Antibacterial and antifungal sesquiterpenoids from aerial parts of anvillea garcinii

Article abstract of DOI:10.3390/molecules25071730

Two new sesquiterpenoids belonging to the guaiane, 4α,9α,10α-trihydroxyguaia- 11(13)en-12,6α-olide (1), and germacrane, 9β-hydroxyparthenolide-9-O-β-D-glucopyranoside (2), classes have been isolated fromthe leaves of the Saudi medicinal plant Anvillea garcinii along with seven known compounds (3-9). The structures of the new metabolites were elucidated by spectroscopic analysis, including one-dimensional (1D) and two-dimensional (2D) Nuclear Magnetic Resonance (NMR) and high-resolution electrospray ionization mass spectrometry (HR-ESIMS). The antimicrobial properties of 1-9 were screened against seven different pathogenic microbes, and compounds 1-3 showed a potent antifungal activity.

Full text of DOI:10.3390/molecules25071730

molecules
Article
Antibacterial and Antifungal Sesquiterpenoids from
Aerial Parts of Anvillea garcinii
1
,
1,2
1
1
Shagufta Perveen *, Jawaher Alqahtani
, Raha Orfali , Hanan Y. Aati
,
1
1
3
4
Areej M. Al-Taweel , Taghreed A. Ibrahim , Afsar Khan , Hasan S. Yusufoglu ,
Maged S. Abdel-Kader 4 and Orazio Taglialatela-Scafati *
,5
2,
1
Department of Pharmacognosy, College of Pharmacy, King Saud University. P. O. Box 22452,
Riyadh 11495, Saudi Arabia; jalqahtani@ksu.edu.sa (J.A.); rorfali@ksu.edu.sa (R.O.);
hati@ksu.edu.sa (H.Y.A.); amaltaweel@ksu.edu.sa (A.M.A.-T.); tshehata@ksu.edu.sa (T.A.I.)
Department of Pharmacy, School of Medicine and Surgery, University of Naples Federico II, Via D.
2
Montesano 49, 80131 Naples, Italy
Department of Chemistry, COMSATS University Islamabad, Abbottabad Campus,
3
Abbottabad-22060, Pakistan; afsarhej@gmail.com
Department of Pharmacognosy, College of Pharmacy, Prince Sattam Bin Abdulaziz University, P.O. Box 173,
4
Al-Kharj 11942, Saudi Arabia; h.yusufoglu@psau.edu.sa (H.S.Y.); mpharm101@hotmail.com (M.S.A.-K.)
Department of Pharmacognosy, Faculty of Pharmacy, Alexandria University, Alexandria 21215, Egypt
5
*
Correspondence: shakhan@ksu.edu.sa (S.P.); scatagli@unina.it (O.T.-S.)
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editors: Pavel B. Drasar and Valeria Patricia Sülsen
Received: 9 March 2020; Accepted: 7 April 2020; Published: 9 April 2020
ꢀ
ꢁꢂꢃꢄꢅꢆ
Abstract: Two new sesquiterpenoids belonging to the guaiane, 4
α
,9
-D-glucopyranoside (
classes have been isolated from the leaves of the Saudi medicinal plant Anvillea garcinii along with seven
known compounds ( ). The structures of the new metabolites were elucidated by spectroscopic
analysis, including one-dimensional (1D) and two-dimensional (2D) Nuclear Magnetic Resonance
NMR) and high-resolution electrospray ionization mass spectrometry (HR-ESIMS). The antimicrobial
properties of were screened against seven different pathogenic microbes, and compounds
α
,10
α
-trihydroxyguaia-
11(13)en-12,6
α
-olide (1), and germacrane, 9β-hydroxyparthenolide-9-O-
β
2),
3–9
(
1–
9
1–3
showed a potent antifungal activity.
Keywords: Anvillea garcinii; Saudi medicinal plants; sesquiterpene lactones; antifungal activity
1
. Introduction
Traditional medicines are a powerful weapon for mankind and they have been used to treat
several health disorders since ancient times. The last few decades have witnessed a renaissance in
the use of natural products, and traditional medicine in general, to prevent or cure several ailments.
While they are considered one of the possible options in several countries, they are practically the main
therapeutic option in many developing countries, including those of the Arabian Peninsula.
