- Email: [email protected]
Integracides F and G: New tetracyclic triterpenoids from the endophytic fungus Fusarium sp.
Phytochemistry Letters 15 (2016) 125–130
Contents lists available at ScienceDirect
Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Integracides F and G: New tetracyclic triterpenoids from the endophytic fungus Fusarium sp. Sabrin R.M. Ibrahima,b,* , Gamal A. Mohamedc,d , Samir A. Rosse a
Department of Pharmacognosy and Pharmaceutical Chemistry, College of Pharmacy, Taibah University, Al Madinah Al Munawwarah 30078, Saudi Arabia Department of Pharmacognosy, Faculty of Pharmacy, Assiut University, Assiut 71526, Egypt Department of Natural Products and Alternative Medicine, Faculty of Pharmacy, King Abdulaziz University, Jeddah 21589, Saudi Arabia d Department of Pharmacognosy, Faculty of Pharmacy, Al-Azhar University, Assiut Branch, Assiut 71524, Egypt e National Center for Natural Products Research, Department of Pharmacognosy, School of Pharmacy, The University of Mississippi, MS 38677, USA b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 18 November 2015 Accepted 21 December 2015 Available online xxx
Two new tetracyclic triterpenoids: integracides F (1) and G (2) have been isolated from the endophytic fungus Fusarium sp. isolated from the roots of Mentha longifolia L. (Labiatae) growing in Saudi Arabia. Their structures were established by UV, IR, 1D (1H and 13C), 2D (1H-1H COSY, HMQC, HMBC, and NOESY) NMR, and HRESIMS spectral data, in addition to comparison with literature data. The isolated compounds were evaluated for their anti-microbial, anti-malarial, anti-leishmanial, and cytotoxic activities. Compound 1 and 2 displayed potent cytotoxic activity towards BT-549 and SKOV-3 with IC50 values of 1.97 and 0.16 mg/mL and 1.76 and 0.12 mg/mL, respectively compared to doxorubicin (IC50 1.61 and 0.095 mg/mL, respectively). Moreover, they exhibited significant anti-leishmanial activity towards Leishmania donovani with IC50 values of 3.74 and 2.53 mg/mL, respectively and IC90 values of 5.11 and 8.89 mg/mL, respectively. ã 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Keywords: Fusarium sp. Integracides Triterpenoids Anti-microbial Anti-malarial Anti-leishmanial Cytotoxic
1. Introduction Endophytic microorganisms are bacteria or fungi that live inside plant tissues, without causing damage or disease symptoms to their hosts (Elkhayat et al., 2015; deSouza et al., 2011). The secondary metabolites produced by these microorganisms are a valuable repository of natural bioactive compounds, many of which have been identified as useful research reagents and potential drug candidates (Ibrahim et al., 2015; Meinwald and Eisner, 2008; Geris dos Santos and Rodrigues-Fo, 2003). Fusarium sp. are a widespread cosmopolitan group of fungi and commonly colonize aerial and subterranean plant parts, either as primary or secondary invaders (El-Kazzaz et al., 2008). Fusarium sp. is well known for the production of integracides, which are a class of a tetracyclic 4,4-dimethylergostane triterpenoids containing a 12-acetyl-D8,14-diene-11-ol moiety. They have been shown to possess elastase, rhinovirus 3C protease, HIV-1 integrase, and cholesteryl ester transfer protein inhibitory activities (Singh et al., 2003a,b; Singh, 2000; Tabata et al., 1999; Brill et al., 1996). As part of an ongoing search for bioactive compounds from endophytic
* Corresponding author. E-mail address: [email protected] (S.R.M. Ibrahim).
fungi, we have identified two new tetracyclic triterpenoids: integracides F (1) and G (2) from Fusarium sp. isolated from the roots of Mentha longifolia L. (Fig. 1). The fungal EtOAc extract was subjected to Sephadex LH-20, silica gel, and RP-18 column chromatography to yield compounds 1 and 2. Herein, we report the isolation and structure elucidation as well as anti-microbial, anti-malarial, anti-leishmanial, and cytotoxic activities of the new compounds. 2. Results and discussion Compound 1 was obtained as colorless powder. Its HRESIMS spectrum gave a pseudo-molecular ion peak at m/z 557.3839 (calcd. for C34H53O6, 557.3842 [M + H]+) compatible with the molecular formula C34H52O6, requiring nine degrees of unsaturation. The IR spectrum showed characteristic absorption bands at 3435 (hydroxyl group), 1723 (ester group), and 1664 and 885 (exocyclic di-substituted double bond) cm 1. The UV spectrum showed an absorption band at lmax 248 nm characteristic for a heteroannular diene system (Singh et al., 2003b). Compound 1 was 43 mass units and one degree of unsaturation more than intergracide B, indicating the presence of an additional acetyl group in 1. The NMR spectral data of 1 were similar to intergracide B previously isolated from Fusarium sp. (Singh et al., 2003a,b). The
http://dx.doi.org/10.1016/j.phytol.2015.12.010 1874-3900/ ã 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
126
S.R.M. Ibrahim et al. / Phytochemistry Letters 15 (2016) 125–130
Fig. 1. Structures of integracides B, F (1), and G (2).
13
C and HSQC NMR spectra of 1 displayed resonances for 34 carbon signals: 9 methyls, 7 methylenes, 9 methines four of them for oxymethine carbons, and 9 quaternary carbons, including 2 carbonyls and three olefinic carbons. In the 1H and 13C NMR spectra, signals for a tri-substituted olefinic double bond were observed at dH 5.52 (brt, J = 2.5 Hz, H-15)/dC 120.7 (C-15) and 147.5 (C-14) (Table 1). It was positioned at C14-C15 based on the HMBC correlations of H-15 to C-8, C-16, and C-17 and H-12, H-16, and H-19 to C-14 and the 1H-1H COSY cross peaks (Figs. 2 and 3) of H-15 to the methylene protons at dH 2.39 (m, H-16A) and 2.00 (m, H-16B). Furthermore, the 1H and 13C NMR showed signals at dH 4.71 (brs, H-28A) and 4.66 (brs, H-28B)/dC 107.0 (C-28) and 156.2 (C-24), indicating the presence of an exomethylene group. Its position was established by the 3J HMBC cross peaks of H-28 to C-23 and C-25, H-22, H-26, and H-27 to C-24, and H-23 and H-25 to C-28. Moreover, four singlet methyl groups at dH 1.21 (H-18)/dC 23.1 (C-18), 0.98 (C-19)/17.0 (C-19), 0.78 (H-29)/17.9 (C-29), and 