Chemical constituents and their antibacterial and antifungal activity from the Egyptian herbal medicine Chiliadenus montanus

Article abstract of DOI:10.1016/j.phytochem.2014.03.027

Phytochemical investigations of the CH₂Cl₂/MeOH (1:1) extract of air-dried aerial parts of Chiliadenus montanus afforded eight metabolites, in addition to eight other previously reported compounds, of which two were isolated for the first time as free acids. Structures were established by spectroscopic methods, including HREIMS, ¹H, ¹³C, DEPT, ¹H-¹H COSY, HMQC and HMBC NMR analysis. Antimicrobial activity against an array of common bacterial and fungal strains was measured via a colorimetric assay with minimal growth inhibition observed in the μg/mL range for one of the tested metabolites.

Full text of DOI:10.1016/j.phytochem.2014.03.027

Phytochemistry xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Chemical constituents and their antibacterial and antifungal activity
from the Egyptian herbal medicine Chiliadenus montanus
Mohamed-Elamir F. Hegazy a, Hisashi Matsuda b, Seikou Nakamura b, Taha A. Hussein c,
Masayuki Yoshikawa b, Paul W. Paré d,
⇑
a Chemistry of Medicinal Plants Department, and Center of Excellence for Advanced Sciences, National Research Centre, El-Tahrir St., Dokki, Giza 12622, Egypt
b Kyoto Pharmaceutical University, Misasagi, Yamashina-ku, Kyoto 607-8412, Japan
c Delta University for Science and Technology, Pharmacognosy Department, Faculty of Pharmacy, Gamasa, Egypt
d Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX 79409, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Phytochemical investigations of the CH2Cl2/MeOH (1:1) extract of air-dried aerial parts of Chiliadenus
montanus afforded eight metabolites, in addition to eight other previously reported compounds, of which
two were isolated for the first time as free acids. Structures were established by spectroscopic methods,
including HREIMS, 1H, 13C, DEPT, 1H–1H COSY, HMQC and HMBC NMR analysis. Antimicrobial activity
against an array of common bacterial and fungal strains was measured via a colorimetric assay with min-
Received 7 November 2013
Received in revised form 7 February 2014
Available online xxxx
Keywords:
Chiliadenus montanus
Asteraceae
imal growth inhibition observed in the
l
g/mL range for one of the tested metabolites.
Ó 2014 Elsevier Ltd. All rights reserved.
Dioxane
Sesquiterpenes
Flavonoids
Antimicrobial activity
Introduction
antiatherogenic, and antioxidant activities (Al-Howiriny et al.,
2005; Hussein, 2011).
The Sinai Peninsula is an epicenter of medicinal plants in the
Arabian Desert with active plant constituents serving as a focal
point for ecologists, taxonomists and phytochemists alike from
around the globe (Abd El-Wahab et al., 2004; Bailey and Danin,
1981; Boulos, 1983; Bown, 1995; Batanouny et al., 1999; Hanafi
and Abdel-Wahab, 2000). Chiliadenus montanus (Vahl.) Brullo
[=Jasonia montana, Varthemia montana (Vahl.) Boiss.], an herb
endogenous to the North Sinai region of Egypt, is a member of
the Asteraceae (Boulos, 2002). Previous phytochemical investiga-
tions of it have established the presence of monoterpenes (Ahmed
and Jakupovic, 1990), sesquiterpenes (Ahmed, 1991; Ahmed and
Jakupovic, 1990; Ahmed et al., 1988; Ahmed et al., 2004; Mah-
moud, 2006; Mohamed, 2007), diterpenes (Al-Howiriny et al.,
2005), triterpenes, sterols (Eid et al., 1987) and flavonoids (Ahmed
et al., 1989; El-Din et al., 1985; Soliman et al., 2009; Zaghloul,
1989). Locally known as Haneida (Täckholm, 1974), this threa-
tened medicinal plant is used as a herbal tea for the treatment of
renal troubles and select chemical components have been
shown to exhibit anti-diabetic, antimicrobial, anti-obesity,
Herein, elucidation of new compounds 1–8 isolated from the
dry aerial parts of C. montanus is reported, in addition to previously
reported compounds 9–16, two of which (11–12) were isolated as
free acids for the first time (Fig. 1). Chemical structures were estab-
lished by comprehensive spectroscopic analysis and comparisons
with previous literature reports when possible. Antimicrobial
activity for all purified metabolites was assayed against an array
of wide-spread bacterial and fungal species.
Results and discussion
The methylene chloride/methanol (1:1) extract of the air-dried
aerial parts of C. montanus was subjected to normal and reversed
phase chromatography to afford a series of metabolites as reported
here, in addition to previously reported compounds 9–16 (Fig. 1).
Compound 1 was isolated as colorless needles, ½a D25
ꢀ
= +8.3 (c 0.3,
CHCl3) and its IR spectrum showed absorption bands at 3250 (OH),
3020 (OH), 1725 (C@O), and 1650 (C@C) cmꢁ1. The low resolution
mass spectrum had a molecular ion peak [M]+ at m/z 410. Its HRE-
IMS exhibited a molecular ion peak [M]+ at m/z 410.1946 (calcd. for
⇑
C
21H30O8: 410.1941). The molecular formula of C21H30O8 was con-
Corresponding author. Tel.: +1 806 742 3062; fax: +1 806 742 1289.
E-mail address: paul.pare@ttu.edu (P.W. Paré).
firmed by 1H and 13C NMR spectroscopic analysis (Table 1). Acid
http://dx.doi.org/10.1016/j.phytochem.2014.03.027
0031-9422/Ó 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Hegazy, M.-E.F., et al. Chemical constituents and their antibacterial and antifungal activity from the Egyptian herbal
medicine Chiliadenus montanus. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.03.027

2
Mohamed-Elamir F. Hegazy et al. / Phytochemistry xxx (2014) xxx–xxx
OH
14
5
14
5
1
1
9
9
R2
R1
OH
13
3
7
3
7
7
11 13
11 13
O
H
12
12
OH
15
15
O
O
OH
OR
4
2
1
R1 = OCH3 R2 =H
R = Glc
R = H
9
3
R
1 = H
R2 = OCH3
R2 = OAc
10 R = CH3
11
R1 = H
OH
12
O
15
15
14
OH
OH
OH
OH
H3CO
12
O
O
12
11
13
1
1
3
5
7
11
9
3
5
13
9
OH
7
5
14
7
O
O
OH
OH
O
OH
O
OH
6
13
8
OH
OH
HO
O
OH
HO
O
H
H
O
H
O
OH
OH
OH
AcO
O
OH
14
OH
O
OH
16
OH
OH
15
Glc: β-D-glucopyranosyl
Fig. 1. Isolated metabolites from Chiliadenus montanus.
hydrolysis generated 3-oxo-
c
-costic acid (9) and a sugar. Seven su-
methoxy group functionality at dH 3.41 (s) was observed in 2.
The relative configuration of C-3 and C-7 was based on comparable
NMR spectroscopic data for a published eudesmane sesquiterpene
gar signals were observed including: H-60 protons that appeared at
dH 3.81 (dd, J = 12.0, 2.0, H-60) and 3.67 (dd, J = 12.0, 5.4, H-60); an
anomeric proton at dH 5.56 (d, J = 7.5 Hz); and other sugar signals
at dH 3.33–3.43 (4H, m). Sugar proton resonances showed correla-
tions in the HMQC spectrum with carbon signals at dC 94.7 (C-10),
72.7 (C-20), 76.8 (C-30), 69.7 (C-40), 77.6 (C-50), and 60.9 (C-60).
