Chemical constituents of the aerial parts of Algerian Galium brunneum: Isolation of new hydroperoxy sterol glucosyl derivatives

Article abstract of DOI:10.1016/j.phytol.2020.05.002

The liposoluble extract of Galium brunneum aerial parts from North-eastern Algeria was chemically investigated. The EtOAc soluble portion contained a series of glycosyl cucurbitacins and sterols including three new glucosyl hydroperoxy sterols 1–3 among other phenolic components whereas the BuOH soluble fraction was dominated by glycosyl derivatives of flavonoids, iridoids and lignans, according to the chemistry reported in the literature for the genus Galium. The structure of new oxidized sterols 1–3 was determined by spectroscopic methods as well as by comparison with related known metabolites. Selected main compounds from both extracts, which revealed moderate antibacterial activities, were tested for their growth inhibitory properties against Gram-positive and Gram-negative bacteria. This is the first report of cucurbitacins in plants of genus Galium.

Full text of DOI:10.1016/j.phytol.2020.05.002

Phytochemistry Letters 38 (2020) 39–45
Contents lists available at ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
Chemical constituents of the aerial parts of Algerian Galium brunneum:
Isolation of new hydroperoxy sterol glucosyl derivatives
a,b
a,c
a,
a
d
Abla Smadi , Fatma Bitam , Maria Letizia Ciavatta *, Marianna Carbone , Anis Bertella ,
a
Margherita Gavagnin
b
a
Consiglio Nazionale Delle Ricerche (CNR), Istituto Di Chimica Biomolecolare (ICB), Via Campi Flegrei, 34, Pozzuoli, 80078 Napoli, Italy
Faculté Des Sciences De La Matière, Département De Chimie, Laboratoire De Chimie Et Chimie De l’Environnement (LCCE), Université De Batna 1, 05000, Algeria
c
Faculté De Médecine, Département De Pharmacie, Université De Batna 2, 05000, Algeria
d
Faculté Des Sciences De La Matière Et De La Vie, Départment De Biologie, Laboratoire De La Microbiologie Appliquée, Université d’Oran 1, 31100, Algeria
A R T I C L E I N F O
A B S T R A C T
Keywords:
The liposoluble extract of Galium brunneum aerial parts from North-eastern Algeria was chemically investigated.
The EtOAc soluble portion contained a series of glycosyl cucurbitacins and sterols including three new glucosyl
hydroperoxy sterols 1–3 among other phenolic components whereas the BuOH soluble fraction was dominated
by glycosyl derivatives of flavonoids, iridoids and lignans, according to the chemistry reported in the literature
for the genus Galium. The structure of new oxidized sterols 1–3 was determined by spectroscopic methods as well
as by comparison with related known metabolites. Selected main compounds from both extracts, which revealed
moderate antibacterial activities, were tested for their growth inhibitory properties against Gram-positive and
Gram-negative bacteria. This is the first report of cucurbitacins in plants of genus Galium.
Galium brunneum
Rubiaceae
β-Sitosterol
Hydroperoxysterols
Cucurbitacins
NMR
1
. Introduction
The Rubiaceae family is one of the largest in the Magnoliopsida
North Africa (Algeria, Morocco and Tunisia). The plant is a perennial
herb growing on calcareous rocks and is characterized by brown or
reddish flowers. Inflorescences are grouped in short and axillary cymes,
in the upper portion of the stems (Quezel and Santa, 1963). Neither
phytochemical nor pharmacological studies have been reported in the
literature to date for this plant.
class with a cosmopolitan distribution (Robbrecht, 1988). According to
recent phylogenetic molecular studies, this family is divided into three
subfamilies, Rubioideae, Cinchonoideae, and Ixoroideae, including
numerous tribes due to high species diversity (Bremer, 2009). The
genus Galium consisting of about 400 herbaceous plant species re-
presents a large genus of the subfamily Rubioideae (tribe Rubieae) that
are world-wide distributed, mostly from the tropics to the temperate
zones (Ehrendorfer et al., 2018, 1976; The Plant List, 2013). Many
Galium species are used in traditional medicine to treat various
pathologies such as epilepsy, hepatitis, phlebo phlogosis, kidney dis-
orders and skin infections and as diuretic and analgesic (Jarić et al.,
In continuing our recent phytochemical investigations on Algerian
plants (Bitam et al., 2008; Bouzergoune et al., 2016; Boumaraf et al.,
2017; Zergainoh et al., 2018; Djebara et al., 2019), we have analyzed
the content of EtOAc and BuOH soluble parts from the hydromethanolic
extract of the aerial parts of G. brunneum collected in Ain Touta region,
Batna (Algeria) in May of 2012 (Smadi, 2018). The chemical study
revealed a secondary metabolite pattern characterized by flavonoids,
iridoids and lignans, according to the chemistry reported in the litera-
ture for the genus Galium. Three unreported hydroperoxy glycosyl
sterols (1-3, Fig. 1) were found in the EtOAc extract along with a series
of known compounds (4-14, see Supplementary material). Among them
known glycosyl triterpenoids (compounds 7, 8, and 10) with cucurbi-
tane nucleus, that was never reported to date in genus Galium, were
identified. Previously reported compounds (15-25, see Supplementary
material) were isolated from the BuOH extract. Due to moderate anti-
bacterial properties exhibited by the G. brunneum extracts against
Gram-positive and Gram-negative bacteria strains, selected main
2
2
007; Chinese Materia Medica, 1977; Bolivar et al., 2011; Shah et al.,
006; Kaval et al., 2014). A large number of phytochemical investiga-
tions have been conducted on several Galium species from different
geographical areas including Algeria. These studies have reported the
occurrence of iridoids, triterpenoids, anthraquinones, lignans and fla-
vonoids displaying a variety of interesting biological (Martins and
Nunez (2015); Bradic et al., 2018; Mocan et al., 2016; Chaher et al.,
2
016; Camero et al., 2018; Gaamoune et al., 2014).
Galium brunneum Munby is a species with a few records limited to
⁎
Corresponding author.