0.96 (H-30)/29.1 (C-30) and doublet methyl group at dH 0.86 (d, J = 6.2 Hz, H-21)/dC 18.3 (C-21) were observed. The HMBC spectrum (Fig. 3) showed cross peaks from H-18 to C-1, C-5, C-9, and C-10, H-19 to C-12, C-13, C-14, and C-17, H-29 and H-30 to C-3, C-4, and C-5, and H-21 to C-17, C-20, and C-22, establishing the locations of the methyl groups at C-10, C-13, C-4, and C-20, respectively. The presence of an isopropyl moiety in 1 was evident from the signals at dH 0.99 (3H, d, J = 6.5 Hz, H-26)/dC 22.2 (C-26), 1.00 (3H, d, J = 6.5 Hz, H-27)/dC 22.1 (C-27), and 2.19 (1H, m H-25)/ 33.5 (C-25) and confirmed by 1H-1H COSY correlations (Fig. 2) of H-26 and H-27 with H-25. The connectivity of isopropyl moiety at C-24 was confirmed by the HMBC correlations of H-25 to C-23, C-24, and C-28. The signals at dH 3.76 (1H, ddd, J = 11.4, 10.1, 4.0 Hz, H-2)/dC 67.3 (C-2), 3.59 (1H, d, J = 10.1 Hz, H-3)/88.5 (C-3), 4.09 (1H, brs, H-11)/67.9 (C-11), and 4.97 (1H, d, J = 2.1 Hz, H-12)/78.2 (C-12) indicated the presence of four oxygen-bonded methine groups. They were positioned at C-2, C-3, C-11, and C-12, respectively based
on the observed 1H-1H COSY and 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-17 and H-19 to C-12 (Fig. 3). The 13C NMR spectrum displayed signals at dC 124.2 and 139.9 characteristic for the presence of a tetrasubstituted olefinic double bond. Its placement 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 1H NMR spectrum showed two singlet methyl signals at dH 2.05 (H-32) and 2.01 (H-34), correlating to the carbon signal resonating at dC 21.4 (C-32, 34) in the HSQC spectrum. Also, they showed HMBC cross peaks to the carbonyl carbons at dC 170.5 (C-31) and 170.3 (C-33), respectively, indicating the presence of two acetoxy moieties in 1. This was confirmed by the ESIMS fragment ion peaks at m/z 514 [M + H-COCH3]+ and 471 [M + H-2 COCH3]+. The HMBC cross peaks (Fig. 3) of H-3 to C-33 and H-12 to C-31 established the connectivity of the acetoxy groups at C-3 and C-12, respectively. The 1HNMR spectrum of 1 showed two singlet signals at dH 5.32 and 5.38 which were assigned to 2-OH and 11-OH groups, respectively (Table 1). Their assignment was secured by the HMBC cross peaks of 2-OH to C-1, C-2, and C-3 and 11-OH to C-9, C-11, and C-12. The relative configuration of 1 was assigned based on the comparison of the 1H and 13C chemical shifts as well as coupling constant values of 1 with literature and further confirmed by the NOESY experiment (Singh et al., 2003a,b). The NOESY spectrum showed correlations of H-3 to H-5, H-11 and H-17 to H-5, and H-11 to H-21, indicating that these protons occurred on the same side of the molecule. Moreover, the NOESY cross peaks of H-2 to H-12 and H-18 and H-12 to H-20 positioned these protons on the other side of the molecule (Fig. 2). On the basis of these evidences and by comparison of NMR data of 1 with those of the previously reported integracides, the structure of 1 was unambiguously elucidated and named integracide F. Compound 2 was isolated as white amorphous powder and its molecular formula was determined as C42H68O7 by the HRESIMS
S.R.M. Ibrahim et al. / Phytochemistry Letters 15 (2016) 125–130
127
Table 1 NMR spectral data of compounds 1 and 2 (CDCl3, 400 and 100 MHz). No.
1
2
dH [mult., J (Hz)]
dC (mult.)
HMBC
dH [mult., J (Hz)]
dC (mult.)
HMBC
1
2.23 m
43.8CH2
2, 3, 10
43.8CH2
2, 3, 18
2 3 4 5 6
3.76 ddd (11.4, 10.1, 4.0) 3.59 d (10.1) – 1.70 dd (11.9, 3.1) 1.69 m 1.60 m 2.32 m 2.20 m – – – 4.09 brs 4.97 d (2.1) – – 5.52 brt (2.5) 2.39 m 2.00 m 1.83 dt (10.3, 7.3) 1.21 s 0.98 s 1.60 m 0.86 d (6.2) 1.54 m 1.14 m 2.05 m 1.88 m – 2.19 m 0.99 d (6.5) 1.00 d (6.5) 4.71 brs 4.66 brs 0.78 s 0.96 s – 2.05 s – 2.01 s – – – – – 5.32 brs 5.38 brs
67.3CH 88.5CH 39.5C 50.7CH 18.2CH2
1, 3, 10 1, 2, 5, 29, 30, 33 – 3, 4, 7, 10 5, 8, 10
67.3CH 88.4CH 39.4C 50.8CH 18.1CH2
1, 3, 4, 10 1, 4, 29, 30, 33 – 3, 4, 6, 10 4, 5, 8
26.6CH2
5, 8, 9
26.6CH2
5, 8, 9
124.2C 139.9C 37.7C 67.9CH 78.2CH 46.9C 147.5C 120.7CH 35.3CH2
– – – 8, 9, 12, 13 9, 11, 13, 14, 31 – – 8, 14, 16, 17 14, 15, 17, 20
124.7C 139.6C 37.7C 67.8CH 78.4CH 46.6C 147.1C 120.6CH 35.4CH2
– – – 8, 9, 12, 13 9, 11, 14, 17, 19, 31 – – 8, 14, 17 13, 15, 17, 20
49.0CH 23.1CH3 17.0CH3 33.2CH 18.3CH3 34.4CH2
12, 13, 21 1, 5, 9, 10 12, 13, 14, 17 17, 21, 22 17, 20, 22 17, 20, 24
49.1CH 23.1CH3 17.0CH3 33.4CH 18.3CH3 34.4CH2
13, 15, 22 1, 5, 9, 10 12, 13, 17 17, 21, 23 17, 20, 22 20, 21, 23
30.9CH2
24, 25, 28
31.7CH2
20, 24, 25, 28
156.2C 33.5CH 22.2CH3 22.1CH3 107.0CH2
– 23, 24, 24, 23,
156.5C 33.6CH 22.2CH3 22.1CH3 107.0CH2
– 23, 24, 24, 23,
17.9CH3 29.1CH3 170.5C 21.4CH3 170.3C 21.4CH3 – – – – – – –
3, 4, 5, 30 3, 4, 29 – 31 – 33 – – – – – 1, 2, 3 9, 11, 12
2.27 m 1.06 m 3.76 ddd (11.2, 10.0, 4.0) 3.59 d (9.5) – 1.12 dd (11.6, 3.2) 1.69 m 1.60 m 2.32 m 2.08 m – – – 4.10 brs 4.98 brd (2.0) – – 5.52 brt (2.0) 2.35 m 2.02 m 1.87 dt (10.6, 7.5) 1.21 s 0.98 s 1.61 m 0.86 d (6.3) 1.52 m 1.15 m 2.08 m 1.89 m – 2.18 m 0.99 d (6.5) 1.00 d (6.5) 4.72 brs 4.67 brs 0.78 s 0.95 s – 1.98 s – 2.32 t (7.2) 1.17 m 1.26–1.22 m 1.61 m 1.25 m 3.35 t (6.8) 5.33 brs 5.38 brs
17.9CH3 29.1CH3 170.3C 21.4CH3 170.6C 34.0CH2 23.7CH2 29.5–29.1 (CH2)4 24.8CH2 30.9CH2 63.1CH2 – –
3, 4, 5, 30 3, 4, 5, 29 – 31 – 33, 35 33, 34 – – 40, 42 40, 41 – –
7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36–39 40 41 42 2-OH 11-OH
24, 25, 25, 24,
26, 27, 28 27 26 25
pseudo-molecular ion peak at m/z 685.5039 (calcd. for C42H69O7, 685.5043 [M + H]+), requiring nine degrees of unsaturation. Compound 2 is 128 mass units more than 1. The IR and NMR data of 2 were quite similar to those of 1, but the signals associated with the acetoxy group at dH 2.01(H-34)/dC 21.4 (C-34) and 170.3 (C-33) in 1 were absent. Instead, new signals for multiple aliphatic protons at dH 1.26–1.22 (m, H-36 to H-39), four methylenes groups at dH 2.32 (t, J = 7.2 Hz, H-34), 1.17 (m, H-35), 1.61 (m, H-40), and 1.25 (m, H-41), and a terminal oxymethylene group at dH 3.35 (t, J = 6.8 Hz, H-42), suggesting the presence of a long chain fatty acid moiety with terminal hydroxyl group were observed (Carballeira et al., 1999). This was confirmed by the presence of additional 1 H-1H COSY spin system extended (Fig. 2) from H-34 to H-42 and HMBC correlations of H-34 to C-33 and C-35, H-35 to C-33 and C-34, H-41 to C-40 and C-42, and H-42 to C-40 and C-41 and further secured by the ESIMS fragment ion peak at m/z 514 [M + HC10H19O2]+. Alkaline hydrolysis of 2 gave fatty acid methyl ester (FAME), which was identified to be 10-hydroxydecanoic acid methyl ester by the GCMS molecular ion peak at m/z 202. The