(3b,10a)-3-methoxyeudesma-4,11(13)-dien-12-oic acid (Zhang
et al., 2013) equivalent to compound 2 at H-3 (dH 3.41 (s) versus
dH 3.39 (s), respectively) and H-7 (dH 2.54, J = 13, 2.8 versus dH
2.44 J = 11.6, respectively). The relative beta methyl configuration
at C-10 was assigned based on NOESY correlations between H-7a
and H-6a and H-6b and CH3-14b. These spatial correlations
The carbohydrate unit was confirmed to be
HPLC retention time comparison with a -glucose authentic stan-
D-glucose based on
D
dard (retention time 7.1 min) and positive optical rotation. The
established a 3b-methoxy, 7a-H, and a 10b-methyl configuration.
b-anomeric configuration for glucose was determined from a rela-
tively large JH1,H2 coupling constant (J = 7.5 Hz) (Ye et al., 2002).
Based on a correlation in the HMBC spectrum between the ano-
meric resonance at dH 5.56 and a carbonyl group at C-12 (dC
165.5) (Fig. 3), the sugar-terpene linkage was identified and 1
Therefore 2 is assigned to 3b-methoxy isocostic acid, a new natural
3
product.
Compound 3 was isolated as a yellowish oil, (½a D25
= ꢁ13.6 (c
ꢀ
0.03, CHCl3)) and its IR spectrum showed absorption bands at
3020 (OH), 1715 (C@O), and 1670 (C@C) cmꢁ1. The 1H- and 13C-
NMR spectra of 2 and 3 (Table 1) indicated the presence of epimers
with a differing configuration at C-3. Relative to 2, 3 has a down-
field H-3 shift of dH 3.61 appearing as a triplet and H-15 appears
up field at dH 1.67. Coupling constant (J = 6.8 Hz) and NOESY data
was named 3-oxo-c-costic acid b-D-glucopyranoside ester, a new
natural product.
Compound 2 was isolated as a yellowish oil, (½a D25
ꢀ
= +73.0 (c
0.01, CHCl3)) and its IR spectrum showed absorption bands at
3020 (OH), 1715 (C@O), and 1670 (C@C) cmꢁ1. HREIMS exhibited
a molecular ion peak [M]+ at m/z 264.1734 (calcd. for C16H24O3:
264.1725). The 1H- and 13C-NMR spectrum (Table 1) confirmed
the molecular formula C16H24O3 and carbon signals were classified
by DEPT NMR as follows: three methyl carbon resonances at dC
17.4, 23.0 and 56.9; six methylene carbon signals at dC 22.0, 28.0,
31.2, 34.2, 42.0 and 124.7; two methine carbon resonances dC
40.0 and 79.3; and five quaternary carbon signals at dC 35.0,
124.0, 140.2, 145.4, and 170.8. Based on NMR spectroscopic com-
parisons with other metabolites previously characterized from this
for C-3 was consistent with methoxy placement in an
(Jakupovic et al., 1988). Compound 3 was assigned to 3
a
orientation
-methoxy
a
isocostic acid, a new natural product.
Compound 4 was isolated as a yellowish oil, ½a D25
= ꢁ24.3 (c
ꢀ
0.03, CHCl3) and its IR spectrum showed absorption bands at
3566 (OH), 3020 (OH), and 1670 (C@C) cmꢁ1. The low resolution
mass spectrum had a molecular ion peak [M]+ at m/z 254. Its HRE-
IMS exhibited a quasi-molecular ion peak [M]+ at m/z 254.1880
(calcd. for C15H26O3: 254.1882). The 1H- and 13C-NMR spectra
(Table 1) confirmed the molecular formula C15H26O3 and the
fifteen carbon signals were classified by DEPT as: three methyl
carbon resonances at dC 11.7, 19.2, and 29.9; six methylene carbon
signals at dC 26.8, 31.0, 31.2, 34.6, 39.8 and 109.1; two methine
species, 2 was very similar to 3-oxo-c-costic acid (9) with the exo-
methylene carboxylic acid group attached to C-7 in a b-configura-
tion. However, in contrast to a C-3 carbonyl group present in 9, a
Please cite this article in press as: Hegazy, M.-E.F., et al. Chemical constituents and their antibacterial and antifungal activity from the Egyptian herbal
medicine Chiliadenus montanus. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.03.027

Table 1
1H NMR and 13C NMR spectroscopic data of 1–4 and 11–12 (600 MHz, d-ppm).a
No
1 (CD3OD)
dH (J in Hz)
1.78 m
2
3
4
11
12
dC
dH (J in Hz)
dC
dH (J in Hz)
dC
dH (J in Hz)
dC
dC
dC
*
1
2
3
37.1 t
1.56 m
1.31 m
34.2 t
1.56 m
1.30 m
1.58 m
35.9 t
3.33 dd (11.5, 4.0)
79.4 d
35.0
38.2
2.32 ddd (16.5, 4.0, 4.0)
2.54 m
–
33.3 t
1.87 m
22.0 t
79.3 d
23.4 t
80.0 d
1.88 m
1.62 br dd (7.0, 4.0)
1.69 m
26.8 t
39.8 t
25.0
74.1
26.4
34.8
1.58 m*
200.2 s
3.39 br s like
3.61 t (6.8)
1.57 m
4
5
6
–
–
128.5 s
163.1 s
33.2 t
124.0 s
140.2 s
31.2 t
124.8 s
139.4 s
31.7 t
–
71.5 s
44.7 d
31.0 t
141.1
124.0
31.8
41.3
75.8
28.1
–
–
1.57 m
2.89 br d (14.0)
2.13 t like (14.0)
2.58 m
2.62 br dt (13.0, 2.8)
1.75 br t (13.0)
2.44 br tt (13.0, 2.8)
1.68 m
2.62 br d (13.8)
1.79 m
2.39 m
1.63 m
1.84 m*
1.52 br dt (12.0, 1.3)
–
1.84 m
7
8
40.2 d
26.9 t
40.0 d
28.0 t
40.3 d
27.9 t
74.3 s
31.2 t
40.3
27.7
34.5
17.1
*
1.78 m
1.52 ddd (13.0, 13.0, 3.5)
1.41 ddd (13.0, 13.0, 2.7)
1.44 m
1.67 m
1.49 br dd (13.0, 4.0)
–
9
1.72 m
41.6 t
1.56 m*
1.32 m
–
42.0 t
1.49 m
1.31 m
–
42.0 t
34.6 t
41.3
38.0
1.50 ddd (13.5, 13.5, 4.0)
10
11
12
13
–
–
–
35.7 s
35.0 s
35.0 s
38.9 s
36.0
36.8
144.0 s
165.5 s
124.5 t
–
–
145.4 s
170.8 s
124.7 t
–
–
144.6 s
170.2 s
126.4 t
–
151.4 s
19.2 q
109.1 t
144.4
171.4
125.1
145.9
171.8
125.1
1.83 s
6.37 s
5.83 s
1.26 s
6.27 s
5.58 s
1.01 s
1.71 s
–
6.31 s
5.68 s
1.08 s
1.67 s
–
5.06 br s
4.81 br d (1.3)
1.03 s
1.13 s
–
14
15
10
21.3 q
9.7 q
23.0 q
17.4 q
–
24.7 q
15.6 q
–
11.7 q
29.9 q
–
24.5
15.2
16.8
38.2
1.73 br d (1.3)*
5.56 d (7.5)
3.33–3.43m*
94.7 d
72.7 d
76.8 d
69.7 d
77.6 d
60.9 t
20
30
40
50
60
3.81 dd (12.0,2.0)
3.67 dd (12.0, 5.4)
–OCH3
3.41 s
56.9 q
3.33 s
56.0 q
a
Recorded in CDCl3.
