Fitoterapia 112 (2016) 161–167

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Fitoterapia

journal homepage: www.elsevier.com/locate/fitote

Integracides H-J: New tetracyclic triterpenoids from the endophytic

fungus Fusarium sp.
Sabrin R.M. Ibrahim a,b,⁎, Hossam M. Abdallah c,d, Gamal A. Mohamed c,e, Samir A. Ross f
a
Department of Pharmacognosy and Pharmaceutical Chemistry, College of Pharmacy, Taibah University, Al Madinah, Al Munawwarah 30078, Saudi Arabia
b
Department of Pharmacognosy, Faculty of Pharmacy, Assiut University, Assiut 71526, Egypt
c
Department of Natural Products and Alternative Medicine, Faculty of Pharmacy, King Abdulaziz University, Jeddah 21589, Saudi Arabia
d
Department of Pharmacognosy, Faculty of Pharmacy, Cairo University, Cairo 11562, Egypt
e
Department of Pharmacognosy, Faculty of Pharmacy, Al-Azhar University, Assiut Branch, Assiut 71524, Egypt
f
National Center for Natural Products Research, Department of Pharmacognosy, School of Pharmacy, The University of Mississippi, MS 38677, USA

a r t i c l e i n f o a b s t r a c t

Article history: Three new tetracyclic triterpenoids namely, integracides H (1), I (4), and J (5), along with integracides B (3) and F
Received 6 May 2016 (2) have been isolated from the endophytic fungus Fusarium sp. isolated from the roots of Mentha longifolia L.
Received in revised form 1 June 2016 (Labiatae) growing in Saudi Arabia. The structure elucidation of the isolated compounds was achieved by spec-
Accepted 5 June 2016
troscopic analysis including UV, IR, 1D (1H and 13C) and 2D (1H\\1H COSY, TOCSY, HSQC, HMBC, and NOESY)
Available online 07 June 2016
NMR as well as HRESIMS and comparison with literature data. Integracides H (1) and J (5) showed significant
Keywords:
anti-leishmanial activity towards Leishmania donovani with IC50 values of 4.75 and 3.29 μM, respectively com-
Fusarium sp. pared to pentamidine (IC50 6.35 μM). Moreover, they displayed potent cytotoxic activity towards BT-549,
Integracides SKOV-3, and KB cell lines with IC50 values of 1.82, 1.32, and 0.18 μM and 2.46, 3.01, and 2.54 μM, respectively.
Triterpenoids © 2016 Elsevier B.V. All rights reserved.
Anti-leishmanial
Cytotoxic

1. Introduction
Fungi are eukaryotic microorganisms that inhabit almost all types of
environments in nature where they play key roles in the maintenance of
ecological balance. They continue to serve as valuable sources of a di-
verse variety of bioactive secondary metabolites, some of which have
found applications as important pharmaceuticals and agrochemicals
[1,2]. Endophytic fungi can be found inhabiting the living internal tis-
sues of some plants, often without causing any obvious negative effects
or external symptoms [3–5]. They are a promising source of new and bi-
ologically active natural products such as alkaloids, terpenoids, steroids,
quinones, isocoumarins, lignans, phenylpropanoids, phenols, and lac-
tones [6–8]. Fusarium sp. a widespread cosmopolitan group of fungi,
that can be isolated from different plant organs, plant debris, and soil
[9]. Also, it is one of the major contributors to the secondary metabolites
of fungal origin. Fusarium sp. has been isolated from both marine and
terrestrial sources. Integracides, a relatively small and under-explored
class of oxygenated tetracyclic 4,4-dimethylergostane triterpenoids,
possess a 12-acetyl-Δ8,14-diene-11-ol moiety. They have been obtained
only from Fusarium sp. and have not been reported from any other
fungal source [3,10–14]. We have previously identified two new
integracide derivatives from Fusarium sp. isolated from the roots of
Mentha longifolia L., which showed significant anti-leishmanial and
cytotoxic activities [3]. In a continuation of our interest to search for
more integracide derivatives from the same fungus, a new culture of
Fusarium sp. was investigated. Chromatographic separation of the
EtOAc extract of Fusarium sp. using VLC, silica gel, sephadex LH-20,
RP-18, and HPLC yielded three new integracide derivatives: integracides
H (1), I (4), and J (5), along with the known integracides B (3) and F (2)
(Fig. 1). These compounds were assigned by extensive spectroscopic
methods. The known integracides were identified by comparison of
their spectroscopic and physical data with the literature. Moreover,
the new integracides were evaluated for their anti-leishmanial and cy-
totoxic activities.
2. Experimental
2.1. General experimental procedures

