (Σ) of the number of computed absolute error values (Δδ), normalized to the number of chemical shifts considered (n) (Tables 1S–6S, Supporting Information). DP4+ probabilities for each accounted isomer of 1, 2, and 4 were computed considering both 13C and 1H NMR chemical shift values and the related experimental data. The chemical shift
datasets obtained using only TMS as a reference compound were employed for the calculation
of the DP4+ probabilities, manually selecting the sp2
atoms in the available DP4+ Toolbox (Excel file) following the “multi-standard” approach.
Antibacterial activity
Gram-positive and gram-negative bacterial strains were Escherichia coli, Staphylococcus aureus, Klebsiella sp., Streptococcus mutans, Citrobacter
sp., Salmonella sp., Bacillus subtilis, Shigella sp., Enterococcus fecalis, Bacillus
clausii, Pseudomonas aeruginosa, Acinetobacter baumannii, Streptococcus epidermidis, and Lysteria monocitogenes and were purchased from ATCC. MICs were determined by the reference protocol of the
“Agar and broth dilution methods to determine MIC of antimicrobial substances” [41]. All strains were grown aerobically in brain heart infusion (BHI) broth-rich medium
at 37 °C. The samples were dissolved in 100% DMSO at different concentrations (extract:
from 500 to 30 µg/mL), added to each well and bacterial suspensions (0.5 × 105 CFU/mL), and then incubated at 37 °C for 24 h. Cell absorbance was measured at 600 nm
using a Tecan Infinite 200 PRO spectrophotometer. A blank control (sterile culture
medium, without compounds and suspensions of microorganisms), a vehicle control (sterile
culture medium with DMSO), and an antimicrobial agent, chlorhexidine gluconate (CHX),
were used. The MIC was determined as the lowest extract concentration that inhibited
visible bacterial growth. Each experiment was performed with duplicate samples at
each time point.
Contributorsʼ Statement
Data collection: M. Di Stasi, V. Parisi, V. Hernandez, E. Gazzillo, G. Bifulco, N.
De Tommasi, G. Donadio Conception and design of the work: M. Chini, G. Bifulco, A.
Braca, N. De Tommasi Statistical analysis: M. Di Stasi, V. Parisi, V. Hernandez, E.
Gazzillo, M. Chini, G. Donadio Analysis and interpretation of the data: M. Di Stasi,
V. Parisi, V. Hernandez, E. Gazzillo, M. Chini, G. Bifulco, A. Braca, N. De Tommasi,
G. Donadio Drafting the manuscript: A. Braca, M. Di Stasi, V. Parisi, V. Hernandez,
E. Gazzillo Critical revision of the manuscript: M. Chini, G. Bifulco, A. Braca, N.
De Tommasi, G. Donadio