Phytochemistry Letters 23 (2018) 100–105

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Phytochemistry Letters

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Panduramides A-D, new ceramides from pandurata fruits T ⁎ Amgad I.M. Khedra, Sabrin R.M. Ibrahimb,c, , Gamal A. Mohamedd,e, Samir A. Rossf, Koji Yamadag a Department of Pharmacognosy, Faculty of Pharmacy, Port Said University, Port Said 42526, Egypt b Department of Pharmacognosy and Pharmaceutical Chemistry, College of Pharmacy, Taibah University, Al Madinah Al Munawarah 30078, Saudi Arabia c Department of Pharmacognosy, Faculty of Pharmacy, Assuit University, Assuit 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, Assuit Branch, Assuit 71524, Egypt f National Center for Natural Products Research, Department of Pharmacognosy, School of Pharmacy, The University of Mississippi, MS 38677, USA g Garden for Medicinal , Graduate School of Biomedical Sciences, Nagasaki University, Bunkyo-machi 1-14, Nagasaki 852-8521, Japan

ARTICLE INFO ABSTRACT

Keywords: Re-investigation of the methanolic extract of Ficus pandurata Hance () fruits, afforded four new cer- Ficus pandurata amides namely, panduramides A-D (1-3 and 5), along with newbouldiamide (4). Their structures were de- Moraceae termined on the basis of various spectroscopic and chemical methods, as well as comparison with literature. The Panduramide isolated compounds were evaluated for their antimicrobial, antimalarial, anti-leishmanial, and cytotoxic ac- Ceramides tivities. In addition, their radioligand displacement affinity on opioid and cannabinoid receptors was assessed. Opioid and cannabinoid receptors Compound 1 exhibited good affinity towards cannabinoid receptor 1 (CB1) with displacement value of 69.9%. Antimalarial Anti-leishmanial

1. Introduction Edward and Dennis, 1993). It is applied in TCM to treat gout, hyper- uricemia, arthritis, and indigestion. It is also used for strengthening the The family Moraceae (mulberry family) comprises over 73 genera spleen and removing dampness (Khedr et al., 2016; Chen et al., 2005). and 1000 species. The plants belonging to this family are widely found Former investigation of F. pandurata led to the isolation of sterols and in subtropical and tropical regions and less common in temperate areas triterpenes (Khedr et al., 2016, 2015; Ramadan et al., 2009). In con- (Shukla and Misra, 1997; Pandey, 1997; Wunder, 1997; Singh et al., tinuation of our search for bioactive constituents from F. pandurata, 1994). The genus Ficus (fig genus) represents more than 800 species of four new ceramides: panduramide A-D (1-3 and 5) and a known one (4) shrubs and woody trees, mostly distributed in tropical regions (Khedr were isolated from the methanolic extract of its fruit (Fig. 1). This work et al., 2015, 2016). Triterpenes and sterols were the major constituents reported the isolation and the structural characterization of these reported from this genus (Khedr et al., 2015; Khedr et al., 2016; compounds using various spectroscopic and chemical methods. The Parveen et al., 2009; Ramadan et al., 2009; Sisy and Abeba, 2005; antimicrobial, antimalarial, anti-leishmanial, and cytotoxic activities of Chiang et al., 2005, 2001; Kuo and Lin, 2004; Chiang and Kuo, 2002, the isolated compounds were evaluated. In addition, their radioligand 2001; Ragasa et al., 1999; Kuo and Chiang, 1999; Tuyen et al., 1998). displacement affinity on cannabinoid and opioid receptors was as- Some Ficus species are grown for their edible fruits and as ornamental sessed. plants (Baily, 1963; Lawrence, 1962; Metcalf and Chalk, 1950). They are widely used for treating various diseases as inflammation, diabetes, 2. Results and discussion tumor, and malaria in Ayurvedic and traditional Chinese medicine (TCM) (Khedr et al., 2016; Lansky et al., 2008). Also, they are used in The fruits were extracted with 70% MeOH. The MeOH extract was the treatment of certain skin diseases and respiratory disorders, as well submitted to repeated column chromatography (CC) and further pur- as for hypotensive, anti-diabetic, and anti-cough applications in tradi- ification using semi-preparative HPLC to afford five pure ceramides tional Egyptian medicine (Khedr et al., 2016). Ficus pandurata Hance (1-5). (fiddle leaf fig) indigenous to central and West Africa, is an evergreen Compound 1 was obtained as a white amorphous powder. The tree with thick dull green, fiddle-shaped attractive leaves (Riffle, 1998; molecular formula C40H79NO5 was determined by the HRFABMS