E-mail address: lciavatta@icb.cnr.it (M.L. Ciavatta).
https://doi.org/10.1016/j.phytol.2020.05.002
Received 16 December 2019; Received in revised form 6 May 2020; Accepted 11 May 2020
1874-3900/ © 2020 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.

A. Smadi, et al.
Phytochemistry Letters 38 (2020) 39–45
Fig. 1. Structures of hydroperoxy sterols 1-3 and related known sterol 9 from G.brunneum.
metabolites 11 and 13 from EtOAc and compounds 15, 16, and 18–22
spectra (Tables 1 and 2) confirmed the presence of an additional hy-
droperoxy function in all three compounds and revealed that the
structures of 1–3 differed from 9 only in the fragment C-4/C-7 being the
remaining part of the molecules the same as 9.
from BuOH extracts were evaluated for this activity.
2
. Results and discussion
Compound 1 was determined to be (24R)-24-ethyl-7α-hydroperoxy-
cholest-5-en-3-O-β-D-glucopyranoside. The double bond was located at
C-5/C-6 the same as 9 and the hydroperoxy substituent was positioned
The extracts of dried aerial parts of G. brunneum, obtained as de-
scribed in details in Experimental, were analyzed by TLC chromato-
graphy using different eluent systems. Only EtOAc and BuOH extracts
were considered in this study. Aliquots of these extracts were submitted
1
1
at C-7 as it was easily evidenced by analysis of the H- H COSY spec-
trum. In fact, the sequence H
ring B an additional deshielded proton at δ
carbon bearing the –OOH substituent was observed to be coupled with
both olefinic proton H-6 (δ 6.07, dd, J = 4.7, 1.5 Hz) and angular
methine H-8 (δ 1.60, m) (Table 1). Consistent with this, the C-NMR
spectrum (Table 2) of 1 contained a CH signal at δ 77.8 (C-7) replacing
2
-1/H
2
-4 was the same as in 9 whereas in
to a series of sequential fractionation steps on SiO
2
, LH-20, C18 chro-
H
4.36 (H-7) attached to a
matography as well as HPLC (see Experimental). Twenty-five pure
compounds belonging to different structural classes and including three
unprecedented hydroperoxy sterols 1-3 (Fig. 1) were finally obtained.
Known metabolites 4–25 were identified by comparison of their
spectroscopic data (NMR, MS and optical rotation) with the literature
as: syringaresinol (4) (Dickey, 1958; Abe and Yamauchi, 1988), bala-
nophonin (5) (Haruna et al., 1982; Sy and Brown, 1999), lariciresinol
H
1
3
H
C
the CH
2
signal at δ 32.0 observed in the carbon spectrum of 9.
C
The α-orientation of 7-OOH was inferred by analysis of H-7 multi-
plicity (dd, J = 4.0, 4.0 Hz) which is consistent with an equatorial
1
3
(
6) (Badawi et al., 1985; Wang et al., 2010), hexa-nor-cucurbitacin D 2-
orientation. In addition, the C-NMR values of C-9 (δ 43.9) and C-14
C
O-β-D-glucoside (7) (Chen et al., 2009), deacetoxycucurbitacin B 2-O-β-
D-glucoside (8) (Stuppner and Wagner, 1989), β-sitosterol-3-β-O-D-
glucoside (9) (Sakakibara et al., 1983; Cheng and Cao (1992)), cu-
curbitacin D 2-O-β-D-glucoside (10) (Yamada et al., 1977), phlorizine
(δ
C
49.4) appeared significantly at low ppm with respect to the corre-
50.0 and
sponding carbons in β-sitosterol-3-β-O-D-glucoside (9) (δ
C
56.9, respectively) (Table 2) due to expected γ-gauche effect of the axial
1
13
substituent at C-7. The H- and C-NMR assignment of 1 was in
agreement with that reported for the corresponding 7α–OH derivative
(Chaurasia and Wichti, 1987; Agnihotri et al., 2008).
(
11) (Rui-Lin et al., 1982), pinellic acid (12) (Nagai et al., 2002), da-
phyllosid (13) (Demіrezer et al., 2006), quercetin (14) (Mabry et al.,
970), 10-O-trans-p-coumaroyl-scandoside (15) (Otsuka et al., 1991),
asperulosid (16) (Otsuka et al., 1991), deacetyl asperulosidic acid (17)
1
Compound 2 was identified as (24R)-24-ethyl-5α-hydroperoxy-
6
,7
cholest-6-en-3-O-β-D-glucopyranoside. It exhibited the
ubstituted double bond and 5–OOH group as it was
indicated by two double doublets of an AB system at δ
Δ
dis-
(
El-Naggar and Beal, 1980), an inseparable mixture of dihydro-de-
hydro-diconiferyl alcohol 4-O-β-D-glucoside (18) (Ouyang et al., 2011)
and dehydro-diconiferyl alcohol 4-O-β-D-glucoside (19) (Salama et al.,
H
5.92 (J =
10.0 and 2.5 Hz, H-6) and δ
H
5.68 (J = 10.0 and 1.5 Hz, H-7), in the
1
1
981), isorhamnetin 3-O-β-rutinoside (20) (Ogawa et al., 2001), quer-
H-NMR spectrum, as well as two CH signals at δ
C
131.8 (C-6) and
1
3
cetin 3-O-β-rutinoside (21) (Mabry et al., 1970), phloretin 3′,5′-di-C-β-
D-glucoside (22) (Ogawa et al., 2001), (7S,8R,8′R)-(-)-lariciresinol
133.6 (C-7) and a C signal at δ
C
82.8 (C-5), in the C-NMR spectrum.
This arrangement was confirmed by diagnostic HMBC correlations be-
tween C-5 and H -19 (δ 0.92), H -1b (δ 1.30), H -4a (δ 3.16), H-6
(δ 5.92) and H-7 (δ 5.68). The α-configuration of 5-OOH substituent
was suggested from the signals observed in the C-NMR for C-6 (δ =
4
−4'-bis-O-D-glucoside (23) (El-Gamal et al., 1997), deacetylasper-
3
H
2
H
2
H
ulosid (24) (Otsuka et al., 1991), and, finally, deacetyldaphyllosid (25)
H
H
1
3
(
Otsuka et al., 1991). The structure of unprecedented hydroperoxy
C
sterols 1–3, established by detailed spectroscopic analysis, mainly 1D
131.8) and C-7 (δ = 133.6) that were comparable with those reported
C
and 2D-NMR techniques, was described as follows.
for the corresponding 5α-OH derivative (Agnihotri et al., 2008; Holland
and Jahangir, 1983).