Optical rotations were determined with a Perkin-Elmer Model 341

⁎ Corresponding author at: Department of Pharmacognosy and Pharmaceutical
Chemistry, College of Pharmacy, Taibah University, Al Madinah, Al Munawarah 30078,
Saudi Arabia.
E-mail addresses: sabrinshaur@gmail.com, sribrahim@taibahu.edu.sa
(S.R.M. Ibrahim).
LC polarimeter (Perkin-Elmer, Waltham, MA, USA). UV spectra were
measured in MeOH on a Shimadzu 1601 UV/VIS spectrophotometer
(Shimadzu, Kyoto, Japan). The IR spectra were measured on a Shimadzu
Infrared-400 spectrophotometer (Shimadzu, Kyoto, Japan). HRESIMS

http://dx.doi.org/10.1016/j.fitote.2016.06.002
0367-326X/© 2016 Elsevier B.V. All rights reserved.
162 S.R.M. Ibrahim et al. / Fitoterapia 112 (2016) 161–167
Fig. 1. Structures of integracides H (1), F (2), B (3), I (4), and J (5).
was recorded on a LTQ Orbitrap (ThermoFinnigan, Bremen, Germany).
ESIMS spectra were obtained with a LCQ DECA mass spectrometer
(ThermoFinnigan, Bremen, Germany) coupled to an Agilent 1100
HPLC system equipped with a photodiode array detector. 1D and 2D
NMR spectra were performed on BRUKER Unity INOVA 850 instruments
(850 MHz for 1H and 214 MHz for 13C NMR) (Bruker BioSpin, Billerica,
MA, USA) using DMSO-d6 as solvent. Chromatographic separations
were performed on SiO2 60 (0.04–0.063 mm, Merck, Darmstadt,
Germany), sephadex LH-20 (0.25–0.1 mm, Merck, Darmstadt,
Germany), and RP-18 (0.04–0.063 mm, Merck, Darmstadt, Germany).
Semi-preparative HPLC was performed on a Develosil C30-UG-5 column
(250 × 4.6 mm, Nomura Chemical Co., Aichi, Japan) and a Wakosil-II
5sil-100 column (150 × 4.6 mm ‘B′, Wako Pure Chemical Industries
Ltd., Japan), using acetonitrile:H2O (80:20) isocratic elution at a flow
rate of 5 mL/min, equipped with a TOSOH RI-8020 detector and a
JASCO BIP-I HPLC pump. TLC analysis was performed on pre-coated
TLC plates with SiO2 60 F254 (0.2 mm, Merck, Darmstadt, Germany).
The solvent systems used for TLC analyses were CHCl3:MeOH (95:5,
S1) and CHCl3:MeOH (90:10, S2). The compounds were detected by
UV absorption at λmax 255 and 366 nm followed by spraying with
anisaldehyde:H2SO4 and heating at 110 °C for 1–2 min.
2.2. Isolation and identification of the fungal material
Mentha longifolia L. (Labiatae) was collected in March 2014 from
Abyar Al-Mashy, Al Madinah Al Munawwarah. The plant sample was
authenticated by Dr. Emad Alsherif, Associate Professor of Plant Ecology,
Dept. of Biology, Faculty of Science & Arts, Khulais, King Abdulaziz
University, Saudi Arabia. A herbarium specimen was deposited at the
herbarium of the Department of Natural Products and Alternative
Medicine, Faculty of Pharmacy, King Abdulaziz University (ML-1-
2014). Fusarium sp. was isolated from the internal tissue of M. longifolia
roots. The inner root tissues were carefully dissected under sterile con-
ditions and placed on potato dextrose agar plates (PDA, Difco), contain-
ing chloramphenicol and gentamicin as antibacterial agents to prevent
bacterial growth.
The dishes were incubated at 27 °C for 4–6 weeks. Then, hyphal tips
of the fungi were periodically removed and transferred to fresh PDA
plates. The fungi were identified on the basis of their colonial morpho-
logical trait and microscopic observation using light microscopy
(CX31RBSF, Olympus). The fungus was deposited at the Department
of Microbiology, Faculty of Pharmacy, Taibah University, Al Madinah
Al Munawwarah, Saudi Arabia (FS No. MAR2014).
2.3. Cultivation of the fungal material
For isolation and identification of secondary metabolites, the fresh
fungal culture was transferred into 25 Erlenmeyer flasks (1 L each), con-
taining rice solid cultures (100 mL of distilled water were added to 100 g
commercially available rice and kept overnight prior to autoclaving).
The cultures were then incubated at room temperature for 30 days
under septic conditions.
 S.R.M. Ibrahim et al. / Fitoterapia 112 (2016) 161–167 163