Overlapped.
*

4
Mohamed-Elamir F. Hegazy et al. / Phytochemistry xxx (2014) xxx–xxx
2
4
Fig. 2. Observed HMBC (solid arrows) and NOESY correlations (dotted arrows) for 2 and 4.
14
5
Compound 5 was isolated as a yellowish oily substance,
= +18 (c 0.03, CHCl3) and its IR spectrum showed absorption
a 2D5
12
½ ꢀ
1
9
14
15
OH
OH
OH
H3CO
13
bands at 3550 (OH), 2972 (OH) and 1684 cmꢁ1 (C@C). Its low res-
olution mass spectrum had a quasi-molecular ion peak at m/z 218
[Mꢁ(2H2O+CH3OH)]+. Its HREIMS exhibited a quasi-molecular ion
peak [Mꢁ(2H2O+CH3OH)]+ at m/z 218.1677 (calcd. for C15H22O:
218.1677). The molecular formula of C16H30O4, was consistent with
1H- and 13C-NMR spectroscopic data (Table 2) and DEPT data
established the presence of sixteen carbon signals assigned to: five
methyls (one methoxy), four methylenes (one olefinic), four
methines (three olefinic and one oxygen bearing), and three oxy-
gen-bearing quaternary carbons respectively. A methoxy group at
dH 3.14 (H3-16, s) correlated with carbons at dC 74.9 (C-11), 25.8
(C-12) and 26.1 (C-13) as observed from the HMBC data (Fig. 3).
The C-11 also correlated with an overlapping singlet at dH 1.25
(H6-12/13), and correlated with an olefinic proton at dH 5.48 (H-
10, d, J = 16.0 Hz), in the HMQC and HMBC spectra, respectively.
The signal at dH 5.61 (H-9, ddd, J = 16.0, 8.2, 6.2 Hz) showed a cor-
relation with an olefinic proton at dH 5.48 (H-10, d, J = 16.0 Hz) and
methylene protons at dH 2.10 (H-8, dd, J = 13.7, 8.2 Hz) and 2.31 (H-
8, dd, J = 13.7, 6.2 Hz); the appearance of a vinyl ABX system ap-
peared at dH 5.85 (H-2, dd, J = 17.2, 10.5 Hz), 5.15 (H-1, dd,
J = 17.2, 1.3 Hz), and 4.97 (H-1, dd, J = 10.5, 1.3 Hz). Additionally,
the resonance at dH 3.79 (H-6, dd, J = 6.8, 6.8 Hz) correlated with
a multiplet at dH 1.71 and 1.88 (H2-4, m) and with a methyl singlet
at dH 1.18 (H3-14, s) in the COSY spectrum. The H-6 also correlated
with dC 25.9 (C-5) in the HMQC spectrum and with dC 84.8 (C-6)
and methylene carbons at dC 40.9 (C-8), in the HMBC spectrum.
HMBC connectivities were also observed between: vinyl methy-
lene protons at dH 5.15 (H-1a, J = 17.2, 1.3 Hz) and 4.97 (H-1b,
dd, J = 10.5, 1.3 Hz,) and an oxygenated signal dC 73.3 (C-3); vinyl
methine protons at dH 5.85 (H-2, dd, J = 17.2, 10.5 Hz) and dC
111.4 (C-1), 83.0 (C-3), and 26.8 (C-15); a methyl singlet at dH
1.18 (H3-14, s) and dC 72.9 (C-7), 84.8 (C-6), and 40.9 (C-8). In
addition, a correlation between a methoxy proton signal at dH
3.14 (H3-16, s) and dC 74.9 (C-11) was observed.
9
3
7
1
5
7
3
13
11
11
12
O
15
5
O
HO
O
O
O
OH
OH
H
OH
OH
6
1
OH
H
H
O
O
7
Fig. 3. HMBC correlations of 1, 5–7.
carbon resonances dC 44.7 and 79.4; and four quaternary carbon
signals at dC 38.9, 71.5, 74.3, and 151.4. NMR comparisons with
previous isolated compounds by Wang et al. (2007) indicated the
presence of an eudesmane (sesquiterpene) framework. The 1H
NMR indicated the presence of exomethylene protons at dH 4.81
(H-13, d, J = 1.3 Hz) and 5.06 (H-13, s), an olefinic methyl at dH
1.83 (H-12, s), and a tertiary methyl at dH 1.03 (H-14, s). HMBC
identified the position of the olefinic methyl protons at dH 1.83
(H3-12, s) that correlated with signals at dC 151.4 (C-11), 109.1
(C-13), and 74.3 (C-7); tertiary methyl protons at dH 1.03 (H3-14)
that correlated with carbons at dC 79.4 (C-1), 44.7 (C-5), 34.6 (C-
9), and 38.9 (C-10); and methyl protons at dH 1.13 (H3-15, s) corre-
lated with carbons at dC 71.5 (C-4), 39.8 (C-3), and 44.7 (C-5)
(Fig. 2). HMBC was also used to infer the position of the three hy-
droxyl groups at C-1 (dC 79.4), C-4 (dC 71.5), and C-7 (dC 74.3). The
relative stereochemistry of 4 was assigned on the basis of coupling
constants and NOESY data (Fig. 2). The OH group at C-1 was placed
in a beta position, according to coupling constants for H-1 [dH 3.12
(dd, J = 11.5, 4.0 Hz)] (Sung et al., 1992, 1988; Wang et al., 2007).
The NOESY spectrum exhibited a correlation between dH 3.33
(H-1, dd, J = 11.5, 4.0 Hz) and dH 1.57 (H-5, m), indicating these
The relative stereochemistry assignment was based on coupling
constants and NOESY data. Correlations between dH 3.79 (H-6, dd,
J = 6.8, 6.8 Hz) and dH 1.18 (H-14, s), dH 2.10 (H-8a, dd, J = 13.7,
6.2 Hz) and dH (H-5a, m), and dH 1.30 (H-15, s) and dH 1.88 (H-4a
and H-5a, m, 2H) indicated that the secondary hydroxyl group is
situated on the same side as the other hydroxyl groups at C-3
and C-7. These chemical data confirmed the structure of 5, 3,6,7-
trihydroxy-11-methoxy-3,7,11-trimethyldodeca-1,9-diene,
signed as a new natural product, Chiliadenol A.
as-
protons were in an
a configuration. The correlation between dH
1.03 (H3-14, s) and dH 1.83 (H3-12, s), together with dH 1.84
(H-8b, m), indicated that the hydroxyl at C-7 is positioned on the
opposite side relative to the hydroxyl groups at C-1 and C-4. There-
fore 4 was assigned to eudesm-11,13-ene-1b,4b,7a-triol, a new
natural product.