The structure of compound 3 was established as (24R)-24-ethyl-6β-
2.1. Structure elucidation of compounds 1-3
hydroperoxy-cholest-4-en-3-O-β-D-glucopyranoside. The sequence H -
2
1
1
1
1/H-4 was easily deduced by H- H COSY experiment allowing the
location of the double bond at C-4. In fact H-3 (δ 4.48, m) showed a
diagnostic vicinal correlation with vinyl proton H-4 (δ 6.18, br s)
along with the expected coupling with H -2 (δ 2.16 and 1.90). The
hydroperoxy substituent was positioned at C-6 (δ 75.0, δ 4.63) as it
Preliminary H-NMR analysis of compounds 1–3, conducted in both
pyridine-d
5
and methanol-d , clearly indicated that all three compounds
4
H
belong to the sterol class being oxidized derivatives of co-occurring β-
H
sitosterol-3-β-O-D-glucoside (9). The HR-ESI-MS spectra of 1–3 showed
2
H
+
the same sodium adduct ion peak at 631.4187 m/z (M + Na) corre-
C
H
was evidenced by analysis of COSY spectrum where H-6 was coupled to
sponding to the molecular formula C35
H
60
8
O with two oxygen atoms
methylene H
2
H
-7 (δ 1.92 and 1.11) further correlated to angular me-
H
more with respect to sterol 9 thus suggesting the presence of a hydro-
peroxy function in their structures. Comparison of 1H- and 13C-NMR
thine H-8 (δ 1.65). Long-range correlations observed in the HMBC
40

A. Smadi, et al.
Phytochemistry Letters 38 (2020) 39–45
Table 1
Table 2
1
H NMR dataa in Pyr-d
13
C NMR dataa in Pyr-d
5
for compounds 1-3 and 9.
5
for compounds 1-3 and 9.
2 δ , m 3 δ
b
9b
1
2
3
9
C
1 δ
C
, m
C
C
, m
C
δ , m
δ
H
, m (J in Hz)
δ
H
, m (J in Hz)
δ
H
, m (J in
H
δ , m (J in Hz)
Hz)
1
37.7, CH
30.1, CH
2
2
29.2, CH
29.5, CH
75.2, CH
33.7, CH
82.8, C
2
2
36.9, CH
27.7, CH
72.9, CH
2
2
37.3, CH
30.0, CH
77.9, CH
39.1, CH
140.9, C
2
2
2
1
2
1.71, m
.07, m
2.08, m
1.78, m
1.30, m
2.30, m
1.95, m
4.85, m
3.16, dd
(13.0, 4.5)
1.78, m
1.82, m
1.02, m
2.16, m
1.90, m
4.48, m
6.18, br s
1.73,m
3
78.53, CH
1
0.99, m
4
39.5, CH
146.5, C
2
2
129.6, CH
143.3, C
75.0, CH
2
2.14, m
5
1
.73, m
1.75, m
6
122.7, CH
77.8, CH
37.4, CH
43.9, CH
37.8, C
131.8, CH
133.6, CH
39.14, CH
44.3, CH
38.7, C
121.7, CH
3
4
3.84, m
2.77, dd
3.93, m
7
39.9, CH
30.1, CH
54.6, CH
37.0, C
2
32.0, CH
31.8, CH
50.1, CH
37.3, C
2
2.71, br dd 13.0, 2.4
8
(
13.0, 4.5)
9
5
6
7
2.53, dd
13.0, 11.7)
6.07, dd
4.7, 1.5)
4.36, dd
2.46, dd 13.0, 11.0
5.34, br s
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
1′
(
20.0, CH
39.5, CH
42.5, C
2
2
21.2, CH
40.3, CH
43.7, C
2
2
21.3, CH
40.0, CH
42.7, C
2
2
21.1, CH
39.7, CH
42.3, C
2
2
5.92, dd
(10.0, 2.5)
5.68, dd
(10.0, 1.5)
1.88, m
4.63, m
(
1.92, m
1.11, m
1.65, m
0.80, m
1.41, m
1.90, m
1.54, m
1.38, m
0.91, m
1.43, m
49.4, CH
24.7, CH
28.6, CH
56.1, CH
11.5, CH
24.7, CH
36.4, CH
18.2, CH
34.2, CH
26.3, CH
46.1, CH
29.5, CH
19.1, CH
19.2, CH
23.4, CH
12.2, CH
53.8, CH
26.4, CH
28.6, CH
56.1, CH
12.2, CH
15.4, CH
36.4, CH
18.9, CH
34.2, CH
24.0, CH
46.1, CH
29.4, CH
19.9, CH
19.2, CH
23.4, CH
12.2, CH
56.5, CH
25.0, CH
28.2, CH
56.3, CH
11.9, CH
20.0, CH
36.8, CH
18.9, CH
34.0, CH
26.9, CH
46.1, CH
30.0, CH
19.3, CH
19.3, CH
23.0, CH
11.8, CH
56.6, CH
24.3, CH
28.3, CH
56.0, CH
11.7, CH
19.2, CH
36.1, CH
19.0, CH
34.0, CH
26.2, CH
45.8, CH
29.2, CH
19.7, CH
18.8, CH
23.2, CH
11.9, CH
(
4.0, 4.0)
2
2
2
2
2
2
2
2
8
9
1
1.60, m
1.62, m
1.43, m
1.97, m
1
2
1.30, m
3
3
3
3
3
3
3
3
1
.18, m
1
1.97, m
1.90, m
0.99, m
0.99, m
1.21, m
2.18, m
1.10, m
1.01, m
1.63, m
1.98, m
1.11, m
0.97, m
1.57, m
1.05, m
1.85, m
1
.14, m
3
2
2
3
2
2
3
2
2
3
2
2
1
1
4
5
1.84, m
2.15, m
1
.23, m
1
6
1.87, m
1.24, m
1.82, m
1
.26, m
3
3
2
3
3
3
2
3
3
3
2
3
3
3
2
3
1
1
1
2
2
2
7
8
9
0
1
2
1.13, m
0.68, s
1.02, m
0.64, s
1.10, m
0.70, s
1.11, m
0.66, s
0.91, s
0.92, s
1.33, s
0.94, s
1.40, m
0.96, d (6.2)
1.08, m
1.36, m
0.93, d (6.2)
1.36, m
1.37, m
0.98, d (6.2)
1.38, m
1.40, m
0.99, d (6.6)
1.41, m
1.09, m
1.25, m
102.9, CH
75.3, CH
78.6, CH
71.6, CH
78.57, CH
102.9, CH
75.3, CH
78.6, CH
71.6, CH
78.5, CH
103.1, CH
75.2, CH
78.7, CH
71.9, CH
78.6, CH
102.4, CH
75.1, CH
78.4, CH
71.5, CH
78.3, CH
2′
3′
1
.01, m
4′
2
3
1.23, m
1.51, m
1.34, m
5′
1
.14, m
6′
62.9, CH
2
62.9, CH
2
62.8, CH
2
62.6, CH
2
2
2
2
2
2
4
5
6
7
8
0.97, m
0.95, m
0.98, m
1.00, m
a
b
1.65, m
1.67, m
1.67, m
1.67, m
Assignments aided by HSQC edited and HMBC (J =7 Hz) experiments.
