Anti-inflammatory metabolites from endophytic fungus Fusarium sp

Article abstract of DOI:10.1016/j.phytol.2018.11.024

Three new ergosterol derivatives, namely, fusaristerols B [(22E,24R)-3-palmitoyl-19(10→6)-abeo-ergosta-5,7,9,22-tetraen-3β-ol] (1), C [(22E,24R)-ergosta-7,22-diene-3β,6β,9α-triol] (3), and D [(22E,24R)-ergosta-7,22-diene-3β,5α,6β,9α-tetraol 6-acetate] (4), along with (22E,24R)-5β,8β-epidioxyergosta-22-en-3β-yl decanoate (2) and (22E,24R)-ergosta-7,22-dien-3β-ol (5), were isolated and characterized from the endophytic fungus Fusarium sp. isolated from Mentha longifolia L. (Labiatae) roots growing in Saudi Arabia. The structures of the isolated metabolites were verified based on UV, IR, NMR (1D and 2D), HRMS, and a comparison with the literature. All the metabolites were assessed for 5-lipoxygenase (5-LOX) inhibitory potential. Compounds 1 and 2 possessed 5-LOX inhibitory potential with an IC₅₀s of 3.61 and 2.45 μM, respectively, compared to that of indomethacin (IC₅₀ 1.17 μM). In addition, their structure-activity relationship was discussed.

Full text of DOI:10.1016/j.phytol.2018.11.024

PhytochemistryLetters29(2019)104–109
Contents lists available at ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
Anti-inflammatory metabolites from endophytic fungus Fusarium sp
⁎
Maan T. Khayata, Sabrin R.M. Ibrahimb,c, Gamal A. Mohamedd,e, , Hossam M. Abdallahd,f
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, King Abdulaziz University, Jeddah 21589, Saudi Arabia
b Department of Pharmacognosy and Pharmaceutical Chemistry, College of Pharmacy, Taibah University, Al Madinah Al Munawwarah 30078, Saudi Arabia
c Department of Pharmacognosy, Faculty of Pharmacy, Assiut University, Assiut 71526, Egypt
d Department of Natural Products and Alternative Medicine, Faculty of Pharmacy, King Abdulaziz University, Jeddah 21589, Saudi Arabia
e Department of Pharmacognosy, Faculty of Pharmacy, Al-Azhar University, Assiut Branch, Assiut 71524, Egypt
f Department of Pharmacognosy, Faculty of Pharmacy, Cairo University, Cairo 11562, Egypt
A R T I C L E I N F O
A B S T R A C T
Keywords:
Three new ergosterol derivatives, namely, fusaristerols B [(22E,24R)-3-palmitoyl-19(10→6)-abeo-ergosta-
Fusarium sp.
5,7,9,22-tetraen-3β-ol] (1), C [(22E,24R)-ergosta-7,22-diene-3β,6β,9α-triol] (3), and D [(22E,24R)-ergosta-7,22-
diene-3β,5α,6β,9α-tetraol 6-acetate] (4), along with (22E,24R)-5β,8β-epidioxyergosta-22-en-3β-yl decanoate (2)
and (22E,24R)-ergosta-7,22-dien-3β-ol (5), were isolated and characterized from the endophytic fungus Fusarium
sp. isolated from Mentha longifolia L. (Labiatae) roots growing in Saudi Arabia. The structures of the isolated
metabolites were verified based on UV, IR, NMR (1D and 2D), HRMS, and a comparison with the literature. All
the metabolites were assessed for 5-lipoxygenase (5-LOX) inhibitory potential. Compounds 1 and 2 possessed 5-
LOX inhibitory potential with an IC50s of 3.61 and 2.45 μM, respectively, compared to that of indomethacin (IC50
1.17 μM). In addition, their structure-activity relationship was discussed.