Compound 6 was isolated as a yellowish oily substance,
½
a 2D5
= +3.67 (c 0.03, CHCl3) and its IR spectrum showed absorption
ꢀ
bands at 3550 (OH), 2972 (OH) and 1684 cmꢁ1 (C@C). Its low res-
olution mass spectrum gave a molecular ion peak [MꢁH2O]+ at m/z
Please cite this article in press as: Hegazy, M.-E.F., et al. Chemical constituents and their antibacterial and antifungal activity from the Egyptian herbal
medicine Chiliadenus montanus. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.03.027

Mohamed-Elamir F. Hegazy et al. / Phytochemistry xxx (2014) xxx–xxx
5
Table 2
1H NMR and 13C NMR spectroscopic data of compounds 5–8 (600 MHz, d-ppm).a
No
5
6
7
8
dH (J in Hz)
dC
dH (J in Hz)
dC
dH (J in Hz)
dC
dH (J in Hz)
dC
1
4.97 dd (10.5, 1.3)
5.15 dd (17.2, 1.3)
5.85 dd (17.2, 10.5)
–
111.4 t
5.07 dd (11.0, 1.3)
5.22 dd (17.0, 1.3)
5.90 dd (17.0, 11.0)
–
111.9 t
5.04 dd (10.5, 1.3)
5.22 dd (17.2, 1.3)
5.84 dd (17.2, 10.5)
–
111.8 t
5.03 dd (10.5, 1.5)
5.21 dd (17.0, 1.5)
5.87 dd (17.0, 10.5)
–
111.8 t
2
3
4
143.7 d
83.0 s
37.4 t
144.9 d
73.3 s
23.1 t
145.4 d
72.7 s
39.0 t
145.2 d
72.7 s
37.5 t
1.88 m*
2.05 m
1.75 br dd (14.0, 7.0)
1.68 m
1.71 m
1.69 m*
5
1.88 m*
25.9 t
1.56 m
41.7 t
1.62 m
30.1 t
1.81 m
29.1 t
1.82 m
1.62 m
6
7
8
3.79 dd (6.8, 6.8)
84.8 d
72.9 s
40.9 t
5.24 br t (7.5)
–
3.00 s
129.6 d
129.1 s
54.3 t
4.40 br t (5.5)
–
2.64 ddd (15.0, 6.2, 1.4)
2.31 m
4.65 ddd (8.2, 8.2, 6.2)
5.19 m
80.5 d
151.8 s
39.8 t
4.28 br t (2.0)
–
2.60 dd like (15.1, 5.0)
2.26 m
4.52 ddd (10.5, 8.2, 5.0)
5.23 m
80.7 d
151.7 s
40.3 t
2.31 dd (13.7, 6.2)
2.10 dd (13.7, 8.2)
5.61 ddd (16.0, 8.2, 6.2)
5.48 d (16.0)
9
10
125.2 d
139.0 d
–
209.4 d
50.9 d
73.7 d
124.9 d
75.2 d
124.7 d
2.26 d (7.0)
11
12
13
14
–
74.9 s
25.8 q
26.1 q
24.3 q
2.12 m
0.89 d (6.9)
0.89 d (6.9)
1.59 s
24.5 s
22.6 q
22.6 q
16.6 q
–
136.7 s
25.9 q
18.3 q
105.0 q
–
137.3 s
25.9 q
18.4 q
104.4 t
*
1.25 s*
1.25 s*
1.18 s
1.70 br s
1.72 d (1.4)
1.68 br s
4.95 dd (4.8, 2.0)
4.80 dd (4.8, 2.0)
1.26 s
1.68 br s*
4.97 dd (4.8, 2.0)
4.83 dd (4.8, 2.0)
1.25 s
*
15
–OCH3
1.30 s
3.14 s
26.8 q
50.4 q
1.27 s
–
28.1 q
–
28.6 q
–
28.3 q
–
–
–
a
Recorded in CDCl3.
Overlapped.
*
220. Its HREIMS exhibited a quasi-molecular ion peak [MꢁH2O]+ at
m/z 220.1833 (calcd. for C15H24O: 220.1827). The molecular for-
mula of C15H26O2 was supported by 1H- and 13C-NMR spectro-
scopic analysis (Table 2). DEPT NMR analysis showed the
presence of fifteen carbon signals assigned to four methyls, five
methylenes (one olefinic), three methines (two olefinic), and three
quaternary carbons (one olefinic, one oxygen-bearing, and one free
keto group), respectively. An isopropyl group appeared as one
methyl doublet signal integrating for six protons at dH 0.89 (H-
12/13, d, J = 6.9 Hz) that was correlated with a methane at dH
2.12 (H-11, m). The methyl protons also correlated with dC 24.5
(C-11) in the HMBC spectrum. Two singlet signals established the
presence of two methyl groups which appeared at dH 1.59 (H-14,
s) with dC 16.6 (C-14) and 1.27 (H-15, s) with dC 28.1 (C-15) that
showed correlations with a quaternary olefinic carbon at dC 129.1
(C-7) and a quaternary oxygenated carbon at dC 73.3 (C-3), respec-
tively. Additionally, a vinyl ABX system appeared as three double
doublets at dH 5.90 (H-2, dd, J = 17.0, 11.0 Hz), 5.22 (H-1, dd,
J = 17.0, 1.3 Hz), 5.07 (H-1, dd J = 11.0, 1.3 Hz); a doublet signal at
dH 2.26 (H2-10, d, J = 7.0 Hz); isolated methylene protons appearing
as a singlet at dH 3.00 (H2-8, s) and correlating with a keto group
and a quaternary olefinic carbon at dC 209.4 (C-9) and 129.1 (C-
7), respectively; and two methylenes appearing as multiplets at
dH 1.56 (H2-5) and 2.05 (H2-4). Examination of the connectivities
in the HMBC spectrum (Fig. 3) indicated: a vinyl methine at dH
5.90 (H-2, dd, J = 17.0, 11.0 Hz) correlated with dC 111.9 (C-1),
73.3 (C-3), and 28.1 (C-15); an oxygenated quaternary carbon at
dC 73.3 (C-3) correlated with dH 1.27 (H3-15, s), 2.05 (H2-4, m),
and 1.56 (H2-5, m); an olefinic methyl singlet at dH 1.59 (H3-14,
s) correlated with dC 129.1 (C-7), 129.6 (C-6), and 54.3 (C-8); and
dH 3.00 (H2-8, s) correlated with dC 16.6 (C-14), 129.1 (C-7), and
209.4 (C-9). Additionally a correlation was observed between dH
2.26 (H2-10, d, J = 7.0 Hz) and dC 209.4 (C-9), 24.5 (C-11), and
22.6 (C-12/13). The above chemical data confirmed the structure
of 6 as a new natural product, 3-hydroxy-3,7,11-trimethyl-1,6-
dodecadien-9-one, Chiliadenol B.