26, 25, 25, 24,
27, 28 27 26 25
HMBC cross peak (Fig. 3) of H-3 to C-33 established the attachment of the fatty acid moiety at C-3. The relative configuration of 2 was established based on the observed NOESY correlations (Fig. 2). From the above data and by comparison with literature, the structure of 2 was assigned to integracide G. The isolated compounds were evaluated for their anti-microbial activity against Candida albicans,Candida glabrata, Candida krusei, Asperigillus fumigates, methicillin-resistant Staphylococcus aureus (MRSA), Cryptococcus neoformans, Staphylococcus aureus, Escherichia coli, Pseudomonus aeruginosa, and Mycobacterium intracellulare, anti-leishmanial activity against Leishmania donovani promastigotes, and anti-malarial activity against chloroquine sensitive (D6, Sierraleon) and resistant (W2, Indo-china) strains of Plasmodium falciparum. In addition, cytotoxicity against SK-MEL, KB, BT-549, SKOV-3, LLC-PK11, and VERO cell lines was tested. Compound 1 and 2 displayed potent cytotoxic activity towards BT-549 and SKOV-3 with IC50 values of 1.97 and 0.16 mg/mL and 1.76 and 0.12 mg/mL, respectively compared to doxorubicin (IC50 1.61 and 0.095 mg/mL, respectively) and anti-leishmanial activity
128
S.R.M. Ibrahim et al. / Phytochemistry Letters 15 (2016) 125–130
Fig. 2. 1H–1H COSY and NOESY correlations of 1 and 2.
towards L. donovani with IC50 values of 3.74 and 2.53 mg/mL, respectively and IC90 values of 5.11 and 8.89 mg/mL, respectively compared to pentamidine (positive control, IC50 2.1 mg/mL). On the other hand, they exhibited no anti-malarial and antimicrobial activities. 3. Experimental 3.1. General experimental procedures Optical rotations were measured on a PerkinElmer Model 341 LC polarimeter (PerkinElmer, Waltham, MA, USA). UV spectra were recorded 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 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 1100HPLC system equipped with a photodiode array detector. 1D and 2D NMR spectra were determined on BRUKER Unity INOVA 400 instruments (400 MHz for 1H and 100 MHz for 13C NMR) (Bruker BioSpin, Billerica, MA, USA) using CDCl3 as solvent, with TMS as the internal reference. Solvents were distilled prior spectroscopic measurements. A GCMS was performed on Clarus 500 GC/MS (PerkinElmer, Shelton, CT). The software controller/ integrator was turbo mass, version 4.5.0.007 (PerkinElmer). An elite 5 MS GC capillary column (30 0.25 mm 0.5 mm, PerkinElmer) was used. The carrier gas was helium at a flow rate
Fig. 3. HMBC correlations of compounds 1 and 2.
S.R.M. Ibrahim et al. / Phytochemistry Letters 15 (2016) 125–130
of 2 mL/min (32 p.s.i., flow initial 55.8 cm/s, split; 1:40). Temperature conditions were: inlet line temperature, 200 C; source temperature, 150 C; trap emission, 100 C; and electron energy, 70 eV. The column temperature program was: 50 C for 5 min, increased to 220 C (rate, 20C/min), and held for 5 min. The injector temperature was 220 C. MS scan was from 50 to 650 m/z. Column 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). 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 lmax 255 and 366 nm followed by spraying with anisaldehyde:H2SO4 and heating at 110 C for 1–2 min. 3.2. Isolation and identification of the fungal material M. longifolia L. (Labiatae) was collected in March 2014 from Al Madinah Al Munawwarah, Saudi Arabia. Fusarium sp. was isolated from the internal tissue of M. longifolia roots. The roots were cut into very small pieces 0.5–1.0 cm and washed with sterilized water to remove dust and debris. The surface was thoroughly treated with 70% EtOH for 1–2 min and air-dried under the flow hood. The outer layers of root were removed and the inner tissues were carefully dissected under sterile conditions and placed on potato dextrose agar plates (PDA, Difco) containing antibiotics to suppress 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 morphological 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).
129
powder). The other fractions were retained for further investigation. 3.5. Spectral data Integracide F (1). Colorless powder, Rf 0.77, Si 60F254 (S2); [a]D +9.7 (c 0.6, MeOH; UV lmax (MeOH): 248 nm; IR (KBr) nmax 3435, 1723, 1664, 885 cm 1; NMR data: see Table 1; HRESIMS m/z 557.3839 (calcd. for C34H53O6 [M + H]+, 557.3842). Integracide G (2). White amorphous powder, Rf 0.70, Si 60 F254 (S2); [a]D +11.3 (c 0.5, MeOH); UV lmax (MeOH): 249 nm; IR (KBr) nmax 3426, 2835, 1715, 1659, 885, 725 cm 1; NMR data: see Table 1; HRESIMS m/z m/z 685.5039 (calcd. for C42H69O7, [M + H]+, 685.5043). 3.6. Alkaline hydrolysis of compound 2 A solution of 2 (4 mg) in 3% KOH/MeOH (4 mL) was left to stand for 15 min at room temperature then neutralized with 1 N HCl/ MeOH. The solution was extracted with CHCl3. The solvent was evaporated and the residue obtained was chromatographed on a silica gel column using n-hexane:EtOAc gradient to furnish the methyl ester of 10-hydroxydecanoic acid, which was identified by GCMS (El-Shanawany et al., 2015; Al-Musayeib et al., 2013). 3.7. Anti-microbial assay The anti-microbial activity of the isolated compounds was tested against C. albicans ATCC 90028, C. glabrata ATCC90030, C. krusei ATCC 6258, A. fumigates ATCC 90906, methicillin-resistant S. aureus ATCC 33591, C. neoformans ATCC 90113, S. aureus ATCC 2921, E. coli ATCC 35218, P. aeruginosa ATCC 27853, and M. intracellulare ATCC 23068 as described previously (Al-Musayeib et al., 2014; Ibrahim et al., 2012). Ciprofloxacin and amphotericin B were used as positive standards.