2.4. Extraction and isolation
The rice culture was extracted with EtOAc and concentrated under
vacuum. The concentrated extract was mixed with 300 mL distilled
H2O and partitioned between n-hexane and 90% MeOH. The total 90%
MeOH extract (15.6 g) was subjected to VLC using n-hexane, EtOAc,
and MeOH, which were separately concentrated to give FS-1 (4.2 g),
FS-2 (3.1 g), and FS-3 (6.2 g), respectively. Fraction FS-2 (3.1 g) was sub-
jected to normal phase vacuum liquid chromatography (VLC) using
CHCl3:MeOH gradients (100% CHCl3 to 50:50 CHCl3:MeOH), 100 mL
fractions were collected and monitored by TLC to obtain eight main
sub-fractions: FSE2–1 to FSE2–8. Sub-fraction FSE2-2 (105 mg) was
chromatographed over silica gel column (40 g, 50 × 2 cm) using
CHCl3:MeOH (98:2 to 90:10) as an eluent to give impure 1, which was
further purified by repeated chromatography on a silica gel column
using a CHCl3:MeOH gradient to afford 1 (9.5 mg, white amorphous
powder). Silica gel column chromatography (90 g, 50 × 3 cm) of sub-
fraction FSE2-3 (270 mg) using CHCl3:MeOH in order of increasing po-
larity afforded impure compounds 2 and 3. They were submitted to
semi-preparative HPLC (80:20 acetonitrile:H2O, isocratic elution) to
yield 2 (13 mg, colorless powder) and 3 (8.7 mg, white amorphous
powder). Similarly, sub-fraction FSE2-4 (45 mg) was purified on semi-
preparative HPLC to afford 3 and 4 (8.4 mg, white amorphous powder).
Sub-fraction FSE-5 (92 mg) was chromatographed over sephadex LH-
20 (30 g, 50 × 3 cm) using MeOH as an eluent to afford impure 5. The
impure 5 was purified on RP-18 column (0.04–0.063 mm; 40 g,
50 × 2 cm) using H2O:MeOH gradient to give 5 (6.7 mg, white amor-
phous powder). The other fractions were retained for further
investigation.
2.4.1. Integracide H (1)
White amorphous powder, Rf 0.69, Si 60 F254 (S1); [α]25D + 13.5 (c
0.3, MeOH); UV λmax (MeOH) (log ε): 250 (2.25) nm; IR (KBr) νmax
3445, 1725, 1668, and 889 cm− 1; NMR data: see Table 1; HRESIMS
m/z 599.3948 (calcd for C36H55O7 [M + H]+, 599.3942).
2.4.2. Integracides B (3) and I (4)
White amorphous powder, Rf 0.74, Si 60 F254 (S2); UV λmax (MeOH)
(log ε): 248 (2.32) nm; IR (KBr) νmax 3480, 2854, 1732, 1660, 1055,
885 cm−1; NMR data: see Table 2; HRESIMS m/z 515.3741 [M + H]+
(calcd for C32H51O5, 515.3736).

Table 1
NMR spectral data of compounds 1 and 5 (DMSO-d6, 850 and 214 MHz).