3450 (OH), 1675 (C@C), and 1576 (C@C). Its low resolution mass
spectrum had a molecular ion peak [MꢁH2O]+ at m/z 234. Whereas
the HREIMS exhibited a quasi-molecular ion peak [MꢁH2O]+ at m/z
234.1627 (calcd. for C15H22O2: 234.1620). By contrast, the FAB-MS
had a molecular ion peak [M+Na+] at m/z 275. Its molecular for-
mula of C15H22O2 was supported by 1H- and 13C-NMR spectro-
scopic data (Table 2). The DEPT spectrum exhibited 15 carbon
signals including: three methyls, five methylenes, four methines,
and three quaternary carbon resonances. A vinyl ABX system sim-
ilar to 5 and 6 was observed as three double doublets at dH 5.84 (H-
2, dd, J = 17.2, 10.5 Hz), 5.22 (H-1, dd, J = 17.2, 1.3 Hz), 5.04 (H-1,
dd, J = 10.5, 1.3 Hz) that indicated the presence of an allyl unit. A
quaternary oxygenated carbon signal, indicated from DEPT analy-
sis, appeared at dC 72.7 (C-3) and which showed a correlation with
a vinyl group at dC 111.8 (C-1) and 145.4 (C-2), as well as a methyl
carbon at dC 28.6 (C-15) in the HMBC spectrum thereby establish-
ing a methyl group on the oxygenated quaternary carbon (C-3)
(Fig. 3). A broad triplet signal at dH 4.40 (H-6, t, J = 5.5 Hz) had a
correlation with an oxygenated dC 80.5 (C-6) and a methylene pro-
ton at dH 1.62 (H2-5) in HMQC and 1H–1H COSY spectra, respec-
tively. Two doublet of doublets at dH 4.83 (H-14, dd, J = 4.8,
2.0 Hz) and 4.97 (H-14, dd, J = 4.8, 2.0 Hz) were characteristic for
exomethylene protons. These exomethylene protons correlated
with methylene protons at dH 2.31 (H-8, m) and 2.64 (H-8, ddd,
J = 15.0, 6.2, 1.4 Hz) in 1H–1H COSY spectrum, as well as with dC
105.0 (C-14) and dC 151.8 (C-7) in HMQC and HMBC spectra,
respectively. A doublet doublet of doublets at dH 4.65 (H-9,
J = 8.2, 8.2, 6.2 Hz) COSY correlated with a methylene proton dH
2.31 (H-8, m) and 2.64 (H-8, ddd, J = 15.0, 6.2, 1.4 Hz), as well as
an olefinic proton at dH 5.19 (H-10, m). In the HMQC spectrum,
the H-9 signal correlated with an oxygenated dC 73.7 (C-9). Two
broad singlet signals for olefinic methyl groups at dH 1.68 (H3-13,
s), and 1.70 (H3-12, s) correlated in the HMBC spectrum with
low-field olefinic signals at dC 136.7 (C-11, quaternary carbon)
and dC 124.9 (C-10, methine carbon). The position of the 2-
methyl-2-propene unit was confirmed by HMBC analysis, in which
the H-10 correlated with dC 136.7 (C-11), 73.7 (C-9), 25.9 (C-12),
and 18.3 (C-13). These NMR spectroscopic data, combined with
Compound 7 was isolated as an oily material, ½a D25
ꢀ
= +22.6 (c
0.03, CHCl3) and its IR spectrum showed absorption bands at
Please cite this article in press as: Hegazy, M.-E.F., et al. Chemical constituents and their antibacterial and antifungal activity from the Egyptian herbal
medicine Chiliadenus montanus. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.03.027

6
Mohamed-Elamir F. Hegazy et al. / Phytochemistry xxx (2014) xxx–xxx
Table 3
Antimicrobial Minimum Inhibitory Concentration (MIC) for 1–16.
Compounds
Minimum inhibitory concentration (
Gram positive bacteria
l )
g mLꢁ1
Gram negative bacteria
Yeast
Staphylococcus aureus
Bacillus subtilis
Klebsiella pneumoniae
Alcaligenes faecalis
Candida albicans
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
12.500
12.500
12.500
12.500
6.250
12.500
12.500
3.125
12.500
6.250
25.000
12.500
6.250
6.250
3.125
6.250
–
25.000
–
–
–
–
25.000
–
–
–
–
–
25.000
–
<3.125
25.000
–
<3.125
25.000
–
<3.125
12.500
–
<3.125
Chloramphenicol
(–) No inhibition observed at any tested concentration.
HREIMS data, established 7 to be a dioxane derivative. Its relative
stereochemistry was assigned on the basis of coupling constants,
with the coupling constants of H-9 (dH 4.65, ddd, J9,10 = 8.2 Hz,
J9,8eq = 8.2 Hz, J9,8ax = 6.2 Hz) determining the relative stereochem-
istry of H-9 to be axial. Additionally, the coupling constants of H-6
(dH 4.40 Hz, br t, J=5.5 Hz) determined the relative stereochemistry
of H-6 to be in an axial position (Ibrahim et al., 2008; Kobayashi
et al., 1993; Yosief et al.,1998). This is in agreement with NOESY
data which showed correlations between H-6ax and H-5ax and be-
tween H-9ax and H-8ax. The above chemical data confirmed the
dimethyl-6
pound Chiliadenol D. In addition to new compounds (1–8), eight
known compounds were isolated: 3-oxo- -costic acid (9) (Garcez
et al., 2010), 3-oxo- -costic acid methyl ester (10) (Ceccherelli
et al., 1989; Bohlmann and Borthakur, 1982), 3 -acetyl- -costic
acid (11), 5 -hydroxy-4 ,15-dihydrocostic acid (12), 5 -hydrox-
ycostic acid (13) (Ahmed and Jakupovic, 1990), eudesmane-
1b,4b,7
-triol (14) (Sung et al., 1992), kaempferol-3-O-(600-O-acet-
yl)-b- -glucopyranoside (15) (Merfort, 1988), and thymol b-
glucopyranoside (16) (Shimoda et al., 2006). Since metabolites
11–12 were previously isolated as methyl esters, the 13C NMR
spectroscopic data for the isolated acids are given (Table 2).
Among all the isolates, metabolite 15 showed antimicrobial
activity inhibiting growth of Staphylococcus aureus, Bacillus subtilis,
Klebsiella pneumonia, Alcaligenes faecalis and Candida albicans with
a,9a-epidioxy-dodeca-1,10,7(14)-ene, as a new com-
c
c
a
c
a
a
a
a
D
D-
structure of 7, 3-hydroxy-3,11-dimethyl-6b,9a-epidioxy-dodeca-
1,7(14),10-triene, as a new natural product Chiliadenol C.
Compound 8 was isolated as an oily material, ½a D25
= ꢁ8.6 (c
ꢀ
0.03, CHCl3) and its IR spectrum showed absorption bands at
3450 (OH), 1675 (C@C), and 1576 (C@C). Its low resolution mass
spectrum had a molecular ion peak [MꢁH2O]+ at m/z 234, with
the HREIMS exhibits a quasi-molecular ion peak [MꢁH2O]+ at m/z
234.1627 (calcd. for C15H22O2: 234.1620). Its molecular formula
of C15H24O3 was supported by analysis of 1H- and 13C-NMR spec-
troscopic data (Table 2). Its DEPT spectrum exhibited 15 carbon
signals with three methyl resonances at dC 18.4, 25.9, and 28.3; five
methylene signals at dC 29.1, 37.5, 40.3, 104.4 and 111.8; four
methine resonances dC 75.2, 80.7, 124.7 and 145.2; and three qua-
ternary signals at dC 72.7, 137.3 and 151.7. The 1H and 13C NMR
spectroscopic data of 8 was quite similar to 7 (Table 2), except
for a downfield chemical shift of 1.5 ppm (dC 37.5, C-4 and dC
75.2, C-9). This carbon chemical shift was accompanied by an up-
field chemical shift for H-6 to appear as broad triplet at dH 4.28
(J = 2.0). From these data, it appears that 8 is an epimer of 7 at po-
sition C-6. The relative stereochemistry of 8 was assigned on the
basis of study of the coupling constants, where the coupling con-
stants of H-9 (dH 4.52, ddd, J9,10 = 5.0, J9,8eq = 8.2, J9,8ax = 10.5 Hz)
determined the relative stereochemistry of H-9 to be axial. Addi-
MIC values 25, 25, 25, 12.5 and 3.125
lg/mL, respectively (Table 3)
using chloramphenicol (less than 6 g/mL) as a positive control. As
l
with other flavonol structures 15 may contain an appropriate
structural configuration to complex with bacterial cell walls and
inhibit microbial growth (Cowan, 1999).