3.3. Cultivation of the fungal material
3.8. Anti-malarial assay
For isolation and identification of secondary metabolites, the fresh fungal culture was transferred into 15 Erlenmeyer flasks (1 L each) containing 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.
The isolated compounds were evaluated for their anti-malarial activities against chloroquine sensitive (D6, Sierraleon) and resistant (W2, Indo-china) strains of Plasmodium falciparum as previously outlined (Al-Musayeib et al., 2014; El-Shanawany et al., 2011). Artemisinin and chloroquine were used as standard antimalarial drugs.
3.4. Extraction and isolation
3.9. Anti-leishmanial assay
The rice culture was extracted with EtOAc and concentrated under vacuum. The concentrated extract was partitioned between n-hexane and 90% MeOH. The total MeOH extract (7.2 g) was subjected to normal phase vacuum liquid chromatography (VLC) using n-hexane, CHCl3, EtOAc, and MeOH, which were separately concentrated to give FS-1 (1.1 g), FS-2 (0.8 g), FS-3 (1.72 g), and FS-4 (2.94 g), respectively. Fraction FS-2 (0.8 g) was chromatographed over silica gel column (60 g 50 2 cm) using CHCl3:MeOH gradients (100% CHCl3 to 60:40CHCl3:MeOH), 50 mL fractions were collected and monitored by TLC to obtain five main subfractions FSC-1-FSC-4. Silica gel column chromatography (20 g 30 2 cm) of sub-fraction FSC-2 (208 mg) using CHCl3: MeOH (98:2 to 90:10) gave impure 1. Sub-fraction FSC-3 (192 mg) was chromatographed over Sephadex LH-20 chromatography (100 g 75 3 cm) using MeOH as an eluent to afford impure 2. Separately, the impure 1 and 2 were purified on RP-18 column (0.04–0.063 mm; 40 g 25 2 cm) using MeOH:H2O gradient to give 1 (37 mg, colorless powder) and 2 (11 mg, white amorphous
The anti-leishmanial activity of the isolated metabolites was tested in vitro against L. donovani promastigotes as previously described (Al-Musayeib et al., 2014; El-Shanawany et al., 2011). Pentamidine and amphoterecin B were used as positive standards. 3.10. 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 noncancerous kidney cell lines: pig kidney epithelial (LLC-PK11) and monkey 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 procedure of (Borenfreund
130
S.R.M. Ibrahim et al. / Phytochemistry Letters 15 (2016) 125–130
et al., 1990). Doxorubicin was used as a positive control, while DMSO was used as the negative control. 4. Conclusion Two new tetracyclic triterpenoids were isolated from the endophytic fungus Fusarium sp. isolated from the roots of M. longifolia. Their structures were elucidated on the basis of extensive spectroscopic analyses. Compounds 1 and 2 showed significant anti-leishmanial and cytotoxic activities. Conflict of interest There is no conflict of interest associated with the authors of this paper. References Al-Musayeib, N.M., Mohamed, G.A., Ibrahim, S.R.M., Ross, S.A., 2013. Lupeol-3-Odecanoate: a new triterpene ester from Cadaba farinosa Forsk. growing in Saudi Arabia. Med. Chem. Res. 22, 5297–5302. Al-Musayeib, N.M., Mohamed, G.A., Ibrahim, S.R.M., Ross, S.A., 2014. New thiophene and flavonoid from Tagetes minuta leaves growing in Saudi Arabia. Molecules 19, 2819–2828. Borenfreund, E., Babich, H., Martin-Alguacil, N., 1990. Rapid chemosensitivity assay with human normal and tumor cells in vitro. In Vitro Cell. Dev. Biol. 26, 1030– 1034. Brill, G.M., Kati, W.M., Montgomery, D., Karwowski, J.P., Humphrey, P.E., Jackson, M., Clement, J.J., Kadam, S., Chen, R.H., McAlpine, J.B., 1996. Novel triterpene sulfates from Fusarium compactum using a rhinovirus 3C protease inhibitor screen. J. Antibiot. 49, 541–546. Carballeira, N.M., Sostre, A., Restituyo, J.A., 1999. Synthesis of racemic 9-methyl-10hexadecenoic acid. Chem. Phys. Lipids 97, 87–91.
deSouza, J.J., Vieira, I.J.C., Filho, E.R., Filho, R.B., 2011. Terpenoids from endophytic fungi. Molecules 16, 10604–10618. El-Kazzaz, M.K., El-Fadly, G.B., Hassan, M.A.A., El-Kot, G.A.N., 2008. Identification of some Fusarium spp. using molecular biology techniques. Egypt. J. Phytopathol. 36, 57–69. El-Shanawany, M.A., Ross, S.A., Ibrahim, S.R.M., Mohamed, G.A., Nafady, A.M., 2011. A new xanthone from the roots of Centaurium spicatum L. Phytochem. Lett. 4, 126– 128. El-Shanawany, M.A., Sayed, H.M., Ibrahim, S.R.M., Fayed, M.A.A., 2015. Stigmasterol tetracosanoate: a new stigmasterol ester from the Egyptian Blepharis ciliaris. Drug Res. 65, 347–353. Elkhayat, E.S., Ibrahim, S.R.M., Mohamed, G.A., Ross, S.A., 2015. Terrenolide S, a new anti-leishmanial butenolide from the endophytic fungus Aspergillus terreus. Nat. Prod. Res. doi:http://dx.doi.org/10.1080/14786419.2015.1072711. Geris dos Santos, R.M., Rodrigues-Fo, E., 2003. Further meroterpenes produced by Penicillium sp., an endophyte obtained from Melia azedarach. Z. Naturforsch. 58c, 663–669. Ibrahim, S.R.M., Mohamed, G.A., Al-Musayeib, N.M., 2012. New constituents from the rhizomes of Egyptian Iris germanica L. Molecules 17, 2587–2598. Ibrahim, S.R.M., Elkhayat, E.S., Mohamed, G.A., Khedr, A.I.M., Fouad, M.A., Kotb, M.H. R., Ross, S.A., 2015. Aspernolides F and G: new butyrolactones from the endophytic fungus Aspergillus terreus. Phytochem. Lett. 14, 84–90. Meinwald, J., Eisner, T., 2008. Chemical ecology in retrospect and prospect. Proc. Natl. Acad. Sci. U. S. A. 105, 4539–4540. Singh, S.B., Ondeyka, J.G., Schleif, W.A., Felock, P., Hazuda, D.J., 2003a. Chemistry and structure-activity relationship of HIV-1 integrase inhibitor integracide B and related natural products. J. Nat. Prod. 66, 1338–1344. Singh, S.B., Zink, D.L., Dombrowski, A.W., Polishook, J.D., Ondeyka, J.G., Hirshfield, J., Felock, P., Hazuda, D.J., 2003b. Integracides: tetracyclic triterpenoid inhibitors of HIV-1 integrase produced by Fusarium sp. Bioorg. Med. Chem. 11, 1577–1582. Singh, S.B., 2000. A new mild PTSA-catalyzed method for sulfate ester hydrolysis and acid-catalyzed rearrangement of 12-acetyl-diene-11-ol tetracyclic triterpenoids involving an angular methyl migration. Tetrahedron Lett. 41, 6973–6976. Tabata, N., Tomoda, H., Yamaguchi, Y., Masuma, R., Bamberger, M.J., Omura, S.J., 1999. Inhibition of cholesteryl ester transfer protein by fungal metabolites, L681, 512. J. Antibiot. 52, 1042–1045.