1 5

No. δH [mult., J (Hz)] δC (mult.) HMBC No. δH [mult., J (Hz)] δC (mult.) HMBC

1 2.28 m 43.4 CH2 2, 3, 5, 10, 18 1 2.26 m 43.3 CH2 2, 3, 5, 10, 18

1.01 m 1.06 m
2 3.86 td (10.6, 4.3) 66.8 CH 1, 3, 5, 35 2 3.74 td (10.4, 4.3) 66.8 CH 3, 4, 10
3 3.58 d (10.6) 88.1 CH 2, 4, 29, 30, 33 3 3.57 d (10.4) 88.0 CH 1, 2, 4, 29, 30, 33
4 – 39.5C – 4 – 39.5C –
5 1.11 dd (12.6, 2.8) 50.2 CH 3, 6, 7, 10, 18 5 1.11 dd (12.8, 2.8) 50.2 CH 6, 10, 18
6 1.72 m 17.8 CH2 5, 8, 10 6 1.68 m 17.7 CH2 5, 10
1.63 m 1.60 m
7 2.32 m 26.4 CH2 5, 8, 9 7 2.32 m 26.2 CH2 5, 6
2.21 m 2.21 m
8 – 123.9C – 8 – 123.8C –
9 – 139.4C – 9 – 139.4C –
10 – 39.9C – 10 – 37.2C –
11 4.08 brs 67.7 CH 8, 9, 12, 13 11 4.07 d (2.4) 67.4 CH 8, 9, 12, 17, 19
12 4.95 brs 77.8 CH 9, 14, 31 12 4.95 d (1.7) 77.7 CH 9, 11, 14, 19, 17, 31
13 – 46.4C – 13 – 46.4C –
14 – 147.1C – 14 – 147.0C –
15 5.50 brs 120.3 CH 8, 13, 14, 17 15 5.50 brs 120.2 CH 8, 14, 16, 17
16 2.35 m 35.0 CH2 14, 15, 17 16 2.38 m 34.8 CH2 13, 15, 17
2.00 m 2.01 m
17 1.85 dt (11.6, 8.5) 48.6 CH 13, 16, 22 17 1.85 dt (11.4, 8.5) 48.5 CH 13, 19, 20, 22
18 1.20 s 22.7 CH3 5, 9, 10 18 1.19 s 22.6 CH3 1, 5, 10
19 0.98 s 16.6 CH3 12, 14, 17 19 0.98 s 16.5 CH3 12, 13, 17
20 1.58 m 32.8 CH 21 20 1.58 m 32.7 CH 21
21 0.84 d (6.4) 17.9 CH3 17, 22 21 0.84 d (6.4) 17.9 CH3 20
22 1.51 m 33.9 CH2 21, 24 22 1.65 m 33.9 CH2 21, 23
1.02 m 1.12 m
23 2.08 m 30.4 CH2 24, 25, 28 23 2.07 m 31.3 CH2 20, 22, 25
1.87 m 1.88 m
24 – 155.3C – 24 – 155.7C –
25 2.20 m 33.1 CH 23, 24, 26, 27 25 2.21 m 33.1 CH 23, 26, 27
26 0.96 d (6.4) 21.8 CH3 24, 25, 27 26 0.96 d (6.6) 21.8 CH3 25, 27
27 0.97 d (6.4) 21.7 CH3 24, 25, 26 27 0.99 d (6.6) 21.7 CH3 25, 26
28 4.70 brs 106.6 CH2 24, 25 28 4.70 d (0.9) 106.5 CH2 23, 24, 25, 26, 27
4.65 brs 4.65 d (0.9)
29 0.74 s 17.5 CH3 3, 4, 5, 30 29 0.75 s 17.4 CH3 3, 4, 5, 30
30 0.95 s 28.5 CH3 3, 4, 5, 29 30 0.95 s 28.5 CH3 3, 4, 5, 29
31 – 169.9C – 31 – 169.8C –
32 1.95 s 21.0 CH3 31 32 1.96 s 21.0 CH3 31
33 – 172.1C – 33 – 170.1C –
34 1.89 s 21.1 CH3 33 34 – 127.9C –
35 – 172.1C – 35, 39 7.00 d (8.5) 130.1 CH 33, 34, 36, 37, 38
36 1.89 s 21.1 CH3 35 36, 38 6.61 d (8.5) 114.8 CH 34, 37, 39
37 – 155.7C –
2-OH 5.31 s – 1, 2
11-OH 5.39 d (6.0) – 9, 11
37-OH 9.13 s – 36, 37, 38
164 S.R.M. Ibrahim et al. / Fitoterapia 112 (2016) 161–167

Table 2
NMR spectral data of compounds 3 and 4 (DMSO-d6, 850 and 214 MHz).