3. Conclusion
Among the Sinai herbal medicinal plants, Jasonia montana, lo-
cally known as Haneida, has a strong aromatic odor and is used
in traditional medicine for diarrhea, stomachaches, and chest dis-
eases (Täckholm, 1974). Haneida owes its therapeutically activity
to an array of metabolites. In this phytochemical investigation of
a closely related species, solvent extraction of the air-dried aerial
parts of C. montana resulted in isolation and characterization of
new compounds 1–8 as well as previously reported compounds
9–16. Antimicrobial activity against an array of common bacterial
and fungal strains was measured via a colorimetric assay with
tionally, the coupling constants of H-6 (dH 4.28, brt, J6,5ax
=
minimal growth inhibition observed in the
lg/mL range for one
J6,5eq = 2.0 Hz) determined the relative stereochemistry of H-6 to
also be axial (Ibrahim et al., 2008; Kobayashi et al., 1993; Yosief
et al., 1998). This suggested that 8 is the 6- axial epimer of 7, which
has a clear effect of the carbon signal of C-9 resonate at dC 75.2 in
compound 8 instead of dC 73.7 in 7 related to the position of hydro-
xyl group at C-3 (dC 72.7) (Ibrahim et al., 2008; Weyerstahl et al.,
1999). This is in agreement with NOESY data that showed correla-
tions between H-6ax and H-5ax and between H-9eq and H-8 eq. The
above chemical data confirmed the structure of 8, 3-hydroxy-3,11-
of the tested metabolites (15).
Experimental
General procedures
Instrumentation included a Horiba SEPA-300 digital polarime-
ter (l = 5 cm) for specific rotation; a Shimadzu FTIR-8100 spec-
Please cite this article in press as: Hegazy, M.-E.F., et al. Chemical constituents and their antibacterial and antifungal activity from the Egyptian herbal
medicine Chiliadenus montanus. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.03.027

Mohamed-Elamir F. Hegazy et al. / Phytochemistry xxx (2014) xxx–xxx
7
trometer for IR analysis; JEOL JMS-GCMATE mass spectrometer for
3b-Methoxy isocostic acid (2)
EI-MS and HR-EI-MS; a JEOL JNM-ECA 600 spectrometer with tet-
ramethylsilane as an internal standard for 1H (600 MHz) and 13C
(150 MHz) NMR spectra; a Shimadzu RID-10A refractive index
detector for HPLC analysis; and a COSMOSIL-Pack type (C18-MS-
II) (250 ꢂ 4.6 mm i.d.) and (250 ꢂ 20 mm i.d.) columns for analyt-
ical and preparative separation, respectively. The following exper-
imental materials were used for chromatography: normal-phase
silica gel column chromatography (cc), silica gel BW-200 (Fuji Sily-
sia Chemical, Ltd., 150–350 mesh); reverse-phase silica gel cc,
Chromatorex ODS DM1020T (Fuji Silysia Chemical, Ltd., 100–
200 mesh); TLC, pre-coated TLC plates with silica gel 60F254 (Merck,
0.25 mm) (ordinary phase) and silica gel RP-18 F254S (Merck,
0.25 mm) (reverse phase); reverse-phase HPTLC, pre-coated TLC
plates with silica gel RP-18 WF254S (Merck, 0.25 mm); and detec-
tion was achieved by spraying with (1:9) H2SO4-MeOH followed
by heating.
Yellowish oily material; ½a D25
ꢀ
= +73.0 (c 0.01, CHCl3); for 1H
(CDCl3, 600 MHz) and 13C (CDCl3, 150 MHz) NMR spectroscopic
data, see Table 1; EIMS m/z 264 [M]+, 249, 232 217, 199, 187,
171; HREIMS m/z 264.1734 (calcd. for C16H24O3: 264.1725); IR
(
mmax cmꢁ1) = 3020, 1715, 1670 cmꢁ1
.
3a-Methoxy isocostic acid (3)
Yellowish oily material; ½a D25
ꢀ
= ꢁ13.6 (c 0.03, CHCl3); for 1H
(CDCl3, 600 MHz) and 13C (CDCl3, 150 MHz) NMR spectroscopic
data, see Table 1; EIMS m/z 264 [M]+, 249, 232 217, 199, 187,
171; HREIMS m/z 264.1734 (calcd. for C16H24O3: 264.1725); IR
(
mmax cmꢁ1) = 3020, 1715, 1670 cmꢁ1
.
Eudesmane-1b,4b,7a-triol-11,13-en (4)
Yellowish oily material; ½a D25
ꢀ
= ꢁ24.3 (c 0.03, CHCl3); for 1H
(CDCl3, 600 MHz) and 13C (CDCl3, 150 MHz) NMR spectroscopic
data, see Table 1; EIMS m/z 254 [M]+, 236, 218, 195, 185, 179,
165; HREIMS m/z 254.1880 (calcd. for C15H26O3: 254.1882); IR
Plant material
(
mmax cmꢁ1) = 3566, 3020, 1670 cmꢁ1
.
Air-dried aerial parts of Chiliadenus montanus (Vahl.) Brullo.
were collected in 2010, from Wadi Gebal, North Sinai, Egypt. Plant
material was identified by Dr. El-Bialy E. Hatab, Egyptian Environ-
mental Affairs Agency, Nature Conservation Sector, Siwa Protected
Area, Siwa, Egypt. A voucher specimen SK-1001 has been deposited
in the Herbarium of St. Katherine Protectorate, Egypt.
Chiliadenol A (5)
Yellowish oily material; ½a D25
ꢀ
= +18.0 (c 0.03, CHCl3); for 1H
(CDCl3, 600 MHz) and 13C (CDCl3, 150 MHz) NMR spectroscopic
data, see Table 2; EIMS m/z 250 [Mꢁ2(H2O)]+, 232 [Mꢁ3(H2O)]+,
218 [Mꢁ[2(H2O)+CH3OH]]+, 203, 178, 155; HREIMS m/z 250.1578
(calcd. for C16H22O2: 250.1933), 218.1677 (calcd. for C15H18O:
218.1671); IR (mmax cmꢁ1) = 3550, 2972, 1684, 1338, 1219, and
771 cmꢁ1
.
Extraction and isolation
Chiliadenol B (6)
Aerial parts (2.0 kg) of C. montana were powdered and extracted
with CH2Cl2:MeOH (1:1) (10 L, 3 days x 2) at room temperature.
The combined extract was concentrated in vacuo to obtain a resi-
due (175 g), which was fractionated on a silica gel column
(6 ꢂ 120 cm) eluting with n-hexane (3 L) followed by a gradient
of n-hexane-CHCl3 up to 100% CHCl3 and then a CHCl3–MeOH stud-
ies at up to 15% MeOH (3 L each of the solvent mixture). The n-hex-
ane:CHCl3 (3:1) fraction was applied to a Sephadex LH-20 column
(3 ꢂ 90 cm) eluted with n-hexane:CH2Cl2:MeOH (7:4:0.25), to give
10 (12 mg). The n-hexane:CHCl3 (1:1) fraction was subjected to sil-
ica gel cc (4 ꢂ 120 cm) eluted with n-hexane:EtOAc (4:1). Fractions
were obtained and combined into three parts A, B and C on the ba-
sis of their TLC profiles. Sub-fraction A, was re-purified using silica
gel cc (6 ꢂ 90 cm) eluted with n-hexane:EtOAc (6:1) to afford 9
(5.5 g). Sub-fraction B was re-purified by reversed phase HPLC,
using MeOH/H2O (75:25 v/v) at a flow rate 3.5 mL/min to afford
2 (7 mg), 3 (3 mg), 7 (5 mg), and 11 (20 mg); sub-fraction C was
re-purified by reversed phase HPLC using MeOH/H2O (70:30 v/v)
to afford 5 (3 mg), 6 (8 mg), 7 (6 mg), and 8 (4 mg) and 1
(15 mg). The CHCl3:MeOH (95:5) fraction was applied to a silica
gel column (6 ꢂ 120 cm) eluted with n-hexane:CHCl3:MeOH
(7:4:1), followed by reversed phase HPLC using MeOH:H2O (1:1)
to afford 4 (10 mg), 12 (12 mg), 13 (10 mg), 7 (20 mg), and 14
(15 mg). The CHCl3:MeOH (75:15) fraction was subjected to silica
gel cc (6 ꢂ 120 cm) eluted with n-hexane:CHCl3:MeOH (7:4:2), fol-
lowed by reversed phase HPLC using MeOH:H2O (1:1) at a flow
rate 5.0 mL/min to afford 15 (30 mg), and 16 (20 mg).