0.85, d (6.8)
0.85, d (6.8)
1.26, m
0.88, d (6.8)
0.85, d (6.8)
1.26, m
0.86, d (6.8)
0.86, d (6.8)
1.32, m
0.86, d (6.7)
0.88, d (6.8)
1.29, m
Assignmens in agreement with the literature.
1
3
C-NMR values of side chain carbons of 1–3 with those of 9 (Table 2)
1
.24,m
2
1
2
3
4
5
6
9
′
0.88, t (6.8)
4.93, d (7.7)
4.05, m
0.88, t (6.8)
5.10, d (7.9)
4.05, m
0.88, t (6.8)
5.11, d (7.6)
4.08, m
0.89, t (7.0)
5.05, d (8.0)
4.05, m
led us to assign the same 24R absolute configuration. The configuration
at C-24 of 9 was established by analysis of the carbon chemical shifts in
chloroform-d of the aglicone obtained by MeOH/HCl hydrolysis of 9
that well fitted with the (24R)-epimer of β-sitosterol (Wright et al.,
′
′
4.28, m
4.25, m
4.30, m
4.27, m
′
4.27, m
4.30, m
4.28, m
4.26, m
1
978). Full assignment of proton and carbon NMR values of 1–3 was
′
3.97, m
3.82, m
4.02, m
3.98, m
′
4.58, m
4.44, m
4.60, m
4.55, ddd (12.0, 7.0,
2.0)
made by 2D-NMR experiments and reported in Tables 1 and 2 (in
4
.42, m
4.42, m
pyridine-d
5
) and in Experimental (in methanol-d ).
4
4
.40, dd (12.0, 7.0)
It should be noted that aglycone hydroperoxy sterols from com-
pounds 1 and 2 have been previously reported from the plant Arum
italicum (Della Greca et al., 1994) whereas the aglycone of 3 has never
been isolated from natural sources. Hydroperoxy sterols belong to the
large class of oxysterols that are widely occurring metabolites of ter-
restrial plants (Xu et al., 2013) as well as marine organisms (Sheu et al.,
a
Assignments aided by COSY, HSQC edited, and HMBC (J =7 Hz) experi-
ments.
Assignmens in agreement with the literature.
b
2
spectrum between quaternary sp carbon C-5 (δ
.82 and 1.02), H-3 (δ 4.48), and H -19 (δ 1.33) as well as between
C-4 (δ 129.2) and H-6 (δ 4.63) supported the proposed structure. The
C
143.3) and H
2
-1 (δ
H
1
997) playing crucial roles in numerous biological processes (Otaegui-
1
H
3
H
Arrazola et al., 2010; Kulig et al., 2016). Oxidized sterols have been
shown to be produced by either enzymatic mechanisms or by auto-
xidation that proceeds via free radical or non-radical pathways (Iuliano,
C
H
β-orientation of 6-OOH was suggested by analysis of the coupling
constants of H-6 which resonated as an apparent triplet (J =3.0 Hz)
according to its equatorial orientation. Consistent with this, the spatial
effect of β-oriented 6-OOH was observed on the chemical shift values of
2
011). However, in the case of allylic hydroperoxy sterols, such as 1–3,
the possibility that they are artifacts arising from autoxidation
(
Beckwith et al., 1989) of co-occurring 9 during drying and storage of
H -19 (δ 1.33) as it was evident by comparing NMR values of model
3
the plant seems to be more plausible. The reaction should involve the
abstraction of an allylic proton by an activated oxygen along with mi-
gration of the carbon-carbon double bond (Beckwith et al., 1989). In
any case, the alternative formation as naturally sensitized photo-
oxygenation products of β-sitosterol in the living plant cannot be ruled
out for compounds 1–3.
compounds exhibiting with 6α–OH or 6β–OH substituent (Shi et al.,
008; Zhao et al., 2005, 2003). In addition, due to the γ-gauche effect of
2
1
3
the axial substituent at C-6, the C-NMR value of C-8 appeared at δ
0.1, in agreement with the carbon values reported for 6β–OH related
compounds.
3
The configuration of the anomeric carbon of compounds 1-3 was
Finally, the antibacterial properties of selected main metabolites
isolated from both extracts were evaluated against some human
defined as β from the coupling constant value of H-1′ (J = 7.6–7.8 Hz)
for all of them. Biogenetical considerations as well as comparison of the
41

A. Smadi, et al.
Phytochemistry Letters 38 (2020) 39–45
Table 3
Antibacterial activity of selected compounds of G. brunneum by disk diffusion method.