Flora of Saudi Arabia is evidently and understandably not as rich and diverse as that of countries
of the Mediterranean basin; however, it has a vital role for various ecosystems, especially in maintaining
the environmental balance and stability [
largest plant families on the planet, which is a family that includes more than one thousand genera and
twenty thousand species [ ]. Asteraceae plants are well known for their biological and pharmacological
1]. The dominating plant family is Asteraceae, one of the
2
effects, largely ascribable to the presence of phytochemicals belonging to polyphenol, flavonoid,
and terpenoid classes [3].
Anvillea is probably the smallest genus of the Asteraceae family, since it includes only four species,
distributed in a large area spanning from North Africa to Iran, including several Middle Eastern
Molecules 2020, 25, 1730; doi:10.3390/molecules25071730
www.mdpi.com/journal/molecules

Molecules 2020, 25, 1730
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countries, such as Egypt, Palestine, and Saudi Arabia [
4]. In Saudi Arabia, Anvillea genus is represented
by the following two species: A. garcinii and A. radiata. A. garcinii DC (Arabic name Nuqd) is one
of the most important ethnomedicinal plants used in the Arabian Peninsula region, indicated for
symptomatic relief of various illnesses such as cold, gastrointestinal disorders, and respiratory system
problems [3]. Traditionally, the dried plant is crushed, mixed with honey or date and olive oil, and used
to treat cold symptoms [4].
Flavonoids and sesquiterpene lactones have been the predominant class of secondary metabolites
obtained by phytochemical studies on A. garcinii [
disclosed the presence of sesquiterpene lactones of the guaianolide- and germacranolide-types,
including the corresponding amino acid adducts, as well as some flavonoids glycosides [11 13].
5–11]. Our previous investigations on this plant
–
The class of Anvillea sesquiterpene lactones is dominated by derivatives with the parthenolide
skeleton, germacranolides endowed with significant biological activities in cancer and inflammation,
as well as in metabolic disorders [14]. Previous examination of the aerial parts of A. garcinii have
afforded several members of the parthenolide class, such as 9α- and 9β-hydroxyparthenolide, 9α-
and 9 -hydroxy-1 ,10 -epoxyparthenolide, parthenolid-9-one, and its cis-isomer [15]. In addition,
β
β
α
guaianolide-type sesquiterpenoids, a class of phytochemicals with a broad range of activities,
including cytotoxic, antiprotozoal, and anti-inflammatory potential [16], also constitute prominent
A. garcinii metabolites. Leucodin and zaluzanin C and their derivatives [17], as well as garcinamine E
and other guaianes [13], have been isolated from this species.
This richness of bioactive secondary metabolites prompted us to continue our phytochemical
and biological investigation of A. garcinii, with the final aim of obtaining a detailed picture of the
metabolome of this plant, providing solid scientific grounds for its use in traditional medicines and
possibly, to develop a new phytotherapeutic drug from this natural source. In this manuscript we
report the isolation of two new sesquiterpenoids belonging to the guaiane (4
1(13)en-12,6 -olide, ( )) and germacranolide (9 -hydroxyparthenolide-9-O-
)) classes, along with seven known compounds (
α
,9
α
,10
α-trihydroxyguaia-
1
α
1
β
β-D-glucopyranoside,
(2
3–
9
) (Figure 1) and the results of screening for
antimicrobial activity on the isolated metabolites.
OH
O
HO
HO
1
4
1'
OH
14
OH
OH O
H
OH
1
1
0
1
5
9
HO
4
6
H
O
11
4
6
11
13
H
15
OH
OH
13
O
1
2
O
15
12
O
O
O
O
3
1
2
HOOC OH
OH
OH
OR
O
O
HO
HO
O
O
OH
OH
O
OH
HO
OR
6
7
R = H
R = CH3
4
R = H
R = CH3
5
OH
R1O
O
8
9
R = β-D-glucose R1 = H
R = H R1 = β-D-glucose
OR
OH
O
Figure 1. Chemical structures of metabolites isolated from Anvillea garcinia.