⁎ Corresponding author at: Department of Pharmacognosy and Pharmaceutical Chemistry, College of Pharmacy, Taibah University, Al Madinah Al Munawwarah 30078, Saudi Arabia. E-mail addresses: [email protected], [email protected] (S.R.M. Ibrahim). https://doi.org/10.1016/j.phytol.2017.11.023 Received 6 October 2017; Received in revised form 24 November 2017; Accepted 28 November 2017 1874-3900/ © 2017 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved. A.I.M. Khedr et al. Phytochemistry Letters 23 (2018) 100–105

Fig. 1. Structures of the isolated compounds 1-5.

Table 1

NMR spectroscopic data of compounds 1 and 5 (C5D5N, 400 and 100 MHz).

No. 1 No. 5

δH [mult., J δC (mult.) δH [mult., J δC (mult.) (Hz)] (Hz)]

1 4.49 dd (10.8, 62.0 (CH2) 1 4.50 dd (10.8, 61.9 (CH2) 5.2) 5.2) 4.43 dd (10.8, 4.45 dd (10.8, 4.6) 4.6) 2 5.04 m 52.9 (CH) 2 5.06 m 52.9 (CH) 3 4.61 m 76.7 (CH) 3 4.61 m 76.7 (CH) 4 4.35 m 72.9 (CH) 4 4.35 m 72.9 (CH) 5 4.27 m 72.4 (CH) 5 4.32 m 72.4 (CH) Fig. 2. Some Key COSY and HMBC correlations of 1-3 and 5.

6 1.70 m 26.6 (CH2) 6 1.66 m 26.7 (CH2) 7–12 1.24–1.29 m 29–30 7–12 1.20–1.35 m 29–30 (CH ) 2 n 2008, 2009; Shin and Seo, 1995). The 1H and 13C NMR spectra of 1 (CH ) 2 n δ 13 2.22 m 32.1 (CH2) 13 2.32 m 32.2 (CH2) showed an amide proton at H 8.58 (d, J = 8.6 Hz), four OH groups at 14 5.48 dt (15.1, 131.1 (CH) 14 5.50 dt (15.3, 130.2 (CH) δH 7.60 (brs, 1-OH), 6.70 (brs, 3, 5-OH), and 6.25 (brs, 4-OH), and a di- 5.8) 5.6) substituted olefinic bond at δH 5.48/δC 131.1 and 5.46/131.3, as well as 15 5.46 dt (15.1, 131.3 (CH) 15 5.44 dt (15.3, 130.5 (CH) the resonance at δ 175.3 and 52.9, implying a ceramide structure to 1 5.8) 5.6) C (Table 1)(Ibrahim et al., 2012, 2009; 2008). The characteristic re- 16 2.22 m 32.9 (CH2) 16 2.32 m 32.9 (CH2) 17–22 1.24–1.29 m 29.1–30.0 17–22 1.20–1.35 m 29.1–30.0 sonances for the 2-amino-1,3,4,5-tetrol moiety were observed at δH (CH2)n (CH2)n 5.04 (m, H-2)/δC 52.9 (C-2), 4.61 (m, H-3)/76.7 (C-3), 4.49 (dd, 23 1.29 m 22.6 (CH2) 1.34 m 22.9 (CH2) J = 10.8, 5.2 Hz, H-1A) and 4.43 (dd, J = 10.8, 4.6 Hz, H-1B)/62.0 (C- 24 0.83 t (6.5) 14.2 (CH ) 0.81 t (6.7) 14.3 (CH ) 3 3 1), 4.35 (m, H-4)/72.9 (C-4), and 4.27 (m, H-5)/72.4 (C-5) (Eltahawy 2-NH 8.58 d (8.6) – 8.58 d (8.6) – 1‘– 175.3 (C) 1‘– 175.3 (C) et al., 2015; Ibrahim et al., 2012; Elkhayat et al., 2012). This was

2‘ 2.46 m 35.2 (CH2)2‘ 2.42 m 33.9 (CH2) confirmed by the observed COSY and HMBC cross peaks (Fig. 2). 3‘ 1.70 m 35.9 (CH )3‘ 1.66 m 35.7 (CH ) 2 2 Moreover, the two primary methyls signals at δH 0.83 (6H, t, 4‘ 1.70 m 23.7 (CH2)4‘ 1.66 m 23.6 (CH2) J = 6.5 Hz, H-24, 16‘)/δC 14.2 (C-24, 16‘) and the methylene groups 5‘–14‘ 1.24–1.29 m 29.1–30.0 5‘–18‘ 1.20–1.32 m 29.1–30.0 cluster at δH 1.24-1.29/δC 29.1-30.0 established the ceramide core (CH2)n (CH2)n