Gram+
Gram−
Compound
nmol/
disk
Staphylococcus
aureus
Staphylococcus
aureus
Bacillus
Pseudomonas
aeruginosa
ATCC 27853
< 7
Escherichia
coli
Klebsiella
cereus
pneunomoniae
ATCC 70603
7.0 ± 0.0
7.7 ± 0.6
7.7 ± 0.6
7.0 ± 0.0
7.7 ± 0.6
7.5 ± 0.9
7.2 ± 0.3
8.0 ± 0.0
18.0 ± 1.0
ATCC 25923
9.8 ± 0.8
7.8 ± 0.8
7.7 ± 0.6
7.2 ± 0.3
15.2 ± 0.8
7.5 ± 0.5
10.2 ± 0.8
8.0 ± 1.0
14.3 ± 05
ATCC 43300
11.0 ± 0.5
7.0 ± 0.0
7.3 ± 0.6
7.2 ± 0.3
10.7 ± 0.6
7.7 ± 0.6
10.0 ± 0.0
7.7 ± 0.8
15.3 ± 0.5
ATCC 11778
13.8 ± 0.3
9.8 ± 0.8
10.0 ± 0.0
9.2 ± 0.3
10.7 ± 0.6
10.7 ± 0.6
10.0 ± 0.0
10.8 ± 0.8
25.0 ± 1.0
ATCC 25922
8.7 ± 0.6
8.3 ± 0.6
9.8 ± 0.8
10.0 ± 0.0
7.7 ± 0.6
8.7 ± 0.6
10.3 ± 1.2
8.2 ± 0.3
15.0 ± 1.0
1
1
1
1
1
2
2
2
1
23
21
19
24
19
16
16
17
8.6
3
< 7
5
< 7
6
< 7
8 + 19
< 7
0
1
2
< 7
< 7
< 7
Streptomycin
21.6 ± 0.5
pathogenic Gram-positive and Gram-negative bacteria strains using the
disk diffusion method. Pure compounds 11, 13, 15, 16, and 20–22
along with the inseparable mixture of 18 and 19 were tested (Table 3).
Compound 11 was the most active compound against three Gram-po-
sitive bacteria. Due to the scarce amount available, new hydroperoxy
sterols 1–3 were not assayed.
evaporated in vacuo to give 500 mL of aqueous phase that was ex-
tracted liquid–liquid subsequently by petroleum ether (300 mL × 3),
EtOAc (300 mL × 3) and BuOH (300 mL × 3). The organic phases were
evaporated to give the corresponding extracts (7.0 g, 10.0 g and 35.0 g,
respectively).
An aliquot (7.0 g) of the EtOAc extract was chromatographed on a
SiO
2
gel column using hexane/EtOAc gradient (100:0 to 0:100, v/v)
and CHCl
3
/MeOH (70:30 to 0:100, v/v), to afford 12 fractions
3
. Experimental
(
FrEtOAcA-L). Selected fractions containing compounds of interest were
considered for subsequent purification steps. Fraction FrEtOAcC was
fractionated by Sephadex LH-20 and eluted in isocratic mode with
3.1. General experimental procedures
CHCl /MeOH (1:1), to give three subfractions (FrEtOAcC1-C3).
3
Optical rotations were measured on a Jasco DIP 370 digital po-
Subfraction FrEtOAcC2 was subjected to C18 cartridge (SPE) and eluted
with a gradient of MeOH in H O (from 0 to 100%) to afford 3 sub-
fractions [FrEtOAcC-I–C-III)]. Subfraction FrEtOAcC-III was purified by
preparative TLC using CHCl -MeOH (95/0.5) mixtures to give com-
pounds 4 (1.8 mg), 5 (1.2 mg), and 6 (1.5 mg). Fraction FrEtOAcE was
submitted to a SiO gel column eluted with a CHCl /MeOH gradient to
aff ;ord 11 subfraction (FrEtOAcE1-E11). Subfraction FrEtOAcE4 was
larimeter. LRESI-MS were performed on a Micromass Q-TOF MicroTM
coupled with a HPLC Waters Alliance 2695. The instrument was cali-
brated by using a PEG mixture from 200 to 1000 MW (resolution spe-
cification 5000 FWHM, deviation < 5 ppm RMS in the presence of a
known lock mass). High resolution mass spectra (HRESI-MS) were ac-
quired on a Q-Exactive hybrid quadrupole-orbitrap mass spectrometer
2
3
2
3
(
Thermo Scientific). NMR experiments were recorded at ICB-NMR
purified on a C18 cartridge (SPE) by using a gradient of MeOH in H O
2
Service Center. Chemical shifts values are reported in ppm and refer-
1
to get 7 subfractions (FrEtOAcE4-I-E4-VII). Subfractions FrEtOAcE-II,
FrEtOAcE-IV, and FrEtOAcE-VII were purified on Sephadex LH-20 CC
enced to internal signals of residual protons (C
5
D
5
N, H δ 7.19, 7.55,
1
3
1
13
8
.71, C 123.5, 135.5, 149.9 ppm; CD
3
OD, H δ 3.34, C 49.0 ppm).