Molecules 2020, 25, 1730
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2
. Results and Discussion
Previous investigations on A. garcinii aerial parts selected the n-butanol fraction of the methanol
extract as the richest in polar sesquiterpenoids. Its chromatographic separation was achieved using
a combination of Sephadex LH-20, silica gel, and RP-18 column chromatography, and yielded
two new (
were elucidated by spectroscopic analysis, mainly one-dimensional (1D) and two-dimensional (2D)
NMR and electrospray ionization mass spectrometry (ESIMS). Compounds were identified as
,4 ,10 -trihydroxy-11 -guai-1-en-12,6 -olide ( ) [18], chlorogenic acid ( ) [19], 3-O-feruloylquinic
acid ( ) [20], 1-O-caffeoyl- -D-glucopyranose ( ) [21], 1-O-feruloyl- -D-glucopyranose ( ) [22],
kaempferol-3-O-glucopyranoside ( ) [23], and kaempferol-7-O-glucopyranoside ( ) [24] by a
1–2) and seven known compounds (3–9) (Figure 1). The structures of these metabolites
3–9
3
α
α
β
β
α
3
4
5
β
6
β
7
8
9
comparison of their spectroscopic data with those reported in the literature. Selected spectra of
these compounds are reported as Supplementary Materials. All these phenolic derivatives are reported
from A. garcinii for the first time.
Compound
1 was isolated as a white solid with the molecular formula C H O , determined
15 22 5
−
by high-resolution electrospray ionization mass spectrometry (HR-ESIMS) (m/z 281.1400 [M–H] ;
1
calculated for C H O , 281.1394), indicating five unsaturation degrees. The H NMR spectrum of 1
1
5
21
5
2
showed the presence of a pair of sp methylene protons (
δ 5.90 and 5.36, each bs); two oxygenated
H
methine protons at
δ 3.58 (1H, d, J = 2.5 Hz) and 4.17 (1H, dd, J = 9.5, 11.0 Hz); three relatively
H
deshielded methines at
methylene protons, and two methyl singlets at
δ 2.00 (1H, brd, J = 11.0 Hz), 2.87 (overlapped), and 2.84 (overlapped); three
H
δ
H
0.95 and 1.10 (see Table 1). The 13C NMR spectral
data of
of one ester carbonyl at
1, which was analyzed with the help of the 2D NMR HSQC spectrum, disclosed the presence
2
2
δ
C
170.8; one sp methylene at δ 118.7, and an additional unprotonated sp
C
carbon at
δ
139.8; two oxymethine carbons at
δ
82.6 and 76.0 and two unprotonated oxygenated
C
C
3
carbons at 79.6 and 76.6. The remaining carbon atoms were assigned as three sp methylenes at
δ
C
C
3
2
2
4.6, 31.6, and 40.3, three sp methines at
1.7 and 22.0.
δ 40.5, 40.7, and 54.6, and two methyl carbon atoms at
C
δ
The guaianolide-type skeleton of compound
1 was assembled on the basis of the 2D NMR COSY
and heteronuclear multiple bond coherence (HMBC) spectra. The COSY spectrum disclosed the single
extended spin system (highlighted in red in Figure 2), which was arranged on the bicyclic system on the
basis of the HMBC correlations from methyl singlets H -14 and H -15. The HMBC correlations from
3
3
the methylene H-13 to C-12, the nuclear Overhauser enhancement spectroscopy (NOESY) correlations
H-6/H -15, H-6/H -14, and H-9/H -14, and the remaining NOESY correlation of 1, shown in Figure 2,
3
3
3
indicated the relative configuration of compound
1. Thus, using all the above-mentioned data,
compound 1 was elucidated as 4α,9α,10α-trihydroxyguaia-11(13)-en-12,6α-olide.
Figure 2. Some key two-dimensional (2D) NMR correlations of
1 (left, correlated spectroscopy (COSY)
and heteronuclear multiple bond coherence (HMBC) and right, nuclear Overhauser enhancement
spectroscopy (NOESY)).

Molecules 2020, 25, 1730
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Interestingly, the presence of
1
as a component of the sesquiterpenoid pool of A. garcinii was
anticipated by us at the time of isolation of garcinamine E [13]. Indeed, garcinamine E, co-occurring in
the n-butanol fraction of the leaves of A. garcinii, is the L-proline adduct at C-13 of 1.