15‘ 1.29 m 22.9 (CH2)19‘ 1.22 m 22.9 (CH2) structure of 1. This was further characterized by comparison of its NMR ‘ ‘ 16 0.83 t (6.5) 14.2 (CH3)20 0.81 t (6.7) 14.3 (CH3) spectral data with those of related ceramide derivatives (Ahmed et al., 1-OH 7.60 brs – 1-OH 7.60 brs – 2008; Krishna et al., 2004; Loukaci et al., 2000). The relative stereo- 3-OH 6.70 brs – 3-OH 6.70 brs – chemistry at C-1, C-2, C-3, C-4, and C-5 was assigned to be 2S,3S,4R, 4-OH 6.25 brs – 4-OH 6.25 brs – 5-OH 6.70 brs – 5-OH 6.70 brs – 5R, since the aforementioned NMR and optical rotation value of 1 were in good agreement with those of phytosphingosine-type ceramides possessing 2S,3S,4R,5R configuration (Mohamed et al., 2013; Elkhayat pseudo-molecular ion peaks at m/z 654.6042 (calcd for C40H80NO5, et al., 2012; Kamga et al., 2010; Bankeu et al., 2010; Eyong et al., + 654.6039 [M + H] ) and 676.5845 (calcd for C40H79NNaO5, 676.5856 2006). The length of the long chain base (LCB) and the structure of the [M + Na]+), suggesting two double bond equivalent (DBE). The IR fatty acid were determined after methanolysis using HCl:MeOH − spectrum showed absorption bands at 3420 cm 1 (OH), 1637 and (Hattori et al., 1998; Tanaka et al., 1998). The fatty acid methyl ester − − 1556 cm 1 (amide), and 2945 cm 1 (aliphatic C-H) (Ibrahim et al., (FAME) isolated from the reaction mixture by extraction with n-hexane

101 A.I.M. Khedr et al. Phytochemistry Letters 23 (2018) 100–105

Fig. 3. EIMS fragmentations of DMDS deri- vatives of 1 and 5.

was subjected to GCMS analysis. The FAME spectrum residue was Table 2 NMR spectroscopic data of compounds 2 and 3 (C D N, 400 and 100 MHz). identified as methyl palmitate (C17H34O2) on the basis of the GC and 5 5 FABMS molecular ion peak at m/z 270 [M]+, which was confirmed by No. 2 No. 3 the positive ion peak at m/z 271 [M + H]+ in the FABMS spectrum.