3 4

No. δH [mult., J (Hz)] δC (mult.) δH [mult., J (Hz)] δC (mult.) HMBC

1 2.26 dd (12.8, 4.3) 43.4 CH2 2.36 dd (9.4, 4.3) 42.3 CH2 5, 10, 18
2 3.74 td (10.2, 4.3) 66.8 CH 3.70 td (11.1, 2.6) 66.8 CH 3, 4
3 3.57 d (10.2) 88.0 CH 3.55 d (11.1) 88.3 CH 1, 2, 4, 29, 30
4 – 39.5C –
5 1.07 dd (12.7, 2.6) 50.3 CH 1.04 dd (11.1, 3.4) 50.5 CH 6, 7, 10, 18
6 1.67 m 17.7 CH2 1.56 m 17.8 CH2 5, 7, 10
1.56 m 1.50 m
7 2.30 m 26.2 CH2 2.30 m 26.2 CH2 5, 6, 10
2.19 m 2.19 m
8 – 123.8C – 123.8C –
9 – 139.4C – 139.4C –
10 – 37.2C – 36.8C –
11 4.07 d (2.6) 67.4 CH 3.93 brs 69.5 CH 8, 9, 10, 12, 13
12 4.95 brs 77.7 CH 4.97 brd (2.0) 79.6 CH 9, 11, 13, 14, 19, 31
13 – 46.4C – 46.7C –
14 – 147.0C – 147.0C –
15 5.50 brs 120.2 CH 5.50 brs 120.2 CH 8, 13, 14, 16
16 2.33 m 34.8 CH2 2.33 m 34.8 CH2 13, 15, 17
1.99 m 1.99 m
17 1.84 dt (11.1, 8.5) 48.5 CH 1.84 dt (11.1, 8.5) 48.5 CH 13, 19, 20, 22
18 1.19 s 22.6 CH3 1.15 s 1, 5, 10
19 0.99 s 16.6 CH3 0.98 s 13, 17
20 1.58 m 32.7 CH 21
21 0.84 d (6.0) 17.9 CH3 0.83 d (6.0) 17, 20
22 1.51 m 33.9 CH2 1.51 m 33.9 CH2 20, 21
1.10 m 1.10 m
23 2.05 m 30.4 CH2 2.07 m 30.6 CH2 22, 25
1.87 m 1.87 m
24 – 155.7C – 155.7C –
25 2.18 m 33.1 CH 2.18 m 33.1 CH 23, 26, 27
26 0.97 d (6.6) 21.8 CH3 0.95 d (6.8) 25, 27
27 0.97 d (6.6) 21.7 CH3 0.95 d (6.8) 25, 26
28 4.70 brs 106.5 CH2 4.68 brs 23, 24, 25, 26, 27
4.65 brs 4.62 brs
29 0.75 s 17.4 CH3 0.73 s 17.5 CH3 4, 5, 30
30 0.95 s 28.6 CH3 0.93 s 28.7 CH3 4, 5, 29
31 – 169.8C – 169.8C –
32 2.00 s 21.0 CH3 1.96 s 21.0 CH3 31
2-OH 5.31 brs – 5.27 brs – 1, 2, 3, 10
11-OH 5.39 d (6.0) – 5.19 d (6.0) – 9, 11, 12

2.4.3. Integracide J (5) procedure of Borenfreund et al. [17]. Doxorubicin was used as a positive
White amorphous powder, Rf 0.56, Si 60 F254 (S2); [α]25D + 18.7 (c control, while DMSO was used as the negative control.
0.6, MeOH); UV λmax (MeOH) (log ε): 253 (2.27), 282 (1.98) nm; IR
(KBr) νmax 3546, 1723, 1615, 1608, 889 cm−1; NMR data: see Table 1; 3. Results and discussion
HRESIMS m/z 635.3937 (calcd for C39H55O7, 635.3942 [M + H]+).
Compound 1 was obtained as white amorphous powder. The molec-
ular formula was C36H55O7 on the basis of the HRESIMS pseudo-molec-
2.5. Anti-leishmanial assay ular ion peak at m/z 599.3946 (calcd for C36H55O7, 599.3948 [M + H]+),
requiring ten degrees of unsaturation. The UV absorption band at λmax
The anti-leishmanial activity of the isolated metabolites was tested
in vitro against Leishmania donovani promastigotes as previously de-
scribed [15,16]. Pentamidine was used as a positive standard.