eluting with CHCl /MeOH (1:1) to provide compounds 7 (3.0 mg), 8
3
1
D- and 2D-NMR spectra were acquired on a Bruker Avance- 400 op-
(
1.0 mg), and 9 (10.0 mg), sequentially. Subfraction FrEtOAcE-V was
further purified by reversed-phase HPLC (C18 Kinetex PFP, 4.6 × 250
mm) with a gradient of MeOH in H O from 50% to 100% to yield
compound 10 (1.0 mg, R 29.8 min). Fraction FrEtOAcE5 was chroma-
erating at 400 MHz, using an inverse probe fitted with a gradient along
the Z-axis, a Bruker Avance III HD spectrometer equipped with a
CryoProbe Prodigy operating at 400 MHz, and a Bruker DRX-600 op-
erating at 600 MHz, using an inverse TCI CryoProbe fitted with a gra-
dient along the Z axis. HPLC separation was performed on a Shimadzu
high-performance liquid chromatography using a Shimadzu liquid
chromatograph LC-10AD equipped with an UV SPD-10A wavelength
detector, with a reversed-phase column (Kinetex PFP, Phenomenex, 4.6
mm i.d. × 250 mm). Silica-gel chromatography was performed using
pre-coated Merck F254 plates (TLC) and Merck Kieselgel 60 powder
2
t
tographed on a C18 cartridge (SPE) using MeOH-H O gradient to give
2
five subfractions (FrEtOAcE5-I-E5-V). Subfraction FrEtOAcE5-V was fur-
ther purified by reversed-phase HPLC (C18 Kinetex PFP, 4.6 × 250
mm) with a MeOH in H O gradient (from 95% to 100% in 40 min) to
2
yield hydroperoxy sterols 1 (0.8 mg, R
min), and 3 (0.5 mg, R 17.8 min). Subfracion FrEtOAcE11 was purified
on a C18 cartridge (SPE) by using a MeOH/H O gradient to get com-
pounds 11 (11.0 mg) and 12 (4.0 mg). Fraction FrEtOAcG was fractio-
nated on VLC C8 eluted with MeOH/ H O gradient to give 7 subfrac-
tions (FrEtOAcG1–G7). Subfraction FrEtOAcG1 was subjected to CC on
C18 cartridge (SPE) using MeOH/H O gradient and following pur-
/MeOH, 1:1) to give compounds
t
11.9 min), 2 (2.0 mg, R 15.2
t
t
(
70–230 mesh). The spots on TLC were visualized under UV light (254
2
nm) and then spraying them with 10% H
heating.
2
SO in water followed by
4
2
2
3
3.2. Plant material
ification on CC Sephadex LH-20 (CHCl
1
3 (3.0 mg) and 14 (8.0 mg). A third of the BuOH extract (10 g) was
The aerial parts of G. brunneum were collected in Ain Touta region,
chromatographed on a SiO
2
gel column using a gradient of MeOH in
Batna (Algeria) in May 2012 and identified by Prof. Bachir Oudjehih,
Institute of Agronomy and Veterinary Sciences, University of Batna 1
CHCl (from 0 to 100%) to afford 13 fractions (FBuOHA-M). Selected
3
fractions containing compounds of interest were considered for further
purification steps. Fraction FrBuOHD was further purified on a SiO gel
column using the same conditions as above to give 7 subfractions
FrBuOHD1–D7). Subfraction FrBuOHD2 and subfraction FrBuOHD5 were
(
Algeria). A voucher specimen is deposited in the herbarium of the
2
department of the same University with code 05/ISAV/DAG/2014.
(
3.3. Extraction and isolation of compounds
purified by Sephadex LH-20 and eluted in isocratic mode with CHCl -
3
MeOH (1/1) to give compounds 15 (40.6 mg) and 16 (47.0 mg), re-
Dried aerial parts (1 kg) were macerated with MeOH/H O, 8:2 (10 L
2
spectively. Fraction FrBuOHE was subjected to open CC on normal phase
×
3), at room temperature. After filtration, the organic solvent was
42

A. Smadi, et al.
Phytochemistry Letters 38 (2020) 39–45
silica gel using CHCl
3
/MeOH gradient as eluent. Eight fractions were
(CH, C-20), 35.0 (CH
2
, C-22), 33.4 (CH , C-4), 30.3 (CH, C-25), 30.0
2
collected (FrBuOHE1–E8). Subfraction FrBuOHE5 was purified by
(CH
(CH
(CH
(CH
2
2
3
3
, C-1), 29.5 (CH
2
, C-16), 29.3 (CH
2
, C-2), 27.1 (CH , C-23), 25.0
2
Sephadex LH-20 (CHCl
Subfraction FrBuOHE7 was fractionated on C18 cartridge (SPE) eluted
with a gradient of MeOH in H O (from 0 to 10%) to give an inseparable
mixture (6.0 mg) constituted by 18 and 19. Selected fractions FrBuOH
and FrBuOHL were combined and subjected to SiO gel column (CHCl
MeOH 99: 0.1 to 90:10) and Sephadex LH-20 column (CHCl /MeOH,
3
/MeOH, 1:1) to yield compound 17 (4.0 mg).
, C-15), 24.3 (CH
2
3
, C-28), 22.1 (CH
, C-21), 15.7 (CH
2
3
, C-11), 20.3 (CH
3
3
, C-26), 19.8
, C-18), 12.3
, C-27), 19.4 (CH
, C-19), 12.5 (CH
+
2
, C-29); ESI-MS (pos. ion mode) m/z 631 [M + Na] ; HR-ESI-MS
+
I
m/z 631.4186 [M + Na] (calcd for C35
H
60
8
O Na 631.4180).
2
3
/
(24R) 24-ethyl-6β-hydroperoxy-cholest-4-en-3-O-β-D-glucopyrano-
2
5
1
13
3
side (3): White powder; [α]
(Pyr-d
D
1
+21.8 (c 0.05, MeOH); H- and C-NMR
1
:1), sequentially, obtaining twelve subfractions (FrBuOHIL1-IL12).
5
) see Table 1 and 2; H-NMR spectral data (in MeOD, 600 MHz)
Subfractions FrBuOHIL6, FrBuOHIL7, and FrBuOHIL8 contained pure
compounds 20 (40 mg), 21 (32 mg), and 22 (16.0 mg), respectively.
Finally, selected subfracions FrBuOHIL9-12 were further purified by C18
δ: 5.79 (1H, br s, H-4), 4.46 (1H, d, J=7.8 Hz, H-1′), 4.30 (1H, m, H-3),
4.28 (1H, dd, J = 3.0, 3.0 Hz, H-6), 3.89 (1H, dd, J = 12.0, 2.0 Hz, H-
6′a), 3.69 (1H, dd, J = 12.0, 5.4 Hz, H-6′b), 3.39 (1H, m, H-3′), 3.32
(1H, m, H-4′), 3.30 (1H, m, H-5′), 3.20 (1H, m, H-2′), 2.16 (1H, m, H-
SPE cartridge using a MeOH/H O gradient to give compounds 23 (8.0
2
mg), 24 (2.0 mg), and 25 (4.0 mg).