Accordingly, the molecular formula of the phytosphingosine base had δH [mult., J δC (mult.) δH [mult., J δC (mult.) to be C24H49NO4. Taking into account the molecular mass of 1 (m/z 654 (Hz)] (Hz)] [M + H]+) and characteristic FABMS fragment ion peak at m/z 415, 1 4.50 dd (10.6, 61.9 (CH2) 1 4.48 dd 61.9 (CH2) the LCB was assigned as (3S,4R,5R,E)-2-aminotetracos-14-ene-1,3,4,5- 4.8) (10.7, 4.9) tetraol. The location of the double bond in the long chain base was 4.45 dd (10.4, 4.41 dd determined by the EIMS analysis of the dimethyl disulfide (DMDS) 5.1) (10.7, 5.2) derivative of 1 that showed a remarkable fragment ion peak at m/z 187 2 5.06 m 52.9 (CH) 2 5.04 m 53.0 (CH) 3 4.62 m 76.7 (CH) 3 4.61 m 76.8 (CH) (C H S) due to the cleavage of the double bond between the carbon 11 23 4 4.42 m 73.0 (CH) 4 4.32 m 72.8 (CH) atoms bearing a methylthio group (Fig. 3), indicating that the double 5 4.28 m 72.4 (CH) 5 4.27 m 72.5 (CH) bond in a long chain base located at C-14 (Inagaki et al., 2004; 6 1.72 m 26.6 (CH2) 6 1.79 m 26.7 (CH2) Kawatake et al., 2002; Yamada et al., 2002; Inagaki et al., 1998). The 7–12 1.24–1.34 m 29.2–30.1 7–12 1.20–1.35 29.2–30.1 geometry of the double bond was deduced to be E from the coupling (CH2)n (CH2)n 13 13 2.25 m 32.3 (CH2) 2.35 m 33.8 (CH2) constant value (J14,15 = 15.1 Hz), as well as from the C chemical 14 5.52 dt (15.3, 130.6 (CH) 5.55 dt (15.3, 130.7 (CH) δ shifts of the methylene carbons [ C 32.1 (C-13) and 32.9 (C-16)] next to 5.6) 5.6) the olefinic bond (Fusetani et al., 1989). Thus, the structure of 1 was 15 5.48 dt (15.3, 130.8 (CH) 5.45 dt (15.3, 130.8 (CH) assigned as N-((E,2S,3S,4R,5R)-1,3,4,5-tetrahydroxytetracos-14-en-2- 5.6) 5.6) yl)palmitamide and named panduramide A. 16 2.10 m 33.3 (CH2) 2.24 m 32.1 (CH2) 17–22 1.24–1.34 m 29.0–30.1 17–22 1.20–1.35 29.0–30.2 Compound 2 was isolated as a white amorphous powder. It had a (CH2)n (CH2)n molecular formula C41H81NO5 on the bases of the HRFABMS pseudo- 23 1.30 m 22.6 (CH2) 23 1.34 m 22.9 (CH2) molecular ion peaks at m/z 668.6198 (calcd for C41H82NO5, 668.6193 24 0.81 t (6.2) 14.3 (CH3) 24 0.86 t (6.5) 14.2 (CH3) – – [M + H]+) and 690.6015 (calcd for C H NNaO , 690.6012 [M 2-NH 8.60 d (8.6) 2-NH 8.58 d (8.6) 41 81 5 ‘– ‘– + 1 175.3 (C) 1 175.2 (C) + Na] ), which is 14 mass units more than 1 that could be due to an 2‘ 2.35 m 33.7 (CH )2‘ 2.20 m 33.8 (CH ) 1 13 2 2 additional methylene. The H and C NMR spectra were virtually 3‘ 1.70 m 35.7 (CH2)3‘ 1.70 m 35.7 (CH2) identical with those of 1 except for the integration of the aliphatic 4‘ 1.70 m 22.9 (CH2)4‘ 1.66 m 22.9 (CH2) 5‘–15‘ 1.24–1.34 m 29.2–30.1 5‘–16‘ 1.20–1.35 29.2–30.1 methylene protons at δH 1.24-1.34 (Table 2). The lengths of fatty acid and LCB were determined by methanolysis followed GC- and FABMS (CH2)n (CH2)n 16‘ 1.30 m 22.9 (CH2)17‘ 1.22 m 22.9 (CH2) analyses of the methanolysis products. The FABMS spectrum of 2 gave 17‘ 0.81 t (6.3) 14.3 (CH3)18‘ 0.86 t (6.5) 14.2 (CH3) fragment ion peaks at m/z 254 and 415 indicating that its LCB was same 1-OH 7.65 brs – 1-OH 7.64 brs – as that of 1 and fatty acid may be heptadecanoic acid. The GCMS 3-OH 6.75 brs – 3-OH 6.73 brs – – – spectrum afforded a molecular ion peak at m/z 284 [M]+, corre- 4-OH 6.25 brs 4-OH 6.28 brs 5-OH 6.75 brs – 5-OH 6.73 brs – sponding to methyl heptadecanoate (C18H36O2), which was further as- sured by the FABMS positive ion peak at m/z 285 [M + H]+. The 1H 13 and C chemical shifts at the stereo-centers C-1, C-2, C-3, C-4, and C-5, was secured by the GCMS and FABMS analyses of the FAME, which α 25 as well as optical rotation value of 2 ([ ]D + 31.3) suggested that 2 gave two peaks at m/z 298 [M]+ and 299 [M + H]+, respectively fi had the same con guration as that of 1. Therefore, 2 was determined to corresponding to a methyl ester of stearic acid. In addition, the location be N-((E,2S,3S,4R,5R)-1,3,4,5-tetrahydroxytetracos-14-en-2-yl)hepta- and geometry of the double, as well as the stereo-centers configuration decanamide. were determined as in 1. Accordingly, 3 was identified as N- Compound 3 was isolated as a white amorphous powder. The ((E,2S,3S,4R,5R)-1,3,4,5-tetrahydroxytetracos-14-en-2-yl)stearamide HRFABMS showed pseudo-molecular ion peaks at m/z 682.6341 (calcd and named panduramide C. + for C42H84NO5, 682.6349 [M + H] ) and 704.6163 (calcd for Compound 5 was obtained as a white amorphous powder. Its + C42H83NNaO5, 704.6169 [M + Na] ), corresponding to the molecular HRFABMS spectrum showed pseudo-molecular ion peaks at m/z formula C H NO . Compound 3 was 28 mass units more than 1, + 42 83 5 710.6657 (calcd for C44H88NO5, 710.6662 [M + H] ) and 732.6478 suggesting that it had two methylene groups more than 1. The IR and + (calcd for C44H87NNaO5, 732.6482 [M + Na] ), which is 56 mass units MR spectral data of 3 were similar to those of 1. The observed fragment greater than 1 which could be due to four additional CH2 groups in the ion peaks at m/z 268 and 415 in the FABMS spectrum indicated that 3 fatty acid residue. Its 1H and 13C NMR spectral data (Table 1) were had the same LCB as that of 1 and fatty acid may be stearic acid. This similar to those of 1, except for the integration of the aliphatic