Ergosterol
Fusaristerol
Endophytic fungi
Anti-inflammatory
5-Lipoxygenase inhibitory
Structural activity relationship
1. Introduction
are bacteria or fungi that live inside plant tissues without causing da-
mage or disease symptoms to their hosts (Elkhayat et al., 2016; Ibrahim
The inflammation process is regulated by a precisely modulated
reaction between the cells and inflammatory mediators (Chen et al.,
2018). The inflammatory mediators include lipoxygenase (LOXs) and
cyclo-oxygenases (COX-1 and 2) enzymes, cytokines, such as tumour
necrosis factor (TNF)-α and the interleukins (ILs), nitric oxide (NO),
prostaglandin E2 (PGE2), and the transcription factor designated as the
nuclear factor (NF)-κB. They are released from the activated in-
flammatory cells (neutrophils, eosinophils, mononuclear phagocytes,
and macrophages) (Al-Attas et al., 2015; Nguyen et al., 2015). The 5-
LOX enzyme catalyses the biosynthesis of leukotrienes (LTs) from ara-
chidonic acid (AA) (Rådmark et al., 2015). The LTs play a significant
role in numerous inflammatory diseases, such as rheumatoid arthritis,
ulcerative colitis, asthma, and atherosclerosis, as well as several types
of cancers (Akdis et al., 2016; Radmark et al., 2007). Therefore, 5-LOX
inhibition has become the focus of many therapeutic approaches to
treat many proliferative and inflammatory diseases (Mashima and
Okuyama, 2015). Recently, the search for bioactive secondary meta-
bolites from edible and medicinal fungi and the study of their me-
chanisms on chronic diseases, such as inflammatory disorders, have
progressively increased (Chen et al., 2017). Endophytic microorganisms
et al., 2015). Early investigations of the genus Fusarium have proven
that it is a rich source of secondary metabolites with diverse bioactiv-
ities (Ibrahim et al., 2016a,b,c; 2018a,b,c,d,e). Our previous study of
Fusarium sp. led to the isolation of tetracyclic triterpenoids, a peptide,
and ergosterol derivatives (Ibrahim et al., 2016b, c; 2018a,c,e). During
our ongoing research to discover potentially active compounds from
Fusarium sp., the chemical investigation of its CHCl3/MeOH extract
resulted in the separation and identification of three new metabolites,
fusaristerols B–D (1, 3, and 4), together with two known compounds (2
and 5). Their structures were verified by various spectroscopic
methods. In addition, the 5-LOX inhibitory capacity of these metabo-
lites was assessed and their structure-activity relationship was dis-
cussed.
2. Results and discussion
2.1. Purification of the metabolites
The endophytic fungus Fusarium sp. was cultured on rice medium.
The rice culture CHCl3/MeOH extract was submitted to various
⁎ Corresponding author at: Department of Natural Products and Alternative Medicine, Faculty of Pharmacy, King Abdulaziz University, Jeddah 21589, Saudi
Arabia.
E-mail address: gahussein@kau.edu.sa (G.A. Mohamed).
https://doi.org/10.1016/j.phytol.2018.11.024
Received 21 October 2018; Received in revised form 21 November 2018; Accepted 27 November 2018
1874-3900/©2018PhytochemicalSocietyofEurope.PublishedbyElsevierLtd.Allrightsreserved.

M.T. Khayat et al.
PhytochemistryLetters29(2019)104–109
Fig. 1. Structures of the new isolated compounds 1, 3, and 4.
chromatographic techniques (SiO2, RP-18, and Sephadex LH-20) to
produce three new (1, 3, and 4) and two known metabolites (2 and 5)
(Fig. 1). Their structures were verified using various spectral data
analyses.
and C-14 were observed (Fig. 2). In addition, the 1H and 13C signals at
δ
H 5.20 (d, J = 15.2, 7.0, H-22)/δC 135.7 (C-22) and 5.25 (d, J = 15.2,
7.0, H-23)/132.2 (C-23) demonstrated the presence of a trans-config-
ured di-substituted olefinic bond (West and Cardellina, 1988). The NMR
data of 1 were comparable to those of an ergosteroid with a rare ar-
omatized ring B: 19(10→6)-abeo-ergosta-5,7,9,22-tetraen-3β-ol, which
was previously reported from the amoeba Acanthamoeba polyphaga
(Bisseret et al., 1987) and the endophytic fungus Colletotrichum sp.
(Wen et al., 2009). In addition, characteristic signals for the carbonyl
group δC 171.0 (C-1`), triplet methylene δH 2.35 (J = 7.2 Hz, H-2`),
multiplet methylenes at δH 1.23–1.29, and a terminal methyl group at
δH 0.84 (t, J = 6.6 Hz, H-16`) indicated the existence of a fatty acyl
moiety in 1. It was assigned to consist of C16 based on the alkaline
hydrolysis of 1, which produced the palmitic acid methyl ester that was
identified by GC- and EIMS analyses and confirmed by the HRESIMS
fragment peak at m/z 394.3227 [M+H-C16H31O]+. The downfield shift
of H-3 (δH 4.45) and the HMBC cross peak of H-3/C-1` revealed the
connectivity of the fatty acyl moiety at C-3 (Fig. 2). The relative con-
figuration at the stereocenters was assigned based on the comparison of
the 1H and 13C NMR chemical shifts, as well as the J values of 1 with the
related derivatives (Wen et al., 2009; Bisseret et al., 1987). Therefore,
the structure of 1 was determined to be (22E,24R)-3-palmitoyl-19(10→
6)-abeo-ergosta-5,7,9,22-tetraen-3β-ol and designated fusaristerol B.