.4. Spectroscopic data
24R) 24-ethyl-7α-hydroperoxy-cholest-5-en-3-O-β-D-glucopyrano-
7a), 2.07 (1H, m, H-12a), 2.05 (1H, m, H-2a), 1.90 (2H, m, H -16), 1.77
2
(
2H, m, H
(2H, m, H
H-22a), 1.36 (1H, m, H-28a), 1.32 (1H, m, H-23a), 1.26 (1H, m, H-28b),
1.23 (1H, m, H-23b), 1.20 (3H, s, H -19), 1.18 (1H, m, H-12b), 1.16
(1H, m, H-17), 1.10 (1H, m, H-7b), 1.06 (1H, m, H-22b), 1.02 (1H, m,
H-14), 0.98 (1H, m, H-24), 0.97 (3H, d, J = 6.6, H -21), 0.90 (3H, t, J
= 7.4, H -29), 0.89 (3H, d, J = 6.9, H -27), 0.87 (3H, d, J = 6.9, H -
2
-1), 1.76 (1H, m, H-2b), 1.72 (2H, m, H-8 and H-25), 1.66
3
2
-15), 1.51 (2H, m, H -11), 1.43 (1H, m, H-20), 1.41 (1H, m,
2
(
3
2
1
5
1
13
side (1): White powder; [α]
D
– 23.5 (c 0.05, MeOH); H- and C-NMR
(
Pyr-d
δ: 5.79 (1H, br dd, J = 4.9, 1.3 Hz, H-6), 4.43 (1H, d, J=7.9 Hz, H-1′),
.08 (1H, dd, J = 4.9, 4.0 Hz, H-7), 3.91 (1H, m, H-6′a), 3.70 (1H, m,
H-3), 3.69 (1H, m, H-6′b), 3.37 (1H, m, H-3′), 3.30 (1H, m, H-5′), 3.28
1H, m, H-4′), 3.18 (1H, m, H-2′), 2.54 (1H, dd, J = 13.0, 4.5 Hz, H-4a),
.37 (1H, dd, J = 13.0, 11.0 Hz, H-4b), 2.15 (1H, m, H-15a), 2.04 (1H,
5
) see Table 1 and 2; H-NMR spectral data (in MeOD, 600 MHz)
3
3
3
3
3
1
3
4
26), 0.79 (1H, m, H-9), 0.76 (3H, s, H -18); C-NMR spectral data (in
MeOD, indirect detection from HSQCed and HMBC) δ: 144.9 (C, C-5),
129.7 (CH, C-4), 102.6 (CH, C-1′), 87.7 (CH, C-6), 77.8 (CH, C-3′), 77.7
(
2
(CH, C-5′), 76.5 (CH, C-3), 74.8 (CH, C-2′), 71.5 (CH, C-4′), 62.8 (CH ,
2
m, H-12a), 1.96 (1H, m, H-23a), 1.93 (1H, m, H-2a), 1.89 (2H, m, H
2
-
C-6′), 57.4 (2 x CH, C-14 and C-17), 55.6 (CH, C-9), 47.3 (CH, C-24),
41.4 (C, C-13), 41.0 (CH , C-12), 37.8 (C, C-10), 37.7 (CH , C-1), 36.5
, C-22), 30.6 (CH, C-8), 30.4 (CH,
, C-2), 26.4 (CH , C-23), 25.0 (CH
, C-19), 19.7 (2 x
, C-18 and C-29);
1
1
), 1.72 (1H, m, H-25), 1.63 (1H, ddd, J = 11.7, 11.7, 4.0 Hz, H-8),
.53 (2H, m, H -11), 1.51 (1H, m, H-14), 1.42 (1H, m, H-20), 1.41 (1H,
-15 and H -28), 1.30 (1H,
2
2
2
(CH
2
, C-7), 37.2 (CH, C-20), 34.4 (CH
2
m, H-9), 1.40 (1H, m, H-22a), 1.32 (4H, m, H
2
2
C-25), 28.9 (CH
C-15), 23.8 (CH
2
2
, C-16), 27.8 (CH
, C-28), 21.6 (CH
2
2
2
2
,
m, H-2b), 1.21 (2H, m, H
2
-16), 1.20 (1H, m, H-22b), 1.19 (1H, m, H-
, C-11), 20.3 (CH
3
3
1
0
0
7), 1.18 (1H, m, H-12b), 1.15 (1H, m, H-23b), 1.04 (3H, s, H
3
-19),
3
CH
3
, C-26 and C-27), 19.0 (CH
3
, C-21), 12.2 (2 x CH
+
.99 (3H, d, J =6.5 Hz, H
3
-21), 0.97 (1H, m, H-24), 0.73 (3H, s, H
-18),
ESI-MS (pos. ion mode) m/z 631 [M + Na] ; HR-ESI-MS m/z 631.4191
.88 (3H, d, J =7.0 Hz, H
3
-27), 0.87 (3H, d, J =7.0 Hz, H
3
-26), 0.90
[M + Na]+ (calcd for C35
H
60
8
O Na 631.4180).
1
3
(
3H, t, J =7.5 Hz, H
3
-29); C-NMR spectral data (in MeOD, 100 MHz)
δ: 148.4 (C, C-5), 122.4 (CH, C-6), 102.5 (CH, C-1′), 79.4 (CH, C-3),
3.5. Hydrolysis of β-sitosterol-3-β-O-D-glucoside (9)
7
9.2 (CH, C-7), 78.1 (CH, C-3′), 77.9 (CH, C-5′), 75.1 (CH, C-2′), 71.7
CH, C-4′), 62.8 (CH , C-6′), 57.2 (CH, C-17), 50.3 (CH, C-14), 47.3
CH, C-24), 44.9 (CH, C-9), 43.4 (C, C-13), 40.7 (CH , C-12), 39.8 (CH
, C-1), 37.8 (C, C-10), 37.5 (CH, C-20),
, C-22), 30.4 (CH, C-25), 29.4 (CH , C-2), 27.2 (CH , C-16),
, C-23), 24.2 (CH , C-15 and C-28), 22.0 (CH , C-11), 20.2
, C-27), 19.36 (CH , C-21), 18.6 (CH , C-19),
, C-29), 11.7 (CH , C-18); ESI-MS (pos. ion mode) m/z 631 [M
(
(
2
Compound 9 (2.5 mg) was dissolved in 1 mL of 1 N HCl in MeOH,
2
2
,
and the obtained solution was stirred for 12 h at 60 °C. After the usual
C-4), 38.4 (CH, C-8), 38.2 (CH
2
workup, the reaction mixture was dried and partitioned between CHCl
3
3
5.1 (CH
5.3 (CH
2
2
2
2
and H O. The organic and aqueous layers were separately dried and
2
2
2
2
subjected to NMR analysis. The proton spectrum of the organic part
indicated to contain 3-β-sitosterol, whereas the aqueous fraction
showed to be a mixture of. Benzoyl chloride (0.5 mL) was added to a
dry pyridine solution (1 mL) of the α- and β-methylglucopyranosyl
mixture, and the reaction was stirred for 12 h at room temperature.