102 A.I.M. Khedr et al. Phytochemistry Letters 23 (2018) 100–105

1 methylene protons at δH 1.20-1.35. The H NMR spectrum of 5 dis- Sephadex LH-20 (0.25-0.1 mm, Pharmacia Fine Chemical Co. Ltd, played resonances for several multiplets from 4.32-5.50 ppm, together Piscataway, NJ) and silica gel 60 (0.04-0.063 mm, Merck, Darmstadt, with an amide NH at δH 8.58 (d, J = 8.6 Hz) characteristic for a cer- Germany) were used for column chromatography. Pre-coated silica gel amide framework. This was established by analysis of 2D NMR data. plates Kieselgel 60 F254 (0.25 mm, Merck, Darmstadt, Germany) were Methanolysis, followed by GC and FABMS analyses identified the FAME used for thin-layer chromatographic (TLC) analysis. Semi-preparative of 5 as methyl icosanoate. Therefore, 5 was assigned as N- HPLC was performed on a Develosil C-30-UG-5 (250 × 4.6 mm i.d ((E,2S,3S,4R,5R)-1,3,4,5-tetrahydroxytetracos-14-en-2-yl)icosanamide Nomura Chemical Co., Aichi, Japan) at a flow rate of 1.5 mL/min, and named panduramide D. equipped with a TOSOH RI-8020 detector and a JASCO BIP-I HPLC Newbouldiamide (4) was identified by the analysis of spectroscopic pump. data (NMR and FABMS) and comparison of those data with literature (Kuete et al., 2007; Eyong et al., 2006). 3.2. material The isolated compounds 1-5 were evaluated for their antimicrobial activity against C. albicans, C. glabrata, C. krusei, A. fumigates, methi- Fruits of F. pandurata were collected in May 2013 from the au- cillin-resistant S. aureus (MRSA), C. neoformans, S. aureus, E. coli, P. thorized trees growing in garden of the Faculty of Pharmacy, Assiut aeruginosa, and M. intracellulare. Also, anti-leishmanial activity against University. The plant was taxonomically identified by Prof. Dr. Salah L. donovani promastigotes and antimalarial activity against chlor- EL-Nagar (Professor of Botany, Department of Botany, Faculty of oquine-sensitive (D6, Sierra Leone) and resistant (W2, Indochina) Science, Assiut University, Egypt). A voucher specimen (FPF-2013) was strains of Plasmodium falciparum of the isolated compounds were as- deposited at the Department of Pharmacognosy, Faculty of Pharmacy, sessed. Furthermore, they were tested for cytotoxicity towards VERO Al-Azhar University, Assiut, Egypt. cell line. The results showed that none of the tested compounds had activity in these assays. In addition, these compounds were evaluated using in vitro radi- 3.3. Extraction and isolation oligand binding affinity assays of cannabinoid receptors (CB1 and CB2) and opioid subtypes receptors (δ, κ, and μ)(León et al., 2013; Thomas The air-dried powdered fruits (1.1 kg) were exhaustively extracted et al., 2005). It is noteworthy that 1 (conc. 10 μM) selectively inhibited by cold percolation with 70% MeOH (4 × 4 L) at room temperature. ff 69.9% of the specific binding of [3H]-CP-55,940 to HEK cell membranes The alcoholic extract was evaporated under reduced pressure to a ord expressing human CB1 (Table 3). Meanwhile, compounds 2-5 exhibited a dark brown residue (95 g). The residue was mixed with 500 mL dis- low radioligand binding displacement at 10 μM, with affinities of 25.1, tilled H2O and subjected to solvent fractionation using EtOAc and n- 39.5, 38.9, and 32.8%, respectively for the CB1 