2.6. Cytotoxicity assay

The in vitro cytotoxic activity was determined against a panel of four

human cancer cell lines: malignant melanoma (SK-MEL), epidermoid
(KB), ductal (BT-549), and ovarian (SKOV-3) carcinomas and two non-
cancerous kidney cell lines: pig kidney epithelial (LLC-PK11) and mon-
key kidney fibroblast (VERO). All cell lines were obtained from the
American Type Culture Collection (ATCC, Rockville, MD). Cells were
seeded at a density of 25,000 cells/well and incubated for 24 h. Test
samples were added at different concentrations and cells were again
incubated for 48 h. At the end of incubation, the cell viability was
determined using Neutral Red dye according to a modification of the Fig. 2. Possible fragmentation pattern of 1.
 S.R.M. Ibrahim et al. / Fitoterapia 112 (2016) 161–167 165

Fig. 3. Some key 1H\

\1H COSY and NOESY correlations of 1, 4, and 5.

250 nm suggested the presence of a heteroannular diene system in 1 [3,
11]. Its IR spectrum showed absorption bands at 3445, 1725, 1668, and
889 cm−1, suggested the presence of hydroxyl, ester carbonyl, and exo-
cyclic di-substituted double bond, respectively. Compound 1 was one
degree of unsaturation and 43 mass units more than intergracide F
(2), indicating the presence of an additional acetyl group in 1. The
HRESIMS spectrum displayed characteristic fragment ion peaks at m/z
556.3775 [M + H-OAc]+, 513.3594 [M + H-2OAc]+, and 454.3370
[M + H-3OAc + H2O]+ (Fig. 2). The NMR data of 1 were similar to
intergracide F (2), which was previously reported from Fusarium sp.
[3]. The 13C and HSQC NMR spectra displayed 36 carbon resonances:
10 methyls, 7 methylenes, 9 methines four of them for oxymethine
carbons, and 10 quaternary carbons, including 3 carbonyls and three
olefinic carbons. A doublet methyl signal at δH 0.84 (d, J = 6.4 Hz, H-
21) and four singlet methyl groups at δH 1.20 (H-18), 0.98 (H-19),
0.74 (H-29), and 0.95 (H−30), correlating to the carbon resonances at
δC 17.9 (C-21), 22.7 (C-18), 16.6 (C-19), 17.5 (C-29), and 28.5 (C-30)
in the HSQC spectrum were observed. The HMBC cross peaks from H-
21 to C-17 and C-22, H-18 to C-5, C-9, and C-10, H-19 to C-12, C-14,
and C-17, and H-29 and H-30 to C-3, C-4, and C-5 confirmed the position
of the methyl groups at C-20, C-10, C-13, and C-4, respectively. The 1H
and 13C NMR spectra showed signals for an exomethylene group at δH
4.70 and 4.65 (each brs, H-28)/δC 106.6 (C-28) and 155.3 (C-24) and a
tri-substituted olefinic double bond at δH 5.50 (brs, H-15)/δC 120.3 (C-
15) and 147.1 (C-14) (Table 1). The HMBC correlations of H-28 to C-
25, H-22, H-26, and H-27 to C-24, and H-23 to C-28, H-15 to C-8, C-13,
and C-17, and H-12, H-16, and H-19 to C-14 established their location
at C24-C28 and C14-C-15, respectively (Fig. 4). In addition, the 1H\\1H
COSY cross peaks of H-15 to the methylene protons at δH 2.35 (m, H-
16A) and 2.00 (m, H-16B) established the position of C14-C15 olefinic

Fig. 4. HMBC correlations of 1, 4, and 5.