1
13
Table 1. H (500 MHz) and C (125 MHz) NMR data for compounds 1 and 2 in CD OD.
3
1
2
Pos.
δ
(mult., J in Hz)
2.80 (m)
δ
δ
(mult., J in Hz)
δ
C
H
C
H
1
40.5
24.6
5.56 (dd, 9.5, 11.5)
2.61 (m)
129.4
23.5
2α
1.64 (dddd, 1.5, 3.5, 7.5, 12.5)
1.49 (tdd, 2.0, 6.5, 12.5)
1.53 (m)
2
β
2.22 (m)
2.13 (m)
3
40.4
35.9
1.22 (m)
4
5
6
7
-
79.6
54.6
82.6
40.7
31.6
-
76.0
76.6
139.8
170.8
118.7
-
-
61.8
65.9
81.4
48.2
35.0
-
83.1
133.9
41.7
178.7
12.0
2.00 (t, 11.0)
4.17 (dd, 9.5, 11.0)
2.87 (ddd, 9.5, 3.5, 1.5)
1.99 (ddd, 2.5, 3.5, 9.2)
2.79 (d, 9.0)
4.04 (t, 9.0)
2.11 (m)
8
α
2.09 (dd, 2.1, 9.2)
1.98 (bdd, 9.2, 7.5)
4.37 (dd, 7.5, 2.1)
-
2.49 (dt, 2.5, 7.0)
-
8
β
1.53 (ddd, 1.0, 1.5, 9.2)
9
3.58 (dd, 2.5, 1.0)
1
1
1
0
1
2
-
-
-
1
1
3a
3b
5.90 (brs)
5.36 (brs)
0.95 (s)
1.10 (s)
1.27 (d, 7.0)
1
1
4
5
21.8
22.0
1.76 (s)
1.36 (s)
10.0
16.3
98.7
73.5
76.5
70.3
76.7
61.4
0
0
0
0
0
1
2
3
4
5
4.11 (d, 7.5)
3.23 (t, 7.5)
3.17 (m)
3.29 (m)
3.31 (m)
3.67 (m)
3.86 (m)
0
6
6
a
b
0
Compound
2
was obtained as an optically active ([
α
]25 D =
−
55, c = 0.10, CH OH) yellow
3
13
gummy solid with molecular formula C H O , as established by HR-ESIMS. The C NMR spectrum,
21
32
9
which was interpreted taking into account data from the 2D NMR HSQC and HMBC experiments,
indicated the presence of one ester carbonyl ( C 178.7); two olefinic carbons ( C 129.4 and 133.9); one
anomeric carbon ( 98.7); seven additional oxygenated methines ( C 83.1, 81.4, 76.7, 76.5, 73.5, 70.3
61.8); one oxygenated methylene ( C 61.4); and
δ
δ
δ
C
δ
and 65.9); one oxygenated unprotonated carbon (
δ
δ
C
3
eight additional sp carbons, including two methines, three methylenes, and three methyls (Table 1).
1
2
Accordingly, the H NMR spectrum of
2 (Table 1) showed a sp methine signal at δ 5.56, a series of
H
oxymethine and oxymethylene protons between
δ
H
4.37 and 3.17, and three methyl signals, namely a
deshielded singlet at
δ 1.76, a singlet at δ 1.36, and a doublet at δ 1.27. These data were indicative
H H H
of the sesquiterpene lactone glycoside nature for compound 2, whose structure was assigned on
the basis of a detailed inspection of the 2D NMR correlations and comparison with data of known
compounds [12].
In particular, the COSY spectrum of
2
revealed the presence of three spin systems, namely (i) from
2
the sp methine H-1 to H -3, (ii) from H-5 to H-9 including the H-11/H -13 branching, and (iii) the
2
3
0
0
sugar spin system (H-1 to H -6 ). The sugar unit was assigned as a
of the coupling constant H-1 /H-2 (J = 7.5 Hz) and acid hydrolysis of
β-D-glucopyranoside on the basis
2
0
0
2, which afforded the free sugar
unit, identified as D-glucose through co-TLC and optical rotation sign.