Compound 3 was obtained as a white amorphous powder. Its mo-
lecular formula C28H47O3 was established by the HRESIMS pseudo-
molecular ion peak at m/z 431.3522 (calcad for C28H47O3, 431.3525 [M
+H]+) that requires six double bond equivalent (DBE). The IR spec-
trum of 3 showed absorption bands at 3437 (OH), 2961 (C–H aliphatic),
and 1655 (C]C) cm−1. It exhibited UV absorption at 205 and 246 nm.
The 13C and HSQC spectra of 3 revealed the presence of 28 carbons: six
methyls, seven methylenes, eleven methines, including two
2.2. Structural characterization of the isolated metabolites
Compound 1 was obtained as a white amorphous powder. The UV
absorption at λmax 215 and 285 nm revealed the presence of an aro-
matic moiety in 1 (Bisseret et al., 1987; Wen et al., 2009; Yan et al.,
2011). The IR spectrum showed bands at 3005, 2896, 1725, 1605, and
1495 cm−1. Its HRESIMS exhibited a pseudo-molecular ion peak at m/z
633.5604 [M+H]+ (calcd for 633.5611, C44H73O2), corresponding to
the molecular formula C44H72O2. This formula required nine degrees of
unsaturation, which accounted for a benzene moiety, three rings, an
olefinic double bond, and a carbonyl group. The 13C NMR and HSQC
spectra revealed 44 carbon resonances: seven methyls, 21 methylenes,
nine methines, including an aromatic moiety (δC 124.0, C-7) and an
oxymethine (δC 71.3, C-3) carbon, and seven quaternary carbons five of
them for aromatic carbons (Tables 1 and 2). The 1H NMR spectrum
showed four doublet methyls at δH 1.07 (H-21), 0.85 (H-26), 0.87 (H-
27), and 0.94 (H-28), a singlet methyl (0.59, H-18), and an aromatic
methyl (2.10, H-19), correlating to the carbons at δC 21.1, 19.5, 19.9,
17.8, 11.5, and 19.5, respectively in the HSQC. The 1H-1H COSY cross
peaks observed, as well as the HMBC correlations of H-18/C-12, C-14,
and C-17, H-19/C-6, C-5, and C-7, H-21/C-17 and C-22, H-26 and H-27/
C-25 and C-24, and H-28/C-23 and C-25 guaranteed the assignment of
the position of the methyl groups. In addition, an aromatic proton at δH
6.66 (H-7) and an oxymethine proton at δH 4.45 (H-3) were observed
with HSQC cross peaks to the carbons at 124.0 (C-7) and 71.3 (C-3). In
the HMBC, correlations from H-3 to C-5 and C-1 and H-7 to C-5, C-9,
105

M.T. Khayat et al.
PhytochemistryLetters29(2019)104–109
Table 1
Table 2
1H NMR spectral data of 1, 3, and 4 (CDCl3, 400 MHz).
13C NMR spectral data of 1, 3, and 4 (CDCl3, 100 MHz).
No.
1
3
4
No.
1
3
4
δH [mult., J (Hz)]
δH [mult., J (Hz)]
δH [mult., J (Hz)]
δC (mult.)
δC (mult.)
δC (mult.)