Contents lists available at ScienceDirect
Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
Integracides F and G: New tetracyclic triterpenoids from the endophytic fungus Fusarium sp. Sabrin R.M. Ibrahima,b,* , Gamal A. Mohamedc,d , Samir A. Rosse a
Department of Pharmacognosy and Pharmaceutical Chemistry, College of Pharmacy, Taibah University, Al Madinah Al Munawwarah 30078, Saudi Arabia Department of Pharmacognosy, Faculty of Pharmacy, Assiut University, Assiut 71526, Egypt Department of Natural Products and Alternative Medicine, Faculty of Pharmacy, King Abdulaziz University, Jeddah 21589, Saudi Arabia d Department of Pharmacognosy, Faculty of Pharmacy, Al-Azhar University, Assiut Branch, Assiut 71524, Egypt e National Center for Natural Products Research, Department of Pharmacognosy, School of Pharmacy, The University of Mississippi, MS 38677, USA b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 18 November 2015 Accepted 21 December 2015 Available online xxx
Two new tetracyclic triterpenoids: integracides F (1) and G (2) have been isolated from the endophytic fungus Fusarium sp. isolated from the roots of Mentha longifolia L. (Labiatae) growing in Saudi Arabia. Their structures were established by UV, IR, 1D (1H and 13C), 2D (1H-1H COSY, HMQC, HMBC, and NOESY) NMR, and HRESIMS spectral data, in addition to comparison with literature data. The isolated compounds were evaluated for their anti-microbial, anti-malarial, anti-leishmanial, and cytotoxic activities. Compound 1 and 2 displayed potent cytotoxic activity towards BT-549 and SKOV-3 with IC50 values of 1.97 and 0.16 mg/mL and 1.76 and 0.12 mg/mL, respectively compared to doxorubicin (IC50 1.61 and 0.095 mg/mL, respectively). Moreover, they exhibited significant anti-leishmanial activity towards Leishmania donovani with IC50 values of 3.74 and 2.53 mg/mL, respectively and IC90 values of 5.11 and 8.89 mg/mL, respectively. ã 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Keywords: Fusarium sp. Integracides Triterpenoids Anti-microbial Anti-malarial Anti-leishmanial Cytotoxic
1. Introduction Endophytic microorganisms are bacteria or fungi that live inside plant tissues, without causing damage or disease symptoms to their hosts (Elkhayat et al., 2015; deSouza et al., 2011). The secondary metabolites produced by these microorganisms are a valuable repository of natural bioactive compounds, many of which have been identified as useful research reagents and potential drug candidates (Ibrahim et al., 2015; Meinwald and Eisner, 2008; Geris dos Santos and Rodrigues-Fo, 2003). Fusarium sp. are a widespread cosmopolitan group of fungi and commonly colonize aerial and subterranean plant parts, either as primary or secondary invaders (El-Kazzaz et al., 2008). Fusarium sp. is well known for the production of integracides, which are a class of a tetracyclic 4,4-dimethylergostane triterpenoids containing a 12-acetyl-D8,14-diene-11-ol moiety. They have been shown to possess elastase, rhinovirus 3C protease, HIV-1 integrase, and cholesteryl ester transfer protein inhibitory activities (Singh et al., 2003a,b; Singh, 2000; Tabata et al., 1999; Brill et al., 1996). As part of an ongoing search for bioactive compounds from endophytic
* Corresponding author. E-mail address: [email protected] (S.R.M. Ibrahim).
fungi, we have identified two new tetracyclic triterpenoids: integracides F (1) and G (2) from Fusarium sp. isolated from the roots of Mentha longifolia L. (Fig. 1). The fungal EtOAc extract was subjected to Sephadex LH-20, silica gel, and RP-18 column chromatography to yield compounds 1 and 2. Herein, we report the isolation and structure elucidation as well as anti-microbial, anti-malarial, anti-leishmanial, and cytotoxic activities of the new compounds. 2. Results and discussion Compound 1 was obtained as colorless powder. Its HRESIMS spectrum gave a pseudo-molecular ion peak at m/z 557.3839 (calcd. for C34H53O6, 557.3842 [M + H]+) compatible with the molecular formula C34H52O6, requiring nine degrees of unsaturation. The IR spectrum showed characteristic absorption bands at 3435 (hydroxyl group), 1723 (ester group), and 1664 and 885 (exocyclic di-substituted double bond) cm 1. The UV spectrum showed an absorption band at lmax 248 nm characteristic for a heteroannular diene system (Singh et al., 2003b). Compound 1 was 43 mass units and one degree of unsaturation more than intergracide B, indicating the presence of an additional acetyl group in 1. The NMR spectral data of 1 were similar to intergracide B previously isolated from Fusarium sp. (Singh et al., 2003a,b). The
http://dx.doi.org/10.1016/j.phytol.2015.12.010 1874-3900/ ã 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
126
S.R.M. Ibrahim et al. / Phytochemistry Letters 15 (2016) 125–130
Fig. 1. Structures of integracides B, F (1), and G (2).
13
C and HSQC NMR spectra of 1 displayed resonances for 34 carbon signals: 9 methyls, 7 methylenes, 9 methines four of them for oxymethine carbons, and 9 quaternary carbons, including 2 carbonyls and three olefinic carbons. In the 1H and 13C NMR spectra, signals for a tri-substituted olefinic double bond were observed at dH 5.52 (brt, J = 2.5 Hz, H-15)/dC 120.7 (C-15) and 147.5 (C-14) (Table 1). It was positioned at C14-C15 based on the HMBC correlations of H-15 to C-8, C-16, and C-17 and H-12, H-16, and H-19 to C-14 and the 1H-1H COSY cross peaks (Figs. 2 and 3) of H-15 to the methylene protons at dH 2.39 (m, H-16A) and 2.00 (m, H-16B). Furthermore, the 1H and 13C NMR showed signals at dH 4.71 (brs, H-28A) and 4.66 (brs, H-28B)/dC 107.0 (C-28) and 156.2 (C-24), indicating the presence of an exomethylene group. Its position was established by the 3J HMBC cross peaks of H-28 to C-23 and C-25, H-22, H-26, and H-27 to C-24, and H-23 and H-25 to C-28. Moreover, four singlet methyl groups at dH 1.21 (H-18)/dC 23.1 (C-18), 0.98 (C-19)/17.0 (C-19), 0.78 (H-29)/17.9 (C-29), and 0.96 (H-30)/29.1 (C-30) and doublet methyl group at dH 0.86 (d, J = 6.2 Hz, H-21)/dC 18.3 (C-21) were observed. The HMBC spectrum (Fig. 3) showed cross peaks from H-18 to C-1, C-5, C-9, and