receptor. Moreover, 1-5 BuOH which were separately concentrated yielding EtOAc (37.0 g), n- showed low radioligand binding displacement towards cannabinoid BuOH (21.0 g), and aqueous (28.0 g) fractions. The EtOAc fraction was – CB2 and δ and κ opioid receptors at the same concentration. None of the subjected to VLC over silica gel (0.04 0.063) eluting with n- tested compounds had activity in μ opioid receptor. hexane:EtOAc and EtOAc:MeOH gradient to give twenty fractions F1- F20. The fractions from F1-F9 were previously investigated by the au- thors (Khedr et al., 2016). Fraction F10 (1.15 g) was chromatographed 3. Experimental on a sephadex LH-20 CC using CHCl3:MeOH (1:1) to yield four sub- fractions (F10a-F10d). Subfraction F10b (400 mg) was subjected to si- 3.1. General experimental procedures lica gel column using n-hexane:EtOAc gradient elution, which provided four subfractions (F10b-1- F10b-4). Subfraction F10b-2 (130 mg) was Optical rotation was measured on a JASCO DIP-370 digital polari- crystallized from MeOH to give F10b-2 (a single spot on normal silica fi meter (Jasco Co., Tokyo, Japan) at 25 °C at the sodium D line (589 nm). gel). F10b-2 was puri ed on semi-preparative HPLC (column, develosil fl Melting points were determined using an Electrothermal 9100 Digital C-30-UG-5 (4.6 mm × 150 mm); solvent, MeOH; ow rate 0.5 mL/min) ff fi Melting Point apparatus (Electrothermal Engineering Ltd., Essex, to a ord ve compounds: F10b-2-2 (1)(Rt 58.8 min, 14.9 mg, white England). The IR spectrum was measured on a Shimadzu Infrared-400 amorphous powder), F10b-2-3 (2)(Rt 48.3 min, 11.6 mg, white amor- spectrophotometer (Shimadzu, Kyoto, Japan). HRFABMS were re- phous powder), F10b-2-4 (3)(Rt 74.2 min, 13.2 mg, white amorphous corded on JMS DX-303 spectrometer (JEOL Ltd., Japan) using m-ni- powder), F10b-2-5 (4)(Rt 86.2 min, 10.7 mg, white amorphous trobenzyl alcohol or Magic bullet as a matrix. EIMS were recorded on powder), and F10b-2-6 (5)(Rt 103.7 min, 11.9 mg, white amorphous JEOL JMS-SX/SX 102A mass spectrometer. FABMS was determined powder) as pure phytoceramides. using an API 2000 mass spectrometer (ThermoFinnigan, Bremen, Germany). GCMS analysis was carried out on Clarus 500 GCMS (Perkin 3.4. Methanolysis and GC- and FABMS analyses of the FAMEs Elmer, Shelton, CT). The software controller/integrator was Turbo Mass, version 4.5.0.007 (Perkin Elmer). An Elite 5MS GC capillary Compounds 1-3 and 5 (4 mg each) was subjected to methanolysis in column (30 × 0.25 mm × 0.5 μm, Perkin Elmer) was used. The carrier 5 mL of 1 N HCl in MeOH at 80 °C for 18 h. 10 mL H2O were added to gas was helium (purity 99.9999%) at a flow rate of 2 mL/min (32 p.s.i., the reaction mixture (Mohamed et al., 2015). The mixture was ex- flow initial 55.8 cm/s, split; 1:40). The column temperature was tracted with n-hexane (3 × 5 mL) to yield the corresponding FAMEs 100–250 °C (rate of temp. increases 5 °C/min). NMR spectra were re- that were identified by GC and FABMS analyses. The results were as corded with a Unity plus 400 spectrometer (Varian Inc., USA). follows: FAME-1 (methyl palmitate), tR 36.6 min, FABMS m/z: 271 [M

Table 3 Results of binding affinity assay of 1-5 for cannabinoid (subtypes: CB1 and CB2) and opioid (subtypes: δ, κ, and μ) receptors.