1
2
2.73 m
2.75 m
2.00 m
1.75 m
4.45 m
3.07 m
2.58 m
–
1.73 m
1.25 m
1.62 m
1.45 m
1.85 m
1.30 m
1.47 m
1
24.5 CH2
31.5 CH2
71.3 CH
36.5 CH2
129.8 C
31.0 CH2
33.2 CH2
67.9 CH
39.6 CH2
51.9 CH
73.8 CH
117.7 CH
144.2 C
76.1 C
30.7 CH2
32.1 CH2
66.3 CH
39.5 CH2
74.4 C
2
3
4
3
4
4.08 m
2.15 m
1.78 m
1.98 m
3.63 d (3.2)
5.37 d (3.2)
1.78 m
1.32 m
2.07 m
1.35 m
1.93 m
1.49 m
1.28 m
1.58 m
3.74 m
1.99 m
1.31 m
–
5
6
132.4 C
72.9 CH
120.6 CH
143.1 C
76.7 C
7
124.0 CH
137.8 C
5
8
6
–
4.70 d (5.1)
5.45 d (5.1)
1.65 m
1.24 m
2.01 m
9
132.0 C
7
6.66 s
2.70 m
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
1`
133.4 C
37.3 C
40.3 C
11
25.1 CH2
37.0 CH2
40.6 C
28.1 CH2
39.4 CH2
43.6 C
28.5 CH2
39.5 CH2
41.6 C
12
2.22 m
1.65 m
2.67 m
1.45 m
51.9 CH
29.4 CH2
25.7 CH2
55.0 CH
11.5 CH3
19.5 CH3
40.5 CH
21.1 CH3
135.7 CH
132.2 CH
40.5 CH
31.6 CH
19.5 CH3
19.9 CH3
17.8 CH3
171.0 C
54.9 CH
22.2 CH2
23.0 CH2
56.1 CH
12.5 CH3
19.0 CH3
40.6 CH
21.3 CH3
135.5 CH
132.3 CH
43.0 CH
33.1 CH
19.8 CH3
20.1 CH3
17.7 CH3
–
51.3 CH
25.8 CH2
29.0 CH2
55.4 CH
11.4 CH3
20.5 CH3
40.0 CH
22.0 CH3
136.0 CH
131.6 CH
42.0 CH
32.4 CH
19.5 CH3
19.7 CH3
17.2 CH3
171.5 C
22.6 CH3
–
14
15
2.20 m
1.55 m
1.35 m
16
2.02 m
1.80 m
1.15 m
1.29 m
17
1.38 m
1.31 m
1.31 m
18
0.59 s
0.62 s
0.54 s
19
2.10 s
1.11 s
1.13 s
20
2.05 m
2.01 m
2.00 m
21
1.07 d (6.8)
5.20 dd (15.2, 7.0)
5.25 dd (15.2, 7.0)
1.91 m
1.05 d (6.9)
0.99 d (6.8)
22
5.17 dd (15.6, 7.2)
5.21 dd (15.4, 7.0)
23
5.24 dd (15.6, 7.2)
5.23 dd (15.4, 7.6)
24
1.84 m
1.85 m
25
1.52 m
1.81 m
1.45 m
26
0.85 d (6.8)
0.87 d (6.8)
0.94 d (6.7)
–
0.85 d (6.7)
0.81 d (6.6)
27
0.85 d (6.7)
0.83 d (6.6)
2`
33.0 CH2
30.1-29.2 CH2
31.3 CH2
22.7 CH2
14.8 CH3
–
28
0.94 d (6.6)
0.89 d (6.9)
3`-13`
14`
15`
16`
–
6-OH
1`
3.42 brs
–
–
–
–
–
–
–
–
–
–
–
–
–
2`
2.35 t (7.2)
1.23-1.29 m
1.23-1.29 m
1.31 m
1.91 s
–
–
3`-13`
14`
15`
16`
–
–
–
–
0.84 t (6.6)
oxymethines δC 67.9 (C-3) and 73.8 (C-6), and four quaternary carbons.
The 1H and 13C NMR spectra of 3 displayed two tertiary methyl groups
(δH 0.62 (H-18)/δC 12.5 (C-18) and δH 1.11 (H-19)/δC 19.0 (C-19) and
four secondary methyl groups (δH 1.05 (H-21)/δC 21.3 (C-21), 0.85 (H-
26)/δC 19.8 (C-26), 0.85 (H-27)/δC 20.1 (C-27), and 0.94 (H-28)/δC
17.7 (C-28) (Tables 1 and 2). In the HMBC, the cross peaks from H-18 to
C-12, C-13, C-14, and C-17, H-19 to C-1, C-5, C-8, C-9, and C-10, H-26
and H-27 to C-24 and C-25, H-28 to C-23 and C-25, and H-21 to C-17
and C-22 established the assignment of the position of the methyl
groups (Fig. 2). In addition, three olefinic protons at δH 5.17 (dd,
J = 15.6, 7.2 Hz, H-22), 5.24 (dd, J = 15.6, 7.2 Hz, H-23), and 5.37 (d,
J = 3.2 Hz, H-7), characteristic of di- and tri-substituted double bonds,
correlated to the carbons at δC 135.5 (C-22), 132.3 (C-23), and 117.7
(C-7) in the HSQC, respectively, suggesting that 3 was an ergosta-7,22-
diene derivative (West and Cardellina, 1988). This was confirmed by
the COSY cross peaks of H-20/22, H-23/H-24, and H-6/H-7 and further
proven by the HMBC cross peaks of H-22 to C-17, C-21, and C-24, H-23
to C-20, C-25, and C-28, and H-7 to C-5, C-9, and C-14 (Fig. 2). The two
oxymethine signals observed at δH 4.08/δC 67.9 and 3.63/73.8 were
attributable to H-3/C-3 and H-6/C-6. Their placement was established
by the COSY correlations and was proven by the HMBC correlations of
H-1 and H-5 to C-3 and H-4, H-5, and H-7 to C-6 (Fig. 2). In addition, an
oxygen-bonded quaternary carbon was observed at δC 76.1 (C-9). Its
location was proven based on the HMBC cross peaks of H-7, H-12, H-14,
and H-19 to C-9. The large coupling constant (15.6 Hz) between H-22
and H-23 indicated the E configuration of the C22-C23 double bond. A
comparison of the chemical shifts of C-26 (δC 19.8), C-27 (δC 20.1), and
C-28 (δC 17.7) of 3 with those of the reported ergostane-type sterols
Fig. 2. Key 1H-1H COSY and HMBC correlations of compounds 1, 3, and 4.