C-10, H-19 to C-12, C-13, C-14, and C-17, H-29 and H-30 to C-3, C-4, and C-5, and H-21 to C-17, C-20, and C-22, establishing the locations of the methyl groups at C-10, C-13, C-4, and C-20, respectively. The presence of an isopropyl moiety in 1 was evident from the signals at dH 0.99 (3H, d, J = 6.5 Hz, H-26)/dC 22.2 (C-26), 1.00 (3H, d, J = 6.5 Hz, H-27)/dC 22.1 (C-27), and 2.19 (1H, m H-25)/ 33.5 (C-25) and confirmed by 1H-1H COSY correlations (Fig. 2) of H-26 and H-27 with H-25. The connectivity of isopropyl moiety at C-24 was confirmed by the HMBC correlations of H-25 to C-23, C-24, and C-28. The signals at dH 3.76 (1H, ddd, J = 11.4, 10.1, 4.0 Hz, H-2)/dC 67.3 (C-2), 3.59 (1H, d, J = 10.1 Hz, H-3)/88.5 (C-3), 4.09 (1H, brs, H-11)/67.9 (C-11), and 4.97 (1H, d, J = 2.1 Hz, H-12)/78.2 (C-12) indicated the presence of four oxygen-bonded methine groups. They were positioned at C-2, C-3, C-11, and C-12, respectively based
on the observed 1H-1H COSY and 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-17 and H-19 to C-12 (Fig. 3). The 13C NMR spectrum displayed signals at dC 124.2 and 139.9 characteristic for the presence of a tetrasubstituted olefinic double bond. Its placement 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 1H NMR spectrum showed two singlet methyl signals at dH 2.05 (H-32) and 2.01 (H-34), correlating to the carbon signal resonating at dC 21.4 (C-32, 34) in the HSQC spectrum. Also, they showed HMBC cross peaks to the carbonyl carbons at dC 170.5 (C-31) and 170.3 (C-33), respectively, indicating the presence of two acetoxy moieties in 1. This was confirmed by the ESIMS fragment ion peaks at m/z 514 [M + H-COCH3]+ and 471 [M + H-2 COCH3]+. The HMBC cross peaks (Fig. 3) of H-3 to C-33 and H-12 to C-31 established the connectivity of the acetoxy groups at C-3 and C-12, respectively. The 1HNMR spectrum of 1 showed two singlet signals at dH 5.32 and 5.38 which were assigned to 2-OH and 11-OH groups, respectively (Table 1). Their assignment was secured by the HMBC cross peaks of 2-OH to C-1, C-2, and C-3 and 11-OH to C-9, C-11, and C-12. The relative configuration of 1 was assigned based on the comparison of the 1H and 13C chemical shifts as well as coupling constant values of 1 with literature and further confirmed by the NOESY experiment (Singh et al., 2003a,b). The NOESY spectrum showed correlations of H-3 to H-5, H-11 and H-17 to H-5, and H-11 to H-21, indicating that these protons occurred on the same side of the molecule. Moreover, the NOESY cross peaks of H-2 to H-12 and H-18 and H-12 to H-20 positioned these protons on the other side of the molecule (Fig. 2). On the basis of these evidences and by comparison of NMR data of 1 with those of the previously reported integracides, the structure of 1 was unambiguously elucidated and named integracide F. Compound 2 was isolated as white amorphous powder and its molecular formula was determined as C42H68O7 by the HRESIMS
S.R.M. Ibrahim et al. / Phytochemistry Letters 15 (2016) 125–130
127
Table 1 NMR spectral data of compounds 1 and 2 (CDCl3, 400 and 100 MHz). No.
1
2
dH [mult., J (Hz)]
dC (mult.)
HMBC
dH [mult., J (Hz)]
dC (mult.)
HMBC
1
2.23 m
43.8CH2
2, 3, 10
43.8CH2
2, 3, 18
2 3 4 5 6
3.76 ddd (11.4, 10.1, 4.0) 3.59 d (10.1) – 1.70 dd (11.9, 3.1) 1.69 m 1.60 m 2.32 m 2.20 m – – – 4.09 brs 4.97 d (2.1) – – 5.52 brt (2.5) 2.39 m 2.00 m 1.83 dt (10.3, 7.3) 1.21 s 0.98 s 1.60 m 0.86 d (6.2) 1.54 m 1.14 m 2.05 m 1.88 m – 2.19 m 0.99 d (6.5) 1.00 d (6.5) 4.71 brs 4.66 brs 0.78 s 0.96 s – 2.05 s – 2.01 s – – – – – 5.32 brs 5.38 brs
67.3CH 88.5CH 39.5C 50.7CH 18.2CH2
1, 3, 10 1, 2, 5, 29, 30, 33 – 3, 4, 7, 10 5, 8, 10
67.3CH 88.4CH 39.4C 50.8CH 18.1CH2
1, 3, 4, 10 1, 4, 29, 30, 33 – 3, 4, 6, 10 4, 5, 8
26.6CH2
5, 8, 9
26.6CH2
5, 8, 9
124.2C 139.9C 37.7C 67.9CH 78.2CH 46.9C 147.5C 120.7CH 35.3CH2
– – – 8, 9, 12, 13 9, 11, 13, 14, 31 – – 8, 14, 16, 17 14, 15, 17, 20
124.7C 139.6C 37.7C 67.8CH 78.4CH 46.6C 147.1C 120.6CH 35.4CH2
– – – 8, 9, 12, 13 9, 11, 14, 17, 19, 31 – – 8, 14, 17 13, 15, 17, 20
49.0CH 23.1CH3 17.0CH3 33.2CH 18.3CH3 34.4CH2
12, 13, 21 1, 5, 9, 10 12, 13, 14, 17 17, 21, 22 17, 20, 22 17, 20, 24
49.1CH 23.1CH3 17.0CH3 33.4CH 18.3CH3 34.4CH2
13, 15, 22 1, 5, 9, 10 12, 13, 17 17, 21, 23 17, 20, 22 20, 21, 23
30.9CH2
24, 25, 28
31.7CH2
20, 24, 25, 28
156.2C 33.5CH 22.2CH3 22.1CH3 107.0CH2
– 23, 24, 24, 23,
156.5C 33.6CH 22.2CH3 22.1CH3 107.0CH2
– 23, 24, 24, 23,
17.9CH3 29.1CH3 170.5C 21.4CH3 170.3C 21.4CH3 – – – – – – –
3, 4, 5, 30 3, 4, 29 – 31 – 33 – – – – – 1, 2, 3 9, 11, 12
2.27 m 1.06 m 3.76 ddd (11.2, 10.0, 4.0) 3.59 d (9.5) – 1.12 dd (11.6, 3.2) 1.69 m 1.60 m 2.32 m 2.08 m – – – 4.10 brs 4.98 brd (2.0) – – 5.52 brt (2.0) 2.35 m 2.02 m 1.87 dt (10.6, 7.5) 1.21 s 0.98 s 1.61 m 0.86 d (6.3) 1.52 m 1.15 m 2.08 m 1.89 m – 2.18 m 0.99 d (6.5) 1.00 d (6.5) 4.72 brs 4.67 brs 0.78 s 0.95 s – 1.98 s – 2.32 t (7.2) 1.17 m 1.26–1.22 m 1.61 m 1.25 m 3.35 t (6.8) 5.33 brs 5.38 brs
17.9CH3 29.1CH3 170.3C 21.4CH3 170.6C 34.0CH2 23.7CH2 29.5–29.1 (CH2)4 24.8CH2 30.9CH2 63.1CH2 – –
3, 4, 5, 30 3, 4, 5, 29 – 31 – 33, 35 33, 34 – – 40, 42 40, 41 – –
7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36–39 40 41 42 2-OH 11-OH
24, 25, 25, 24,
26, 27, 28 27 26 25
pseudo-molecular ion peak at m/z 685.5039 (calcd. for C42H69O7, 685.5043 [M + H]+), requiring nine degrees of unsaturation. Compound 2 is 128 mass units more than 1. The IR and NMR data of 2 were quite similar to those of 1, but the signals associated with the acetoxy group at dH 2.01(H-34)/dC 21.4 (C-34) and 170.3 (C-33) in 1 were absent. Instead, new signals for multiple aliphatic protons at dH 1.26–1.22 (m, H-36 to H-39), four methylenes groups at dH 2.32 (t, J = 7.2 Hz, H-34), 1.17 (m, H-35), 1.61 (m, H-40), and 1.25 (m, H-41), and a terminal oxymethylene group at dH 3.35 (t, J = 6.8 Hz, H-42), suggesting the presence of a long chain fatty acid moiety with terminal hydroxyl group were observed (Carballeira et al., 1999). This was confirmed by the presence of additional 1 H-1H COSY spin system extended (Fig. 2) from H-34 to H-42 and HMBC correlations of H-34 to C-33 and C-35, H-35 to C-33 and C-34, H-41 to C-40 and C-42, and H-42 to C-40 and C-41 and further secured by the ESIMS fragment ion peak at m/z 514 [M + HC10H19O2]+. Alkaline hydrolysis of 2 gave fatty acid methyl ester (FAME), which was identified to be 10-hydroxydecanoic acid methyl ester by the GCMS molecular ion peak at m/z 202. The