The HMBC correlations of
2
were instrumental to join the above deduced fragments. In particular,
3
2
J cross-peaks of H -14 with C-1, C-9, and the unprotonated sp C-10 and of H -15 with C-3, C-4,
3
3

Molecules 2020, 25, 1730
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and C-5 indicated the presence of the ten-membered ring. Moreover, cross-peaks of both H-6 and
H -13 with the ester carbonyl carbon C-12 indicated the presence of the lactone ring. The linkage
3
3
0
of the D-glucopyranose moiety at position 9 was indicated by the J HMBC correlation of H-1
4.10) with C-9 ( 83.1). The presence of an epoxyde ring at C-4/C5, which accounted for the
(δ
δ
C
H
remaining unsaturation degree, was in perfect agreement with the NMR resonances of the involved
carbons (C-4 = 61.8 ppm and C-5 = 65.9 ppm). The NOESY correlations were, then, used to deduce
the relative configuration of compound 2. In particular, NOESY cross-peak of H-1 with H -14
3
indicated the Z configuration of the endocyclic double bond. The relative configuration of the five
consecutive stereogenic centers (C-4 to C-11) was deduced on the basis of the following NOESY
correlations: H -15/H-6, H-5/H-7, H -3/H-5, and H-7/H -13. Finally, the NOESY cross-peak of H-7
3
2
3
with H-9 pointed to the
α
-orientation of both protons. Thus, compound
2
was identified as the new
-E isomer of has been recently
isolated from Asteriscus graveolens [25] and, accordingly, its reported C NMR resonances for C-1 and
C-10 showed significant differences as compared with those of . Both compounds and are close
1,10
1Z-9
β-hydroxyparthenolide-9-O-
β-D-glucopyranoside. The
∆
2
13
2
1
2
analogues of sesquiterpenoids previously isolated from the same species and we have assumed that
they share the absolute configuration of their co-occurring analogues.
The isolated metabolites (1–9) were evaluated for their antimicrobial activity against pathogenic
bacteria and fungi (Tables 2 and 3). Compounds
pathogenic fungi, with a growth inhibitory activity around 80% at 50
and C. parapsilosis, respectively. The respective minimum inhibitory concentrations (MIC) of
1–3 showed antifungal activities against human
−
1
µg mL against Candida albicans
1–3
−1
were 0.21, 0.26, and 0.38 µg mL against C. albicans and 0.25, 0.31, and 0.34 against C. parapsilosis.
This finding is in agreement with our previously reported results on antifungal activity of guaianolide
sesquiterpenoids [26]. In addition, 1–3 also showed activity against Gram-positive and Gram-negative
−1
pathogenic bacteria with MIC ranging from 2.3 to 6.3 µg mL (Table 3). Chlorogenic acid (4), a
non-sesquiterpenoid, showed a significant inhibition against pathogenic fungi (Table 2) and strong
antibacterial activity against the Gram-negative bacteria E. xiangfangensis and E. fergusonii. Ester
−1
derivatives 6 and 7 showed neither antifungal nor antibacterial activity at 25 µg mL
.
Table 2. Antifungal activity of compounds 1–9.
Growth Inhibition
MIC (µg mL 1)
−
Compound
(%, mean ± SD) *
C. albicans
C. parapsilosis
C. albicans
C. parapsilosis
1
2
3
4
5
6
7
8
83.4 ± 3.3
79.8 ± 5.3
85.0 ± 3.4
61.2 ± 3.3
23.6 ± 5.2
19.5 ± 2.9
15.8 ± 3.2
42.7 ± 4.4
45.3 ± 3.7
54.7 ± 2.6
81.3 ± 2.6
76.5 ± 4.5
80.0 ± 2.7
69.5 ± 2.4
18.9 ± 3.7
21.7 ± 3.4
10.9 ± 4.7
51.8 ± 2.5
49.9 ± 4.8
51.5 ± 4.1
0.21 ± 0.04
0.26 ± 0.07
0.38 ± 0.03
0.89 ± 0.02
0.68 ± 0.01
0.73 ± 0.08
0.97 ± 0.12
0.74 ± 0.05
0.68 ± 0.08
0.29 ± 0.06
0.25 ± 0.05
0.31 ± 0.02
0.34 ± 0.06
0.61 ± 0.08
0.79 ± 0.03
0.86 ± 0.07
0.79 ± 0.06
0.62 ± 0.03
0.74 ± 0.02
0.33 ± 0.04
9
Itraconazole
*
Results expressed as mean ± standard deviation (SD).