established a 24R configuration (Yan et al., 2011; Qin et al., 2009). The
relative β-configuration of the hydroxyl functionalities at C-3 and C-6
was assigned based on the small coupling constants values of H-3 and
H-6, as well as the NOESY cross peaks of H-3 to H-5 and H-6 (Fig. 3). In
addition, the NOESY correlations between H-19 and H-18 suggested
that these protons were in a β-configuration. The 20R configuration was
assigned based on the NOESY correlation of the H-18 with H-20, as well
as the comparison of the NMR data with those of related ergosterol
derivatives (Yaoita et al., 2001, 1998). Biogenetically, the formation of
106

M.T. Khayat et al.
PhytochemistryLetters29(2019)104–109
8
7
6
5
4
3
2
1
0
Fig. 3. Some key NOESY correlations of 3.
Indometh Compd-1 Compd-2 Compd-3 Compd-4 Compd-5
Fig. 4. 5-Lipoxygenase inhibitory activity of compounds 1-5.
the R configured C-20 is common in the fungal ergosteroids (Brown,
1998). The relative stereochemistry for the hydroxyl group at C-9 was
not determined due to a lack of the advantageous NOESY correlations.
It was presumed to be in an α-configuration by comparison to the
previous reports (Mansoor et al., 2006; Moss, 1989) because all natural
sterols have a trans B/C ring fusion (Dewick, 2002). Consequently, the
structure of 3 was assigned as (22E, 24R)-ergosta-7,22-diene-3β,6β,9α-
triol and designated fusaristerol C.
1994; Kobori et al., 2007). More interestingly, steroids with diverse
structures were found to possess anti-inflammatory capacities against
cyclooxygenase-2/1/5-lipooxygenase (COX-2/COX-1/5-LOX) and cy-
tokines (Joy et al., 2017). Zheng et al. (2008) reported that ergosterol
peroxide isolated from Melandrium firmum exhibited 5-LOX inhibitory
activity (Zheng et al., 2008). Thus, the 5-LOX inhibitory activity of 1-5
from Fusarium sp. was assessed in an attempt to explore their activity
against 5-LOX. It is noteworthy that 1 and 2 displayed prominent 5-LOX
inhibitory activity with IC50 values of 3.61 and 2.45 μM, respectively,
compared to that of indomethacin (IC50 1.17 μM), while 3, 4, and 5 had
moderate activity with IC50s 7.01, 4.79, and 4.11 μM, respectively
(Fig. 4).
Compound 4 was obtained as a white amorphous solid. Its mole-
cular formula C30H48O5 was determined on the basis of the HRESIMS
pseudo-molecular ion peak at m/z 489.3587 [M+H]+ (calcd for
489.3580, C30H49O5). Compound 4 was 58 mass units and one degree
of unsaturation more than 3. The IR spectrum of 4 exhibited absorp-
tions at 3386 (OH), 2964 (CeH), 1738 (C]O), and 1648 (C]C) cm−1
.
The 1H and 13C NMR spectral data of 4 were similar to those of 3 with
the exception that the signals for a methine group H-5/C-5 (δH 1.98/δC
51.9) were missing, and new signals for an acetyl moiety [δH 1.91 (H-
2`)/δC 22.6 (C-2`) and δC 171.5] and an oxygen-bonded quaternary
carbon (δC 74.4, C-5) appeared in comparison to 3 (Tables 1 and 2). The
HMBC cross peaks of H-3, H-7, and H-19/C-5 (δC 74.4) assured the
placement of the oxygen-bonded quaternary carbon at position 5., The
acetoxy moiety was located at C-6 (Fig. 2) on the basis of the HMBC
correlation of H-6/C-1` and the downfield shift of H-6 (δH 4.70). The
relative stereochemistry of 4 was determined by comparison of the
NMR data and coupling constant values with those of related metabo-
lites (Qin et al., 2009; Li et al., 2014). Thus, 4 was assigned as
(22E,24R)-ergosta-7,22-diene-3β,5α,6β,9α-tetraol 6-acetate and desig-
nated fusaristerol D.