166 S.R.M. Ibrahim et al. / Fitoterapia 112 (2016) 161–167
double bond (Fig. 3). Moreover, the 1H and 13C NMR spectra displayed
two doublet methyls at δH 0.96 (3H, d, J = 6.4 Hz, H-26)/δC 21.8 (C-
26), 0.97 (3H, d, J = 6.4 Hz, H-27)/δC 21.7 (C-27) and multiplet methine
at δH 2.20 (H-25)/33.1 (C-25), corresponding to an isopropyl moiety.
This was confirmed by 1H\\1H COSY and TOCSY cross peaks of H-26
and H-27 with H-25 and the HMBC cross peaks of H-25 to C-26 and C-
27, H-26 to C-25 and C-27, and H-27 to C-25 and C-26. The HMBC
cross peaks of H-25 to C-23 and H-26 and H-27 to C-24 established
the connectivity of this moiety at C-24. Signals for three acetoxy groups
were observed at δH 1.95 (H-32)/δC 21.0 (C-32) and 1.89 (H-34, 36)/
21.1 (C-34, 36). This was confirmed by the HMBC cross peaks of H-32
to C-31 (δC 169.9) and H-34 and H-36 to C-33 and C-35 (δC 172.1) and
further secured by the observed HRESIMS fragment ion peaks (Figs. 2
& 4). Their attachment at C-2, C-3, and C-12 was secured by the HMBC
correlations of H-2 to C-35, H-3 to C-33, and H-12 to C-31. Additionally,
the four oxymethines at δH 3.86 (td, J = 10.6, 4.3 Hz, H-2), 3.58 (d, J =
10.6 Hz, H-3), 4.08 (brs, H-11), and 4.95 (brs, H-12) showed HSQC cross
peaks to the carbon signals at δC 66.8 (C-2), 88.1 (C-3), 67.7 (C-11), and
77.8 (C-12). The HMBC correlations of H-1 to C-2 and C-3, H-5, H-29,
and H-30 to C-3, H-12 to C-11, and H-19 to C-12 (Fig. 4) proved their lo-
cation at C-2, C-3, C-11, and C-12, respectively. The presence of a tetra-
substituted olefinic double bond was evident by singlet carbon signals
at δC 123.9 and 139.4. Its presence at C8-C9 was secured by the HMBC
cross peaks of H-6, H-11, and H-15 to C-8 and H-7, H-12, and H-18 to
C-9. The relative configuration at the stereo-centers of 1 was assigned
based on the observed NOESY cross peaks, in addition to comparison
of the 1H and 13C chemical shifts and coupling constant values of 1
with literature [3,10,11]. The NOESY correlations of H-3 to H-5 and H-
11 and H-17 to H-5, H-11, and H-21 revealed that these protons present
on the same side of the molecule. Furthermore, the cross peaks of H-2 to
H-12 and H-18 and H-12 to H-20 positioned these protons on the other
side (Fig. 3). On the basis of these findings, the structure of 1 was unam-
biguously elucidated and named integracide H.
Compound 4 was isolated as an approximate 1:2 mixture with
integracide B (3) as shown by paired signals in NMR spectra. They
showed a single spot on silica TLC plate and single peak on semi-prepar-
ative HPLC. Further attempts to purify this peak using different HPLC
mobile phases were fruitless. The HREIMS spectrum gave a single pseu-
do-molecular ion peak at m/z 515.3739 [M + H]+, consistent with the
molecular formula C32H50O5, which required 8 degrees of unsaturation.
The IR spectrum showed absorption bands at 3480 (OH), 1732 (C_O),
1660, and 1055 cm−1. Assignment of individual signals to 3 and 4 was
achieved by 2D NMR (1H\\1H COSY, TOCSY, HSQC, HMBC, and
NOESY). The NMR data of 4 showed features very similar to 3, with dif-
ferences in the 1H and 13C chemical shifts of H-11/C-11 (δH 3.93 (brs)/δC
69.5 for 4 and δH 4.07 (d, J = 2.6 Hz)/δC 67.4 for 3) and H-12/C-12 (δH
4.97 (brd, J = 2.0 Hz)/δC 79.6 for 4 and δH 4.95 (brs)/δC 77.7 for 3).
The observed NOESY cross peaks of H-11 to H-2, H-18, and H-19 and
H-12 to H-3, H-5, H-17, and H-21 in 4 suggested that 4 to be an isomer
of 3, which differed in the configuration at C-11 and C-12. Accordingly, 4
was identified as a 11,12-isomer of the co-occurring integracide B (3)
and named integracide I.
Compound 5 was isolated as white amorphous powder. The
HRESIMS showed a pseudo-molecular ion peak at m/z 635.3945 (calcd
for C39H55O7, 635.3948 [M + H]+), which compatible with the molecu-