Yellowish oily material; ½a D25
ꢀ
= +3.67 (c 0.03, CHCl3); for 1H
(CDCl3, 600 MHz) and 13C (CDCl3, 150 MHz) NMR spectroscopic
data, see Table 2; EIMS m/z 220 [M-H2O]+, 202, 187, 151, 120; HRE-
IMS m/z 220.1833 (calcd. for
C15H24O: 220.1827); IR (mmax
cmꢁ1) = 3550, 3020, 1684 cmꢁ1
.
Chiliadenol C (7)
Yellowish oily material; ½a D25
ꢀ
= +22.6 (c 0.03, CHCl3); for 1H
(CDCl3, 600 MHz) and 13C (CDCl3, 150 MHz) NMR spectroscopic
data, see Table 2; EIMS m/z 234 [MꢁH2O]+, 219, 201 179, 150; HRE-
IMS m/z 234.1627 (calcd. for
C15H22O2: 234.1620); IR (mmax
cmꢁ1) = 3450, 1675, 1576 cmꢁ1
.
Chiliadenol D (8)
Yellowish oily material; ½a D25
ꢀ
= ꢁ8.6 (c 0.03, CHCl3); for 1H
(CDCl3, 600 MHz) and 13C (CDCl3, 150 MHz) NMR spectroscopic
data, see Table 2; EIMS m/z 234 [M-H2O]+, 219, 201 179, 150; HRE-
IMS m/z 234.1627 (calcd. for
C15H22O2: 234.1620); IR (mmax
cmꢁ1) = 3450, 1675, 1576 cmꢁ1
.
Acid hydrolysis of 1
A solution of 1 (1.0 mg) in 5% aq. H2SO4–1,4-dioxane (1:1, v/v,
1.0 mL) was heated under conditions where reflux began, this
being maintained for 3 h. After cooling, the reaction mixture was
neutralized with Amberlite IRA-400 (OHꢁ form) and the resin
was removed by filtration. After removal of the solvent from the fil-
trate, the residue was transferred to a Sep-Pak C18 cartridge with
H2O and MeOH. The H2O eluate was concentrated and the residue
was subjected to HPLC analysis under the following conditions:
HPLC column, Kaseisorb LC NH2-60-5, 4.6 mm i.d. ꢂ 250 mm (To-
kyo Kasei Co., Ltd., Tokyo, Japan); detection, optical rotation [Sho-
dex OR-2 (Showa Denko Co., Ltd., Tokyo, Japan); mobile phase,
CH3CNH2O (85:15, v/v); flow rate 0.8 mL/min]. Identification of
3-Oxo-c-costic acid b-D-glucopyranoside ester (1)
Colorless needles; ½a D25
ꢀ
= +8.3 (c 0.03, CHCl3); for 1H (CD3OD,
600 MHz) and 13C (CDCl3, 150 MHz) NMR spectroscopic data, see
Table 1; EIMS m/z 410 [M]+, 290, 275, 248, 233, 220, 202; HREIMS
m/z 410.1946 (calcd. for
C
21H30O8: 410.1941); IR
(mmax
cmꢁ1) = 3250, 3020, 1725, 1650 cmꢁ1
.
D-glucose was carried out by comparison of its retention time
Please cite this article in press as: Hegazy, M.-E.F., et al. Chemical constituents and their antibacterial and antifungal activity from the Egyptian herbal
medicine Chiliadenus montanus. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.03.027

8
Mohamed-Elamir F. Hegazy et al. / Phytochemistry xxx (2014) xxx–xxx
Ahmed, B., Al-Howiriny, T.A., Al-Rehaily, A.J., Mossa, J.S., 2004. Two germacrane
sesquiterpenes and a bibenzopyran from Jasonia montana. J. Saudi Chem. Soc. 8,
105–114.
Ahmed, A.A., Ali, A.A., Mabry, T.J., 1989. Flavonoid aglycons from Jasonia montana.
Phytochemistry 28, 665–667.
and optical rotation with that of authentic sample (Wako Pure
Chemical Industries, Japan; tR: 7.1 min (D-glucose, positive optical
rotation).
Ahmed, A.A., Jakupovic, J., 1990. Sesqui- and monoterpenes from Jasonia montana.
Phytochemistry 29, 3658–3661.
Antimicrobial assay
Ahmed, A.A., Jakupovic, J., Eid, F., Ali, A.A., 1988. 11-Hydroxyjasionone, a new
sesquiterpene type from Jasonia montana. Phytochemistry 27, 3875–3877.
Al-Howiriny, T.A., Al-Rehaily, A.J., Pols, J.R., Porter, J.R., Mossa, J.S., Ahmed, B., 2005.
Three new diterpenes and the biological activity of different extracts of Jasonia
montana. Nat. Prod. Res. 19, 253–265.
Bailey, C., Danin, A., 1981. Bedouin plant utilization in Sinai and the Negev. Econ.
Bot. 35, 145–162.
Batanouny, K.H., Aboutabl, E., Shabana, M., Soliman, F., 1999. Wild Medicinal Plants
in Egypt. An Inventory to Support Conservation and Sustainable Use. Palm Press,
Cairo, Egypt, p. 207.
Bohlmann, F., Borthakur, N., 1982. Naturally occurring terpene derivatives. Part 424
sesquiterpenes and norsesquiterpenes from Pechuel-Loeschea leibnitziae.
Phytochemistry 21, 1160–1162.
Boulos, L., 1983. Medicinal Plants of North Africa. References Publication, Inc.,
Algonac, Michigan, p. 286.
Boulos, L., 2002. Flora of Egypt, vol. 3. Al hadara publishing, Cairo, Egypt, p. 373.
Bown, D., 1995. Encyclopedia of Herbs and Their Uses. Dorling Kindersley, London.
Ceccherelli, P., Curini, M., Marcotullio, M.C., Rosati, O., 1989. Biogenetic-type
transformation of 3-keto-4,5-epoxyeudesmanes: synthesis of cyperanes,
eremophilanes and spirovetivanes. Tetrahedron 45, 3809–3818.
Cowan, M.M., 1999. Plant products as antimicrobial agents. Clin. Microbiol. Rev. 12,
564–582.