2.4. Structure-activity relationships
Comparing the 5-LOX inhibitory capacities of these groups of er-
gosterol derivatives, the structure-activity relationship of these com-
pounds could be hypothesized, which is consistent with other research
reported (Joy et al., 2017; Aparoy et al., 2012) (Fig. 5). It is noted that
the presence of the C22-C23 olefinic double bond is essential for activity
(Joy et al., 2017). A long chain fatty acyl moiety increases the anti-
inflammatory activity, similar to that in 1 and 2, due to the increase in
the lipophilic character of the compounds, while its reduction to acetyl,
as in 4, or its removal, as in 3, reduces the activity (Joy et al., 2017).
More activity is obtained when a peroxy group is present as in 2. Thus,
the absence of the chain fatty acyl moiety and peroxy group dramati-
cally decreases activity. It is noteworthy that the hydrophobicity is an
important factor in determining the anti-inflammatory activities that
may be due to the hydrophobic interactions between the inhibitor and
the binding pocket of the enzyme.
The known metabolites were identified as fusaristerol
A (2)
(Ibrahim et al., 2018c) and (22E,24R)-ergosta-7,22-dien-3β-ol (3)
(Ishizuka et al., 1997) by comparison of their spectral and physical data
with the literature.
2.3. 5-LOX inhibitory activity of the test compounds
Inflammation is a defence reaction of the body and a local response
of living tissues to injury aimed at eliminating or limiting the spread of
an injurious agent (Al-Attas et al., 2015). Ergosterol is a precursor of
vitamin D2 and steroid hormone drugs, which is a well-known steroid
that plays an important role in the fungal cell membrane (Ivanova et al.,
2013). It is noteworthy that ergosterol and its related derivatives ex-
hibited an anti-inflammatory potential (Yasukawa et al., 1994; Kobori
et al., 2007). Ma et al. (2013) stated that ergosterol and an enriched
ergosterol fraction inhibited the production of nitric oxide (NO), tu-
mour necrosis factor-α (TNF-α) and nuclear factor kappa-light-chain-
enhancer of activated B cells (NF-κB) luciferase activity in the macro-
phage RAW 264.7 cells (Ma et al., 2013). In addition, it was found that
ergosterol suppressed lipopolysaccharide (LPS)-induced pro-in-
flammatory gene expression by inhibiting NF-kB p65, the CCAAT-en-
hancer-binding protein-β (C/EBPβ) transcriptional activity and mi-
togen-activated protein kinase (MAPKs) activation (Yasukawa et al.,
Fig. 5. Structure properties affecting the 5-LOX inhibitory capacity of the tested
ergosterol derivatives.
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3. Experimental
was chromatographed on SiO2 CC (50 g, 50 cm × 3 cm) using n-
hexane:EtOAc 97:3-80:20 to obtain impure 2. A LiChrolut RP-18 solid
phase extraction tube (H2O:acetonitrile gradient) was used for its pur-
3.1. General experimental procedures
ification to yield
2 (8.3 mg). SiO2 CC (70 g × 50 × 3 cm, n-
Optical rotation was measured on a digital polarimeter (JASCO DIP-
370) (Jasco Co., Tokyo, Japan). UV spectra were recorded in absolute
MeOH on a Shimadzu 1601 UV/Vis spectrophotometer. The IR spectra
hexane:EtOAc 95:5 to 70:30) of sub-fraction FSE-5 (709 mg) produced
impure 3 and 4. Their purification was attained on RP-18 CC (40 g,
50 × 2 cm, 0.04-0.063 mm) using an H2O:MeOH gradient to obtain 3
(8.4 mg) and 4 (6.1 mg). Sub-fraction FSE-6 (532 mg) was chromato-
graphed on a SiO2 CC (60 g, 50 cm × 3 cm, n-hexane:EtOAc 97:3-80:20)
to get impure 5, that was purified by repeated SiO2 CC (n-hexane:EtOAc
gradient) to afford 5 (11.9 mg).
were performed on
a Shimadzu Infrared-400 spectrophotometer
(Shimadzu, Kyoto, Japan). JEOL JMS-SX/SX 102 A was utilized to
measure the EIMS (Joel, Peabody, MA, USA). The HRESIMS was re-
corded on an LTQ Orbitrap (Thermo Finnigan, Bremen, Germany).