Compds CB1% Displacement CB2% Displacement δ % Displacement κ % Displacement μ % Displacement

1 69.9 ± 1.23 2.4 ± 0.04 18.3 ± 0.23 22.4 ± 0.18 – 2 25.1 ± 0.46 3.2 ± 0.08 17.9 ± 0.14 9.9 ± 0.06 – 3 39.5 ± 0.71 1.7 ± 0.02 13.3 ± 0.11 21.2 ± 0.11 – 4 38.9 ± 0.56 5.9 ± 0.09 11.6 ± 0.09 25.5 ± 0.09 – 5 32.8 ± 0.09 1.6 ± 0.07 3.9 ± 0.10 32.6 ± 0.21 –

103 A.I.M. Khedr et al. Phytochemistry Letters 23 (2018) 100–105

+ +H] ; FAME-2 (methyl heptadecanoate), tR 39.7 min, FABMS m/z: versions of the CLSI/NCCLS methods (Ibrahim et al., 2012). + 285 [M + H] ; FAME-3 (methyl stearate), tR 46.2, FABMS m/z: 299 + [M + H] ; FAME-5 (methyl icosanoate), tR 58.8 min, FABMS m/z: 327 3.9. Antimalarial assay [M + H]+. The isolated compounds were tested at concentrations of 3.5. Dimethyl disulfide (DMDS) derivatives of 1–3 and 5 4760–528.9 ng/mL on chloroquine sensitive (D6, Sierra Leone) and resistant (W2, Indochina) strains of Plasmodium falciparum. The stan- Separately, 1-3 and 5 (2.0 mg) was dissolved in DMDS (1 mL) and dard antimalarial agents, chloroquine and artemisinin were used as iodine (1.5 mg) was added to the solution. The resulted mixture was positive controls, whereas DMSO was used as a negative control stored at 60 °C for 40 h in a small-volume sealed vial. The reaction was (Ibrahim et al., 2016; Elkhayat et al., 2016; El-Shanawany et al., 2011). subsequently quenched with 5% aqueous Na2S2O3. The mixture was extracted with n-hexane (3 × 5 mL). The extract was concentrated to 3.10. Anti-leishmanial assay give the DMDS derivatives. EIMS was measured for each derivative. The anti-leishmanial activity of the isolated metabolites was tested 3.6. Spectral data in vitro against L. donovani promastigotes as previously described (Ibrahim et al., 2015a,b; Ibrahim et al., 2012). Pentamidine and am- 3.6.1. Panduramide A [N-((E,2S,3S,4R,5R)-1,3,4,5-tetrahydroxytetracos- photericin B were used as positive controls. 14-en-2-yl)palmitamide] (1) 25 White amorphous powder; [α]D + 30.8 (c 0.03, pyridine); IR νmax 3.11. Cytotoxicity assay − (dry film) (cm 1): 3420, 2945, 1637, 1556; FABMS: m/z 654.7 [M +H]+, 676.7 [M + Na]+; HRFABMS: m/z 654.6042 [M + H]+ (calcd The in vitro cytotoxic activity of the isolated compounds was de- + for C40H80NO5, 654.6039), 676.5845 [M + Na] (calcd for termined against noncancerous kidney cell lines (VERO) at concentra- C40H79NNaO5, 676.5856); NMR spectral data, see Table 1. tions of 4760-528.9 ng/mL. The cell line was obtained from the American Type Culture Collection (ATCC, Rockville, MD). The cell 3.6.2. Panduramide B [N-((E,2S,3S,4R,5R)-1,3,4,5-tetrahydroxytetracos- viability was determined using Neutral Red dye according to a previous 14-en-2-yl) heptadecanamide] (2) method with modification (Ibrahim et al., 2015a,b; Mohamed et al., 25 White amorphous powder; [α]D + 31.3 (c 0.03, pyridine); IR νmax 2013; Borenfreund et al., 1990). Doxorubicin and DMSO were used as − (dry film) (cm 1): 3434, 2297, 1640, 1550; FABMS: m/z 668.7 [M positive and negative controls, respectively. +H]+, 690.7 [M + Na]+; HRFABMS: m/z 668.6198 (calcd for + C41H82NO5, 668.6193 [M + H] ), 690.6015 (calcd for C41H81NNaO5, 4. Conclusions 690.6012 [M + Na]+); NMR spectral data, see Table 2. In summary, this work described the separation and structural de- 3.6.3. Panduramide C [N-((E,2S,3S,4R,5R)-1,3,4,5-tetrahydroxytetracos- termination of four new ceramides, panduramides A-D ( 1-3 and 5) and 14-en-2-yl)stearamide] (3) a known one (4) from the MeOH extract of F. pandurata fruits. Their 25 White amorphous powder; [α]D + 33.1 (c 0.03, pyridine); IR νmax structures were verified based on various spectroscopic and chemical − (dry film) (cm 1): 3386, 1645, 1550, 1380; FABMS: m/z 682.9 [M methods. Their antimicrobial, antimalarial, anti-leishmanial, and cy- +H]+, 704.9 [M + Na]+; HRFABMS: m/z 682.6341 (calcd for totoxic activities, as well as radioligand displacement affinity on opioid + C42H84NO5, 682.6349 [M + H] ), 704.6163 (calcd for C42H83NNaO5, and cannabinoid receptors were evaluated. Compound 1 exhibited good 704.6169 [M + Na]+); NMR spectral data, see Table 2. displacement affinity towards CB1 receptor.