26, 25, 25, 24,
27, 28 27 26 25
HMBC cross peak (Fig. 3) of H-3 to C-33 established the attachment of the fatty acid moiety at C-3. The relative configuration of 2 was established based on the observed NOESY correlations (Fig. 2). From the above data and by comparison with literature, the structure of 2 was assigned to integracide G. The isolated compounds were evaluated for their anti-microbial activity against Candida albicans,Candida glabrata, Candida krusei, Asperigillus fumigates, methicillin-resistant Staphylococcus aureus (MRSA), Cryptococcus neoformans, Staphylococcus aureus, Escherichia coli, Pseudomonus aeruginosa, and Mycobacterium intracellulare, anti-leishmanial activity against Leishmania donovani promastigotes, and anti-malarial activity against chloroquine sensitive (D6, Sierraleon) and resistant (W2, Indo-china) strains of Plasmodium falciparum. In addition, cytotoxicity against SK-MEL, KB, BT-549, SKOV-3, LLC-PK11, and VERO cell lines was tested. Compound 1 and 2 displayed potent cytotoxic activity towards BT-549 and SKOV-3 with IC50 values of 1.97 and 0.16 mg/mL and 1.76 and 0.12 mg/mL, respectively compared to doxorubicin (IC50 1.61 and 0.095 mg/mL, respectively) and anti-leishmanial activity
128
S.R.M. Ibrahim et al. / Phytochemistry Letters 15 (2016) 125–130
Fig. 2. 1H–1H COSY and NOESY correlations of 1 and 2.
towards L. donovani with IC50 values of 3.74 and 2.53 mg/mL, respectively and IC90 values of 5.11 and 8.89 mg/mL, respectively compared to pentamidine (positive control, IC50 2.1 mg/mL). On the other hand, they exhibited no anti-malarial and antimicrobial activities. 3. Experimental 3.1. General experimental procedures Optical rotations were measured on a PerkinElmer Model 341 LC polarimeter (PerkinElmer, Waltham, MA, USA). UV spectra were recorded 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 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 1100HPLC system equipped with a photodiode array detector. 1D and 2D NMR spectra were determined on BRUKER Unity INOVA 400 instruments (400 MHz for 1H and 100 MHz for 13C NMR) (Bruker BioSpin, Billerica, MA, USA) using CDCl3 as solvent, with TMS as the internal reference. Solvents were distilled prior spectroscopic measurements. A GCMS was performed on Clarus 500 GC/MS (PerkinElmer, Shelton, CT). The software controller/ integrator was turbo mass, version 4.5.0.007 (PerkinElmer). An elite 5 MS GC capillary column (30 0.25 mm 0.5 mm, PerkinElmer) was used. The carrier gas was helium at a flow rate
Fig. 3. HMBC correlations of compounds 1 and 2.
S.R.M. Ibrahim et al. / Phytochemistry Letters 15 (2016) 125–130
of 2 mL/min (32 p.s.i., flow initial 55.8 cm/s, split; 1:40). Temperature conditions were: inlet line temperature, 200 C; source temperature, 150 C; trap emission, 100 C; and electron energy, 70 eV. The column temperature program was: 50 C for 5 min, increased to 220 C (rate, 20C/min), and held for 5 min. The injector temperature was 220 C. MS scan was from 50 to 650 m/z. Column 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). 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 lmax 255 and 366 nm followed by spraying with anisaldehyde:H2SO4 and heating at 110 C for 1–2 min. 3.2. Isolation and identification of the fungal material M. longifolia L. (Labiatae) was collected in March 2014 from Al Madinah Al Munawwarah, Saudi Arabia. Fusarium sp. was isolated from the internal tissue of M. longifolia roots. The roots were cut into very small pieces 0.5–1.0 cm and washed with sterilized water to remove dust and debris. The surface was thoroughly treated with 70% EtOH for 1–2 min and air-dried under the flow hood. The outer layers of root were removed and the inner tissues were carefully dissected under sterile conditions and placed on potato dextrose agar plates (PDA, Difco) containing antibiotics to suppress 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 morphological 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).
129
powder). The other fractions were retained for further investigation. 3.5. Spectral data Integracide F (1). Colorless powder, Rf 0.77, Si 60F254 (S2); [a]D +9.7 (c 0.6, MeOH; UV lmax (MeOH): 248 nm; IR (KBr) nmax 3435, 1723, 1664, 885 cm 1; NMR data: see Table 1; HRESIMS m/z 557.3839 (calcd. for C34H53O6 [M + H]+, 557.3842). Integracide G (2). White amorphous powder, Rf 0.70, Si 60 F254 (S2); [a]D +11.3 (c 0.5, MeOH); UV lmax (MeOH): 249 nm; IR (KBr) nmax 3426, 2835, 1715, 1659, 885, 725 cm 1; NMR data: see Table 1; HRESIMS m/z m/z 685.5039 (calcd. for C42H69O7, [M + H]+, 685.5043). 3.6. Alkaline hydrolysis of compound 2 A solution of 2 (4 mg) in 3% KOH/MeOH (4 mL) was left to stand for 15 min at room temperature then neutralized with 1 N HCl/ MeOH. The solution was extracted with CHCl3. The solvent was evaporated and the residue obtained was chromatographed on a silica gel column using n-hexane:EtOAc gradient to furnish the methyl ester of 10-hydroxydecanoic acid, which was identified by GCMS (El-Shanawany et al., 2015; Al-Musayeib et al., 2013). 3.7. Anti-microbial assay The anti-microbial activity of the isolated compounds was tested against C. albicans ATCC 90028, C. glabrata ATCC90030, C. krusei ATCC 6258, A. fumigates ATCC 90906, methicillin-resistant S. aureus ATCC 33591, C. neoformans ATCC 90113, S. aureus ATCC 2921, E. coli ATCC 35218, P. aeruginosa ATCC 27853, and M. intracellulare ATCC 23068 as described previously (Al-Musayeib et al., 2014; Ibrahim et al., 2012). Ciprofloxacin and amphotericin B were used as positive standards.
3.3. Cultivation of the fungal material
3.8. Anti-malarial assay
For isolation and identification of secondary metabolites, the fresh fungal culture was transferred into 15 Erlenmeyer flasks (1 L each) containing 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.