GC–MS analysis was conducted on a Clarus 500 GCMS (Perkin Elmer,
Shelton, USA). The software controller/integrator was Turbo Mass
version 4.5.0.007 (Perkin Elmer). An Elite 5MS GC capillary column
(30 × 0.25 mm × 0.5 μm, Perkin Elmer) was used. The carrier gas was
helium (purity 99.9999%) at a flow rate of 2 mL/min (32 psi., flow
initial 55.8 cm/s, split; 1:40). The column temperature was 100–250 °C
(rate of temperature increases 5 °C/min). BRUKER Unity INOVA 400
was used to measure the NMR spectra (Bruker BioSpin, Billerica, MA,
USA). Sephadex LH-20 (0.25–0.1 mm), SiO2 60 (0.04-0.063 mm), and
RP-18 (0.04–0.063 mm, Merck, Darmstadt, Germany) were used for the
chromatographic separations. The TLC analysis utilized TLC pre-coated
plates (SiO2 60 F254, 0.2 mm, Merck, Darmstadt, Germany). The chro-
matograms were developed using the following solvent systems: n-
hexane:EtOAc (95:5, S1) n-hexane:EtOAc (90:10, S2), and n-
hexane:EtOAc (85:15, S3). The compounds were detected by spraying
with p-anisaldehyde/H2SO4 reagent and heating at 110 °C. Linoleic
acid, 5-lipoxygenase kits, and indomethacin were purchased from
Sigma-Aldrich (St. Louis, MO, USA).
3.4. Spectral data
3.4.1. Fusaristerol
B
((22E,24R)-3-palmitoyl-19(10→6)-abeo-ergosta-
5,7,9,22-tetraen-3β-ol) (1)
25
White amorphous powder; Rf 0.72 (S3); [α]D +44.7 (c 0.3,
CHCl3). UV (MeOH) λmax (log ε) nm: 215 (3.77), 285 (3.09) nm; IR
(KBr) γmax: 3005, 2896, 1725, 1605, 1495 cm−1; HRESIMS m/z
633.5604 [M+H]+ (calcd for 633.5611, C44H73O2); See Tables 1 and 2
for the NMR data.
3.4.2. Fusaristerol C [(22E,24R)-ergosta-7,22-diene-3β,6β,9R-triol] (3)
White amorphous powder; Rf 0.54 (S2); [α]D25 +52.4 (c 0.5 CHCl3);
IR (KBr) γmax: 3437, 2961, 1655, 1453, 1028 cm−1; HRESIMS m/z
431.3522 (calcad for C28H47O3, 431.3525 [M+H]+). See Tables 1 and
2 for the NMR data.
3.4.3. Fusaristerol
D
[(22E,24R)-ergosta-7,22-diene-3β,5α,6β,9α-tetraol
6-acetate] (4)
25
3.2. Fungal material isolation, identification, and cultivation
White amorphous powder; Rf 0.61 (S2); [α]D +35.1 (c 0.6,
CHCl3); IR (KBr) γmax: 3386, 2964, 1738, 1648 cm−1; HRESIMS m/z
489.3587 [M+H]+ (calcd for 489.3580, C30H49O5). See Tables 1 and 2
for the NMR data.
Mentha longifolia was collected from Abyar Al-Mashy, Al Madinah Al
Munawwarah in March 2017. It was authenticated by Dr. Emad Alsherif
(Faculty of Science & Arts, King Abdulaziz University). A specimen (ML-
1-2014) was deposited at the Natural Products and Alternative
Medicine Department Herbarium, Faculty of Pharmacy, King Abdulaziz
University. Fusarium sp. was separated from the interior tissues of the
M. longifolia roots. The tissues were precisely dissected under sterile
conditions and placed on potato dextrose agar plates (PDA, Difco), in-
cluding gentamicin and chloramphenicol to prevent the growth of
bacteria. The plates were incubated for four-six weeks at 27 °C. Fungal
hyphal tips were removed periodically and transferred to fresh PDA
plates. The fungi were specified based on their morphological colony
traits and microscopic examination using Olympus CX31RBSF light
microscopy. The fungus (FS No. MAR2014) was maintained at the
Microbiology Department, College of Pharmacy, Taibah University. The
fresh fungal material was cultured over rice solid cultures in
Erlenmeyer flasks (10, 1 L each) (distilled H2O (100 mL) + rice (100 g)
and kept overnight prior to autoclaving) and maintained under aseptic
conditions for 30 days at room temperature.