Assays were performed according to Eloff (1998) with some
modifications. Microorganisms used in the present study were col-
lected from the culture collection of Chemistry of Microbial and
Natural Products Department at the National Research Centre
(NRC), Dokki, Cairo, Egypt. Bacterial strains used were the gram po-
sitive bacteria, Staphylococcus aureus and Bacillus subtilis, and the
gram negative bacteria, Klebsiella pneumoniae, Alcaligenes faecalis
and Escherichia coli. Fungal yeasts used were Saccharomyces cerevi-
siae and Candida albicans. All microorganisms were cultured on
nutrient agar medium (purchased from Lab M limited, Lancashire
BL9 6AS, United Kingdom) at 37 °C for 24 h prior to use, while
the fungi was cultured on potato dextrose agar (purchased from
Becton, Dickinson and company Sparks, MD 21152, USA) at 37 °C
for 24 h then suspended in potato dextrose broth prior to use. After
incubation, the microbial colonies were scrapped off the agar and
transferred to nutrient and potato dextrose broth solutions for bac-
teria and yeast respectively, to prepare 0.5 McFarland of microbial
cultures with turbidity standard 107 CFU/mL (colony-forming
units). Tested pure compounds were prepared by dissolving each
(1 mg) in DMSO (1 mL), and then diluting with deionized H2O to
prepare stock solutions of 10% DMSO. All compounds were serially
Eid, F., El-Dahmy, S., Gupta, P.K., 1987. Constituents from Jasonia montana and
Allagopappus dichotoma. Pharmazie 42, 423–424.
El-Din, A.S., El-Sebakhy, N., El-Ghazouly, M., 1985. Methoxylated flavonoids of
Tanacetum santolinoides and Jasonia montana. Acta Pharm. 35, 283–287.
Eloff, J.N., 1998. A Sensitive and quick microplate method to determine the minimal
inhibitory concentration of plant extracts for bacteria. Planta Med. 64, 711–713.
Garcez, F.R., Garcez, W.S., Hamerski, L., Miranda, A.C.D.M., 2010. Eudesmane and
rearranged eudesmane sesquiterpenes from Nectandra cissifiora. Quim. Nova 33,
1739–1742.
diluted 50% with sterile media from 25 to 3.12
of each compound 100 L to the first well of 96-well micro plates.
Subsequently, inoculated broths (100 L) were added to each well.
l
g mLꢁ1 by addition
l
Hanafi, Y., Abdel-Wahab, M., 2000. Wild medicinal Plants in Sinai. Arabian Gulf of
Est. Egypt, p. 337.
l
Hussein, M.A., 2011. Anti-obesity, antiatherogenic, anti-diabetic and antioxidant
activities of J. montana ethanolic formulation in obese diabetic rats fed high-fat
diet. Free Radic. Antioxid. 1, 49–60.
Ibrahim, S.R., Ebel, R., Wray, V., Müller, W.E., Edrada-Ebel, R., Proksch, P., 2008.
Diacarperoxides, norterpene cyclic peroxides from the sponge Diacarnus
megaspinorhabdosa. J. Nat. Prod. 71, 1358–1364.
Jakupovic, J., Lehmann, L., Bohlmann, F., King, R.M., Robinson, H., 1988.
Sesquiterpene lactones and other constituents from Cassinia, Actinobole and
Anaxeton species. Phytochemistry 27, 3831–3839.
Kobayashi, M., Kondo, K., Kitagawa, I., 1993. Antifungal peroxyketal acids from an
Okinawan marine sponge of Plakortis sp. Chem. Pharm. Bull. 41, 1324–1326.
Mahmoud, A.A., 2006. Jasomontanone, a novel bicyclic sesquiterpene from the
leaves of Jasonia montana. Nat. Prod. Commun. 1, 15–19.
The inoculated 96-well plates were incubated at 37 °C for 24 h. The
chloramphenicol antibiotic (P98%, Sigma–Aldrich) was used as a
positive control reference in the assay. Extract-free solution was
used as a negative control, and 10% DMSO solution was used as
blank control.
As an indicator of microbial growth, p-iodonitrotetrazolium vio-
let salt (40 l
L) (INT, Sigma) (0.2 mg mLꢁ1) dissolved in H2O was
added to the wells. Plates were incubated at 37 °C for 1 h until
the purple color of formazan appeared. MIC values were recorded
as the lowest concentration of the extract that completely inhib-
ited microbial growth, i.e. a clear well.
Merfort, I., 1988. Acetylated and other flavonoid glycosides from Arnica chamissonis.
Phytochemistry 27, 3281–3284.
Mohamed, A.E.H.H., 2007. Jasonone, a nor-sesquiterepene from Jasonia montana. Z.
Naturforsch. B: Chem. Sci. 62, 125–128.
Shimoda, K., Kondo, Y., Nishida, T., Hamada, H., Nakajima, N., Hamada, H., 2006.
Biotransformation of thymol, carvacrol, and eugenol by cultured cells of
Eucalyptus perriniana. Phytochemistry 67, 2256–2261.
Soliman, F.M., Moussa, M.Y., Abdallah, H.M., Othman, S.M., 2009. Cytotoxic activity
of flavonoids of Jasonia Montana Vahl. (Botsch). (Astraceae) growing in Egypt.
Aust. J. Basic Appl. Sci. 3, 148–152.
Sung, T.V., Steffan, B., Steglich, W., Klebe, G., Adam, G., 1992. Sesquiterpenoids from
the roots of Homalomena aromatica. Phytochemistry 31, 3515–3520.
Täckholm, V., 1974. Students’ Flora of Egypt. Cairo University, Cooper Native
Printing Co., Beirut, p. 888.
Wang, Y.F., Wang, X.Y., Lai, G.F., Lu, C.H., Luo, S.D., 2007. Three new sesquiterpenoids
from the aerial parts of Homalomena occulta. Chem. Biodivers. 4, 925–931.
Weyerstahl, P., Marschall, H., Thefeld, K., Rustaiyan, A., 1999. Constituents of the
essential oil of Tanacetum (syn. Chrysanthemum) fruticulosum Ledeb. from Iran.
Flavour Frag. J. 14, 112–120.
Acknowledgements
The research described in this paper was supported by JSPS (Ja-
pan Society for the Promotion of Science) postdoctoral fellowship
(ID No. P10117), National Research Center, Cairo, Egypt, and the
Welch Foundation (D-1478).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.phytochem.2014.
03.027.
Ye, Q., Qin, G., Zhao, W., 2002. Immunomodulatory sesquiterpene glycosides from
Dendrobium nobile. Phytochemistry 61, 885–890.
Yosief, T., Rudi, A., Wolde-ab, Y., Kashman, Y., 1998. Two new C22 1,2-
dioxanepolyketides from the marine sponge Acarnus cf. bergquistae. J. Nat.
Prod. 61, 491–493.
Zaghloul, A.M., 1989. Methoxylated flavonoids from Jasonia montana (Vahl) Botsch.
Mansoura J. Pharm. Sci. 5, 27–35.
Zhang, Z.J., He, J., Li, Y., Wu, X.D., Gao, X., Peng, L.Y., Cheng, X., Li, R.T., Zhao, Q.S.,
2013. Three new sesquiterpenes from Laggera pterodonta. Helv. Chim. Acta 96,
732–737.
References
Abd El-Wahab, R.H., Zaghloul, M.S., Moustafa, A.A., 2004. Conservation of Medicinal
Plants in St. Catherine Protectorate, South Sinai. I. Evaluation of Ecological
Status and Human Impact. Proceedings of First International Conference on
Strategy of Egyptian Herbaria, Giza, Egypt, pp. 231–251.
Ahmed, A.A., 1991. Two geraniol derivatives from Jasonia montana. Pharmazie 46,
362–363.
Please cite this article in press as: Hegazy, M.-E.F., et al. Chemical constituents and their antibacterial and antifungal activity from the Egyptian herbal
medicine Chiliadenus montanus. Phytochemistry (2014), http://dx.doi.org/10.1016/j.phytochem.2014.03.027