Molecules 2020, 25, 1730
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Table 3. Antibacterial activity of compounds 1–9.
MIC (µg mL 1)
−
Compound
Staphilococcus
Bacillus
licheniformis
Escherichia
xiangfangensis
Escherichia
fergusonii
Pseudomonas
aeruginosa
aureus
1
2
3
4
5
6
7
8
9
2.3
3.4
5.2
>25
>25
>25
>25
9.4
2.3
3.1
4.4
>25
>25
>25
>25
>25
7.5
>25
>25
3.8
5.7
6.3
>25
4.6
>25
>25
>25
6.8
>25
>25
>25
>25
>25
>25
>25
>25
>25
0.523
5.2
>25
>25
>25
>25
>25
0.523
>25
0.523
8.4
0.523
Amikacin
0.523
*
Results expressed as mean ± standard deviation (SD).
3
. Materials and Methods
3
.1. General
Optical rotations were measured in analytical grade methanol using a JASCO P-2000 polarimeter
JASCO, 2967-5, Tokyo, Japan). The 1D and 2D NMR data were acquired using a Bruker AVANCE
(
1
13
spectrometer (Bruker, Billerica, MA, USA) (500 MHz for H and 125 MHz for C). Chemical shifts
) in ppm, relative to tetramethylsilane, were calculated basing on the residual solvent signal, and J
(
δ
scalar coupling constants are reported in Hz (Hertz). The ESI-MS analyses were measured on an Triple
Quadrupole 6410 QQQ LC/MS mass spectrometer (Agilent, Santa Clara, CA, USA) with ESI ion source
◦
(
gas temperature was 350 C, nebulizer pressure was 60 psi, and gas flow rate was 12 L/min), operating in
the negative and positive scan modes of ionization through direct infusion method using CH OH H O
\
3
2
(
4:6 v/v) at a flow rate of 0.5 mL/min. Column chromatography procedures were performed using silica
gel 70–230 mesh, RP-18, Sephadex LH-20 (each from; E. Merck, Darmstadt, Germany). TLC analysis
was performed using precoated silica gel 60 F254 and RP-18 (Merck, Darmstadt, Germany) plates,
and spots were visualized via exposure under UV light (254/365 nm) and by spraying with different
spray reagent. Analytical grade solvents and reagents were purchased from Sigma-Aldrich (St. Louis,
MO, USA). Deuterated methanol (CD OD-d) and dimethylsulfoxide (DMSO-d ) were obtained from
3
6
Cambridge Isotope Laboratories (Tewksbury, MA, USA).
3
.2. Plant Material
The aerial parts of A. garcinii were collected in March 2018 in the area 17 km South West of Al-Kharj
city and identified by taxonomist, Dr. M. Atiqur Rahman, College of Pharmacy, Medicinal, Aromatic
and Poisonous Plants Research Center, King Saud University. A voucher specimen (PSAU-CPH-6-2018)
is kept in the herbarium of College of Pharmacy, Prince Sattam Bin Abdulaziz University.