3.5. 5-Lipoxygenase inhibitory assay
The 5-LOX activity of compounds 1-5 at four different concentra-
tions (0.1, 5, 10, and 20 μM) was evaluated as previously described
(Ibrahim et al., 2018d,e,f; Mohamed, 2016; Yawer et al., 2007). A
mixture of 10 μL of each compound (1 mM in MeOH) and 20 μL lipox-
ygenase (70 units) in phosphate buffer (0.1 M aqueous, pH 8.0) to reach
a 160 μL volume was incubated for 10 min at 25 °C. The reaction was
initiated by the addition of 10 μL linoleic acid solution (20 μM) as a
substrate, resulting in the formation of (9Z,11E,13S)-13-hydro-
peroxyoctadeca-9,11-dienoate. The UV absorbance change at 234 nm
was measured over a 6 min period. All the experiments were conducted
in triplicate, and the analysis took a place using a 96-well microplate
reader (Tecan Genios). The positive control was indomethacin. The IC50
values were obtained using linear regression analysis.
3.6. Alkaline hydrolysis of compound 1
3.3. Extraction and isolation
A solution of 1 (4 mg) in KOH/MeOH (3%, 5 mL) was left for 20 min
at room temperature. The solution was neutralized using 1 N HCl/
MeOH. It was extracted with CHCl3 (10 mL × 3). The solvent was
evaporated, and the residue obtained was separated on SiO2 CC (n-
hexane:EtOAc, 99:1 to 90:10) to produce palmitic acid methyl ester (tR
36.5 min) that was identified using EI- and GC–MS (Khedr et al., 2018;
Al-Musayeib et al., 2013; Ibrahim et al., 2013).
The culture was extracted at room temperature with CHCl3/MeOH
(1:1) and concentrated under vacuum. The total extract (14.19 g) was
subjected to VLC using n-hexane, EtOAc, and MeOH, which were con-
centrated separately to obtain FSH (1.71 g), FSE (3.24 g), and FSM
(8.36 g), respectively. Fraction FSE (3.24 g) was subjected to Sephadex
LH-20 column chromatography (CC, CHCl3:MeOH, 70:30), and the
fractions (100 mL) were collected and examined by TLC to produce
eight sub-fractions: FSE-1 to FSE-8. Sub-fraction FSE-2 (359 mg) was
separated on silica gel (SiO2 CC (40 g × 50 × 2 cm) using n-
hexane:EtOAc (98:2 to 70:30) as an eluent to produce impure 1, which
was purified on RP-18 CC (40 g, 50 × 2 cm, 0.04-0.063 mm) using a
H2O:MeOH gradient to obtain 1 (11.4 mg). Sub-fraction FSE-3 (456 mg)
4. Conclusion
New ergosterol derivatives (1, 3, and 4) and two known metabolites
(2 and 5) were isolated from the endophytic fungus Fusarium sp. The
elucidation of their structures was accomplished using extensive
108

M.T. Khayat et al.
PhytochemistryLetters29(2019)104–109
spectroscopic techniques. Compounds 1-5 possessed 5-LOX inhibitory
activity. Thus, Fusarium sp. has proven to be a rich source of ergosterol
derivatives and other metabolites of diverse classes. Ergosterol deriva-
tives might have good potential for future use as a lead drug to develop
5-LOX inhibitory agents to treat various inflammatory disorders.
endophytic fungus Aspergillus terreus. Phytochem. Lett. 14, 84–90.
Ibrahim, S.R.M., Mohamed, G.A., Al Haidari, R.A., El-Kholy, A.A., Asfour, H.Z., Zayed,
M.F., 2018c. Fusaristerol A: a new cytotoxic and antifungal ergosterol fatty acid ester
from the endophytic fungus Fusarium sp. associated with Mentha longifolia roots.
Pharmacog. Mag. 14, 308–311.
Ibrahim, S.R.M., Mohamed, G.A., Al Haidari, R.A., El-Kholy, A.A., Zayed, M.F., Khayat,
M.T., 2018e. Tagetnoic acid, a new lipoxygenase inhibitor peroxy fatty acid from
Tagetes minuta growing in Saudi Arabia. Nat. Prod. Res. https://doi.org/10.1080/
14786419.2018.1488712.
Acknowledgements
Ibrahim, S.R.M., Mohamed, G.A., Khayat, M.T., Zayed, M.F., El-Kholy, A.A., 2018f. Anti-
inflammatory terpenoids from Cyperus rotundus rhizomes. Pak. J. Pharm. Sci. 31
(Suppl), 1449–31456 3.
This project was funded by the Deanship of Scientific Research
(DSR) at King Abdulaziz University, Jeddah, under grant no. (G: 162-
166-1439). Therefore, the authors acknowledge and thank the DSR for
technical and financial support.
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