The isolated compounds were evaluated for their anti-malarial activities against chloroquine sensitive (D6, Sierraleon) and resistant (W2, Indo-china) strains of Plasmodium falciparum as previously outlined (Al-Musayeib et al., 2014; El-Shanawany et al., 2011). Artemisinin and chloroquine were used as standard antimalarial drugs.
3.4. Extraction and isolation
3.9. Anti-leishmanial assay
The rice culture was extracted with EtOAc and concentrated under vacuum. The concentrated extract was partitioned between n-hexane and 90% MeOH. The total MeOH extract (7.2 g) was subjected to normal phase vacuum liquid chromatography (VLC) using n-hexane, CHCl3, EtOAc, and MeOH, which were separately concentrated to give FS-1 (1.1 g), FS-2 (0.8 g), FS-3 (1.72 g), and FS-4 (2.94 g), respectively. Fraction FS-2 (0.8 g) was chromatographed over silica gel column (60 g 50 2 cm) using CHCl3:MeOH gradients (100% CHCl3 to 60:40CHCl3:MeOH), 50 mL fractions were collected and monitored by TLC to obtain five main subfractions FSC-1-FSC-4. Silica gel column chromatography (20 g 30 2 cm) of sub-fraction FSC-2 (208 mg) using CHCl3: MeOH (98:2 to 90:10) gave impure 1. Sub-fraction FSC-3 (192 mg) was chromatographed over Sephadex LH-20 chromatography (100 g 75 3 cm) using MeOH as an eluent to afford impure 2. Separately, the impure 1 and 2 were purified on RP-18 column (0.04–0.063 mm; 40 g 25 2 cm) using MeOH:H2O gradient to give 1 (37 mg, colorless powder) and 2 (11 mg, white amorphous
The anti-leishmanial activity of the isolated metabolites was tested in vitro against L. donovani promastigotes as previously described (Al-Musayeib et al., 2014; El-Shanawany et al., 2011). Pentamidine and amphoterecin B were used as positive standards. 3.10. 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 noncancerous kidney cell lines: pig kidney epithelial (LLC-PK11) and monkey 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 procedure of (Borenfreund
130
S.R.M. Ibrahim et al. / Phytochemistry Letters 15 (2016) 125–130
et al., 1990). Doxorubicin was used as a positive control, while DMSO was used as the negative control. 4. Conclusion Two new tetracyclic triterpenoids were isolated from the endophytic fungus Fusarium sp. isolated from the roots of M. longifolia. Their structures were elucidated on the basis of extensive spectroscopic analyses. Compounds 1 and 2 showed significant anti-leishmanial and cytotoxic activities. Conflict of interest There is no conflict of interest associated with the authors of this paper. References Al-Musayeib, N.M., Mohamed, G.A., Ibrahim, S.R.M., Ross, S.A., 2013. Lupeol-3-Odecanoate: a new triterpene ester from Cadaba farinosa Forsk. growing in Saudi Arabia. Med. Chem. Res. 22, 5297–5302. Al-Musayeib, N.M., Mohamed, G.A., Ibrahim, S.R.M., Ross, S.A., 2014. New thiophene and flavonoid from Tagetes minuta leaves growing in Saudi Arabia. Molecules 19, 2819–2828. Borenfreund, E., Babich, H., Martin-Alguacil, N., 1990. Rapid chemosensitivity assay with human normal and tumor cells in vitro. In Vitro Cell. Dev. Biol. 26, 1030– 1034. Brill, G.M., Kati, W.M., Montgomery, D., Karwowski, J.P., Humphrey, P.E., Jackson, M., Clement, J.J., Kadam, S., Chen, R.H., McAlpine, J.B., 1996. Novel triterpene sulfates from Fusarium compactum using a rhinovirus 3C protease inhibitor screen. J. Antibiot. 49, 541–546. Carballeira, N.M., Sostre, A., Restituyo, J.A., 1999. Synthesis of racemic 9-methyl-10hexadecenoic acid. Chem. Phys. Lipids 97, 87–91.
deSouza, J.J., Vieira, I.J.C., Filho, E.R., Filho, R.B., 2011. Terpenoids from endophytic fungi. Molecules 16, 10604–10618. El-Kazzaz, M.K., El-Fadly, G.B., Hassan, M.A.A., El-Kot, G.A.N., 2008. Identification of some Fusarium spp. using molecular biology techniques. Egypt. J. Phytopathol. 36, 57–69. El-Shanawany, M.A., Ross, S.A., Ibrahim, S.R.M., Mohamed, G.A., Nafady, A.M., 2011. A new xanthone from the roots of Centaurium spicatum L. Phytochem. Lett. 4, 126– 128. El-Shanawany, M.A., Sayed, H.M., Ibrahim, S.R.M., Fayed, M.A.A., 2015. Stigmasterol tetracosanoate: a new stigmasterol ester from the Egyptian Blepharis ciliaris. Drug Res. 65, 347–353. Elkhayat, E.S., Ibrahim, S.R.M., Mohamed, G.A., Ross, S.A., 2015. Terrenolide S, a new anti-leishmanial butenolide from the endophytic fungus Aspergillus terreus. Nat. Prod. Res. doi:http://dx.doi.org/10.1080/14786419.2015.1072711. Geris dos Santos, R.M., Rodrigues-Fo, E., 2003. Further meroterpenes produced by Penicillium sp., an endophyte obtained from Melia azedarach. Z. Naturforsch. 58c, 663–669. Ibrahim, S.R.M., Mohamed, G.A., Al-Musayeib, N.M., 2012. New constituents from the rhizomes of Egyptian Iris germanica L. Molecules 17, 2587–2598. Ibrahim, S.R.M., Elkhayat, E.S., Mohamed, G.A., Khedr, A.I.M., Fouad, M.A., Kotb, M.H. R., Ross, S.A., 2015. Aspernolides F and G: new butyrolactones from the endophytic fungus Aspergillus terreus. Phytochem. Lett. 14, 84–90. Meinwald, J., Eisner, T., 2008. Chemical ecology in retrospect and prospect. Proc. Natl. Acad. Sci. U. S. A. 105, 4539–4540. Singh, S.B., Ondeyka, J.G., Schleif, W.A., Felock, P., Hazuda, D.J., 2003a. Chemistry and structure-activity relationship of HIV-1 integrase inhibitor integracide B and related natural products. J. Nat. Prod. 66, 1338–1344. Singh, S.B., Zink, D.L., Dombrowski, A.W., Polishook, J.D., Ondeyka, J.G., Hirshfield, J., Felock, P., Hazuda, D.J., 2003b. Integracides: tetracyclic triterpenoid inhibitors of HIV-1 integrase produced by Fusarium sp. Bioorg. Med. Chem. 11, 1577–1582. Singh, S.B., 2000. A new mild PTSA-catalyzed method for sulfate ester hydrolysis and acid-catalyzed rearrangement of 12-acetyl-diene-11-ol tetracyclic triterpenoids involving an angular methyl migration. Tetrahedron Lett. 41, 6973–6976. Tabata, N., Tomoda, H., Yamaguchi, Y., Masuma, R., Bamberger, M.J., Omura, S.J., 1999. Inhibition of cholesteryl ester transfer protein by fungal metabolites, L681, 512. J. Antibiot. 52, 1042–1045.