After removal of the solvent under reduced pressure, the usual workup
afforded α- and β-methyl-tetra-benzoyl-glucopyranoses in ratio of 2:1.
The mixture was purified on a Pasteur pipette silica gel column (light
petroleum ether/diethyl ether gradient) to give 0.8 mg and 0.4 mg of
pure α- and β-tetra-benzoate which were identified by comparison with
literature data (Gavagnin et al., 2007).
(
CH
3
, C-26), 19.41 (CH
3
3
3
1
2.3 (CH
3
3
+
+
+
Na] ; HR-ESI-MS m/z 631.4191 [M + Na] (calcd for C35
H
60
8
O Na
6
31.4180).
(
24R) 24-ethyl-5α-hydroperoxy-cholest-6-en-3-O-β-D-glucopyrano-
25 1 13
side (2): White powder; [α]
D
– 9.2 (c 0.2, MeOH); H- and C-NMR
1
(
Pyr-d
5
) see Table 1 and 2; H-NMR spectral data (in MeOD, 600 MHz)
δ: 5.78 (1H, dd, J = 10.0, 1.8 Hz, H-7), 5.64 (1H, dd, J = 10.0, 2.7 Hz,
H-6), 4.44 (1H, d, J=7.9 Hz, H-1′), 4.18 (1H, m, H-3), 3.90 (1H, br d, J
=
11.0 Hz, H-6′a), 3.70 (1H, dd, J = 11.0, 4.0 Hz, H-6′b), 3.39 (1H, m,
H-3′), 3.31 (2H, m, H-4′ and H-5′), 3.17 (1H, m, H-2′), 2.52 (1H, dd, J
13.0, 5.0, H-4a), 2.07 (1H, m, H-12a), 1.99 (1H, m, H-8), 1.98 (1H,
m, H-2a), 1.95 (2H, m, H -16), 1.82 (1H, m, H-9), 1.75 (4H, m, H -15),
.71 (1H, m, H-2b), 1.70 (1H, m, H-25), 1.57 (1H, m, H-1a), 1.50 (1H,
m, H-4b), 1.42 (2H, m, H-20 and H-22a), 1.39 (2H, m, H -11), 1.37
1H, m, H-1b), 1.32 (2 H m, H -28), 1.31 (1H, m, H-14), 1.24 (2H, m,
-23), 1.21 (1H, m, H-12b), 1.19 (1H, m, H-17), 1.08 (2H, m, H-22b),
.00 (3H, s, H -19), 0.97 (3H, d, J =6.5 Hz, H -21), 0.96 (1H, m, H-24),
.90 (3H, t, J =7.5 Hz, H -29), 0.88 (3H, d, J =7.0 Hz, H -26), 0.86
-27), 0.76 (3H, s, H -18); C-NMR spectral data
=
3.6. Antimicrobial activity
2
2
1
Six strains of bacteria were used as test microorganisms. The bac-
terial strains included Gram-positive Staphylococcus aureus ATCC
25923, Staphylococcus aureus ATCC 43300 and Bacillus cereus ATCC
11778 and Gram-negative Pseudomonas aeruginosa ATCC 27853,
Escherichia coli ATCC 25922 and Klebsiella pneunomoniae ATCC 70603.
The modified agar diffusion method was performed as described by
Hossain et al. (Hossain et al., 2012) with some modifications. A stan-
2
(
2
H
2
1
0
3
3
3
3
1
3
(
3H, d, J =7.0 Hz, H
3
3
7
(
in MeOD, indirect detection from HSQCed and HMBC) δ: 135.0 (CH, C-
), 131.6 (CH, C-6), 102.9 (CH, C-1′), 84.0 (C, C-5), 78.2 (2 x CH , C-3′
, C-
′), 57.5 (CH, C-17), 55.1 (CH, C-14), 47.5 (CH, C-24), 45.5 (CH, C-9),
4.3 (C, C-13), 41.4 (CH , C-12), 40.5 (CH, C-8), 38.6 (C, C-10), 37.3
dardized bacterial suspension was adjusted to a density of 1.0 × 10
−
1
7
2
UFC mL
from an overnight culture and poured into sterile petri
and C-5′), 75.8 (CH, C-3), 75.3 (CH, C-2′), 71.7 (CH, C-4′), 62.8 (CH
2
dishes containing Mueller Hinton agar (MHA) using a cotton swab.
EtOAc and BuOH extracts as well as pure compounds were dissolved in
DMSO at a concentration of 500 μg/mL and sterilized by filtration
6
4
2
43

A. Smadi, et al.
Phytochemistry Letters 38 (2020) 39–45
through 0.22 μm sterile Millipore filters; then 10 μl of these solutions
were spotted onto sterile filter paper disks (6 mm in diameter,Whatman
no 1) and deposited in the center of the petri dish. After 24 h of in-
cubation at 37 °C, the inhibition zones were measured in mm. Strep-
tomycin 10 μg/dikc was used as a positive control for the tested bac-
teria, and dimethyl sulfoxide (DMSO) as negative control. All
experiments were performed in triplicate.
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Acknowledgements
The authors thank Mr. A. Esposito of ICB-NMR service and Mrs. D.
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