3.6.4. Panduramide D [N-((E,2S,3S,4R,5R)-1,3,4,5-tetrahydroxytetracos- Acknowledgments 14-en-2-yl)icosanamide] (5) 25 White amorphous powder; [α]D + 34.8 (c 0.03, pyridine); IR νmax We are grateful to Mr. M. Inada, Mr. N. Yamaguchi, and Mr. N. − (dry film) (cm 1): 3425, 2945, 1640, 1559; FABMS: m/z 710.8 [M Tsuda of the Scientific Support Section of Joint Research Center, +H]+, 732.8 [M + Na]+; HRFABMS: m/z 710.6657 (calcd for Nagasaki University for NMR and MS spectra measurements. This work + C44H88NO5, 710.6662 [M + H] ), 732.6478 (calcd for C44H87NNaO5, was supported in part by a Grant-in-Aid for Scientific Research No. 732.6482 [M + Na]+); NMR spectral data, see Table 1. 23590008 and 26460124 from the Japan Society for the Promotion of Science, which is gratefully acknowledged. 3.7. Radioligand displacement for cannabinoid and opioid receptor subtypes References Compounds 1-5 were evaluated in competition binding with can- nabinoid receptor subtypes, CB1 and CB2 as previously described Ahmed, S.A., Khalifa, S.I., Hamann, M.T., 2008. Antiepileptic ceramides from the Red Sea (Khedr et al., 2016; León et al., 2013; Thomas et al., 2005). Also, they sponge Negombata corticata. J. Nat. Prod. 71, 513–515. δ κ μ Baily, L.H., 1963. The Standard Cyclopedia of Horticulture, 11th edition. the MacMillan were tested against the opioid receptor subtypes ( , , and ) as outlined Co., New York, pp. 1229–1233. previously (Khedr et al., 2016; León et al., 2013). Bankeu, J.J.K., Mustafa, S.A.A., Gojayev, A.S., Lenta, B.D., Noungou, D.T., Ngouela, S.A., Asaad, K., Choudhary, M.I., Prigge, S., Guliyev, A.A., Nkengfack, A.E., Tsamo, E., Ali, M.S., 2010. Ceramide and cerebroside from the stem bark of Ficus mucuso 3.8. Antimicrobial assay (Moraceae). Chem. Pharm. Bull. 58, 1661–1665. Borenfreund, E., Babich, H., Martin-Alguacil, N., 1990. Rapid chemosensitivity assay with All the isolated compounds were tested for their antimicrobial ac- human normal and tumor cells in vitro. In Vitro Cell Dev. Biol 26, 1030–1034. tivity at concentrations of 20-0.8 μg/mL against Candida albicans ATCC Chen, S.F., Lu, G.Z., Zhao, W.L., 2005. Traditional Chinese Medicine Processing Standards of Zhejiang Province. Zhejiang science and technology Press, Hangzhou, China. 90028, Candida glabrata ATCC90030, Candida krusei ATCC 6258, Chiang, Y.M., Kuo, Y.H., 2001. New peroxy triterpenes from the aerial roots of Ficus Asperigillus fumigates ATCC 90906, methicillin-resistant Staphylococcus microcarpa. J. Nat. Prod. 64, 436–439. aureus ATCC 33591, Cryptococcus neoformans ATCC 90113, Chiang, Y., Kuo, Y., 2002. Novel triterpenoids from the aerial roots of Ficus microcarpa.J. Org. Chem. 67, 7656–7661. Staphylococcus aureus ATCC 2921, Escherichia coli ATCC 35218, Chiang, Y., Kuan Su, J., Liu, Y., Kuo, Y., 2001. New cyclopropyltriterpenoids from the Klebsiella pneumonia ATCC 13883, Pseudomonus aeruginosa ATCC aerial roots of Ficus microcarpa. Chem. Pharm. Bull. 49, 581–583. 27853, and Mycobacterium intracellulare ATCC 23068 using modified Chiang, Y.M., Chang, J., Kuo, C., Chang, C., Kuo, Y., 2005. Cytotoxic triterpenes from the

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