3
.3. Extraction and Isolation
The shade dried powdered aerial parts of A. garcinii (0.5 kg) were extracted with methanol at room
temperature (3
×
2.5 L). Total methanol extract was concentrated under reduced pressure using a rotary
evaporator (Büchi Rotavapor RII, Flawil, Switzerland). The crude extract (50 g) was suspended in H O
2
(
0.5 L) and extracted successively with chloroform and n-butanol, and then the residual water fraction
was lyophilized. The n-butanol soluble fraction (30 g) was subjected to a silica gel open column and
eluted with a gradient of CH Cl :CH OH (9.5:0.5 1.0:9.0), to afford nine major fractions 1–9 based on
their TLC image. Fraction 3 (0.8 g) was further subjected to RP C-18 column chromatography, eluted
under medium pressure with a gradient of water/methanol (4.0:6.0 9.0:1.0), to obtain two subfractions
which was further loaded on a RP C-18 column and eluted with a gradient mixture of water/methanol
→
2
2
3
→

Molecules 2020, 25, 1730
7 of 10
(
6.0:4.0
RP C-18 column using water/methanol (8.0:2.0
was rechromatographed on a Sephadex LH-20 column with water/methanol (1:1–100:0) to afford two
compounds (10 mg) and (12 mg). Fraction 7 (0.5 g) was separated on a Sephadex LH-20 column with
a gradient mixture of water/methanol (9.0:1.0 7.0:3.0), and finally was divided on a silica gel column
with CHCl /MeOH (8.5:1.5) to afford compounds (6.0 mg) and (11 mg). Subfraction 8 (0.2 g) was
further purified by HPLC (flow rate 1 mL/min, wavelength 254 nm, and CH OH–0.01%HCOOH/H O,
→
1.0:9.0), which yielded compounds
1
(10 mg) and
3
(8 mg). Fraction 5 (0.3 g) was applied to a
→1.0:1.0) to yield compound 2 (15 mg). Fraction 6 (0.6 g)
4
5
→
6
7
3
3
2
4:6) to afford 8 (15 mg, Rt 25.5 min). Subfraction 9 (0.1 g) was purified by HPLC (flow rate 1.0 mL/min,
wavelength 254 nm, CH OH–0.01%HCOOH/H O, 1:1) to afford compound 9 (12 mg, Rt 27.5 min).
3
2
2
5
4
α
,9
α
,10
α
-Trihydroxyguaia-11(13)en-12,6
MeOH); UV (MeOH), λmax 223 nm (
in CD OD) see Table 1; negative ions ESIMS, m/z 281.1400 [M–H] , calculated for C H O , 281.1394.
-Hydroxyparthenolide-9-O-
c 0.10, MeOH); UV (MeOH), λmax 211 nm (
125 MHz, in CD OD) see Table 1; positive ions ESIMS, m/z 451.1948 [M+ Na] , calculated for
α
-olide (
1
): Yellow gummy solid, [
α
]D + 72 (c 0.10,
1
13
ε
4000); H NMR (500 MHz, in CD OD) and C NMR (125 MHz,
3
−
3
15 21
5
25
1
Z-9
β
β
-D-glucopyranoside (
2
): Yellow gummy solid, [
α
]
+ 55
D
1
13
(
(
ε
3200); H NMR (500 MHz, in CD OD) and C NMR
3
+
3
C H NaO , 451.1944.
21
32
9
3
.4. Acid Hydrolysis of 2
Compound
2
(3.0 mg) was dissolved in 0.6 mL of a solution of 1N HCl–methanol (1:1). The mixture
◦
was heated at 65 C for 60 min and concentrated in vacuo, water was added and the whole
was extracted with ethyl acetate. The aqueous portion was filtered, the filtrate was evaporated,
and D-glucose (0.9 mg, 71%) was identified from the sign of its optical rotation ([
25
α
]
+ 52.0) and
D
co-TLC (n-butanol/water/acetic acid, 8:2:10, Rf 0.17) with an authentic sample of D-glucose (Merck)
using anisaldehyde as spray reagent for visualization.
3
.5. Antibacterial Bioassay
The antibacterial activity was determined according the reported method [27]. Muller Hinton agar
plate contained microorganisms after suspension in a nutrient broth for 24 h wells were created in the
plate and loaded with 10 L of the sample solution obtained using DMSO as solvent. Amikacin was used
µ
as standard antibiotic for five different pathogenic bacteria two were Gram-positive, i.e., Staphylococcus
aureus (CP011526.1) and Bacillus licheniformis (KX785171.1) and three were Gram-negative, i.e.,
Enterobacter xiangfangensis (CP017183.1), Escherichia fergusonii (CU928158.2), and Pseudomonas aeruginosa
(
NR-117678.1).
The clear area which was free of microbial growth was measured three times to detect the
diameter of the zone of inhibition and the mean were recorded. The minimal inhibitory concentration
−1
(
MIC, µg mL ) of the tested isolated compounds that inhibited the visible bacterial growth was
calculated using varying concentrations of the tested compounds following the broth microdilution
method [27,28].