lar formula C41H56O8. Compound 5 showed an increase of 36 mass units
and three degrees of unsaturation compared to 1. Also, the HRESIMS
spectrum of 5 revealed the presence of prominent fragment ion peaks
at m/z 592.3799 [M + H-acetyl]+ and 471.3475 [M + H–(acetyl + p-
hydroxy benzoyl)]+. Its UV spectrum showed absorption bands at 253
and 282 nm, which are consistent with the presence of a heteroannular
diene and benzoyl moieties in 5 [3]. The IR spectrum suggested the pres-
ence of OH group (3546 cm−1), ester carbonyl (1723 cm−1), double
bond (1615 cm−1), and aromatic ring (1608 cm−1) [18,19]. The 1H
and 13C NMR spectra of 5 were comparable to those of 1 (Table 2). How-
ever, the acetoxy groups signals at δH 1.89 (H-34, 36)/δC 21.1 (C-34, 36)
Table 3
Results of cytotoxic and anti-leishmanial activities of compounds 1 and 5.
IC50 IC50
Compd no. BT-549 SKOV-3 KB L. donovani
1 1.82 1.32 0.18 4.75
5 2.46 3.01 2.54 3.29
Doxorubicin 2.78 0.16 0.41 –
Pentamidine – – – 6.35
and 172.1 (C-33, 35), which were encountered in 1 were not present.
Instead, new signals for a p-hydroxy benzoyl moiety at δH 7.00 (2H, d,
J = 8.5 Hz, H-35, 39)/δC 130.1 (C-35, 39), 6.61 (2H, d, J = 8.5 Hz, H-
36, 38)/δC 114.8 (C-36, 38), 9.31 (s, 37-OH), 170.1 (C-33), 127.9 (C-
34), and 155.7 (C-37) were observed. This moiety was established by
the 1H\\1H COSY correlations of H-35 and H-39 to H-36 and H-38 and
HMBC cross peaks of H-35 and H-39 to C-33, C-34, and C-37, H-36 and
H-38 to C-34 and C-37, and 37-OH to C-36, C-37, and C-38. This was fur-
ther secured by the HRESIMS fragment ion peak at m/z 471.3475
[M + H–(acetyl + p-hydroxy benzoyl)]+. Its attachment at C-3 was
confirmed by the HMBC correlation of H-3 to C-33. In addition, a singlet
signal for hydroxyl group at δH 5.31 (s, 2-OH) was observed. Its 1H\\1H
COSY with H-2 and HMBC to C-1 and C-2 confirmed its location at C-2.
The relative configuration of 5 was established based on the observed
NOESY correlations (Fig. 3). From the above data and by comparison
with literature, the structure of 5 was assigned to integracide J.
Compounds 1 and 5 were evaluated for their anti-leishmanial activ-
ity towards L. donovani promastigotes and cytotoxicity activity towards
SK-MEL, KB, BT-549, SKOV-3, LLC-PK11, and VERO cell lines.
Integracides H (1) and J (5) displayed potent cytotoxic activity to-
wards BT-549, SKOV-3, and KB with IC50 values of 1.82, 1.32, and
0.18 μM and 2.46, 3.01, and 2.54 μM, respectively compared to doxoru-
bicin (IC50 2.78, 0.16, and 0.41 μM, respectively). Moreover, they exhib-
ited significant anti-leishmanial activity towards L. donovani with IC50
values of 4.75 and 3.29 μM, respectively compared to pentamidine (pos-
itive control, IC50 6.35 μM) (Table 3). On the other hand, they exhibited
no activity towards SK-MEL, LLC-PK11, and VERO cell lines.
4. Conclusion
Fusarium sp. has proven to be a rich source of anti-leishmanial and
cytotoxic integracides and other metabolites of diverse classes. Investi-
gation of the EtOAc extract of the endophytic fungus Fusarium sp. isolat-
ed from the roots of Mentha longifolia afforded three new tetracyclic
triterpenoids, integracides H (1), I (4), and J (5) and two known com-
pounds. Integracides H (1) and J (5) exhibited significant anti-leishman-
ial and cytotoxic activities. So, these compounds could be used as lead
compounds for new potential anti-leishmanial and cytotoxic agents de-
rived from fungi. However, further investigation should be undertaken
to explore the plausible mechanisms by which this class of compounds
exerted their anti-leishmanial and cytotoxic effects.
Conflict of interest
There are no conflicts of interest of all authors with respect to this
work.
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