3
.6. Antifungal Assay
Well diffusion and broth microdilution techniques were used in this study to detect the antifungal
activity of the isolated compounds. According to Gong and Guo [29], in SDA plate the sample solutions
6
−1
(
100
µ
L), approximately 3
×
10 colony-forming units (CFU) mL was smeared of two pathogenic
fungi, Candida albicans and Candida parapsilosis. Wells were created in SDA plates and loaded with the
◦
1
0
µ
g of the tested compounds dissolved in DMSO and incubated at 37 C for 1 day. Itraconazole was
used as a standard antifungal and the diameters (in mm) of zone of inhibition were measured. The rates
of growth inhibition were obtained according to the following formula taking into consideration
as means:
±
SD
%
Growth inhibition rate = (dc − ds) / (dc − d ) × 100
0

Molecules 2020, 25, 1730
8 of 10
where d
c
is the diameter of the untreated control fungus, d
s
is the diameter of the sample-treated
fungus, and d is the diameter of the fungus cut.
0
The minimal inhibitory concentration (MIC) of the isolated compounds 1–9 against Candida
albicans and C. parapsilosis was determined by using varying concentrations of the tested compounds
following the broth microdilution method following the instructions of the Clinical and Laboratory
Standards Institute. Serial dilution of the isolated compounds was prepared into two-fold using sterile
Roswell Park Memorial Institute (RPMI) 1640 medium with MOPS (0.165 mol L-1) and presence of
◦
glucose (2%). The 96-well microplates were performed and incubated at 37 C for 24 h.
3
.7. Statistical Analysis
Data analysis was expressed as mean
±
standard deviation (SD) of three replicates.
Where applicable, the data were subjected to one-way analysis of variance (ANOVA). According to a
Microsoft Excel 2010 statistical package analyses, the significant differences were considered statistically
significant P values < 0.05.
4
. Conclusions
This investigation on the Saudi Arabia plant A. garcinii yielded two additional members of the
sesquiterpene lactone class, a new guaianolide, anticipated as precursor of garcinamine E, and a new
parthenolide glycoside. Both of these compounds have been evidenced to have a significant antifungal
activity, complementing that which had already been revealed for previously isolated congeners of the
same family. Therefore, this study clearly evidences the potential of even a single plant to provide a
countless list of bioactive phytochemicals and, more in general, of the Saudi Arabian flora to enrich
the global phytochemical effort. Although less diverse than others, Saudi Arabian flora is well worth
of being studied in detail, also to provide scientific basis to the traditional use of medicinal plants in
this area.
Supplementary Materials: The following are available online at http://www.mdpi.com/1420-3049/25/7/1730/s1.
Figures S1 and S2: 1H and C NMR spectra of compound
13
1
. Figures S3 and S4: DEPT-90 and DEPT-135 of
. Figure S7: negative ESIMS
. Figures S8 and S9: H and C NMR spectra of compound . Figures S10 and S11:
DEPT-135 and DEPT-90 of compound . Figures S12 and S13: 2D NMR COSY and HSQC spectra of compound
Figure S14: positive ESIMS spectrum of compound
compound
1. Figures S5 and S6: 2D NMR COSY and HSQC spectra of compound 1
1
13
spectrum of compound
1
2
2
2.
1
13
2. Figures S15 and S16: H and C NMR spectra of compound
1
13
3
. Figures S17 and S18: H and C NMR spectra of compound
4
. Figure S19: DEPT-90 of compound
4. Figures S20
and S21: H and 13C NMR spectra of compound
1
5. Figures S22 and S23: H and 2D HSQC spectra of compound
1
6
.
Author Contributions: Investigation, J.A., R.O., H.Y.A., A.M.A.-T., T.A.I., A.K., H.S.Y., M.S.A.-K.; writing—original
draft preparation, S.P. and O.T.S.; writing—review and editing, S.P. and O.T.S.; funding acquisition, S.P. and O.T.-S.
All authors have read and agreed to the published version of the manuscript.
Funding: The authors would like to extend their sincere appreciation to the Deanship of Scientific Research at
King through the “Research Group Project no RGP-221”. J. Alqahtani thanks the supporting of EJSP for her PhD
program at the University of Naples Federico II.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are available from the authors.
©
2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).