Trends Phytochem. Res. 1(4) 2017 235-242 ISSN: 2588-3631 (Online) ISSN: 2588-3623 (Print) Trends in Phytochemical Research (TPR) Trends in Phytochemical Research (TPR) Volume 1 Journal Homepage: http://tpr.iau-shahrood.ac.ir Issue 4 December 2017 © 2017 Islamic Azad University, Shahrood Branch Press, All rights reserved. Original Research Article Characterization of bioactive compounds from Ficus vallis-choudae Delile (Moraceae) Jean Jules Kezetas Bankeu1, 2, , Amadou Dawé3, Marius Mbiantcha4, Guy Raymond Tchouya Feuya5, Iftikhar Ali6, Marthe Aimée Tchuente Tchuenmogne7, Lateef Mehreen8, Bruno Ndjakou Lenta9, , Muhammad Shaiq Ali2 and Augustin Silvère Ngouela7 Department of Chemistry, Faculty of Science, The University of Bamenda, P.O. Box 39, Bambili, Cameroon International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan 3 Department of Chemistry, Higher Teacher Training College, University of Maroua, P.O. Box 55 Maroua, Cameroon 4 Department of Animal Biology, Faculty of Science, University of Dschang, P.O. Box 67, Dschang, Cameroon 5 Department of Chemistry, Faculty of Science, Scientific and Technical University of Masuku, Box. 943, Franceville, Gabon 6 Department of Chemistry, Karakoram International University, 15100-Gilgit, Gilgit-Baltistan, Pakistan 7 Department of Chemistry, Faculty of Science, University of Yaoundé I, P.O. Box 812, Yaoundé, Cameroon 8 Pharmaceutical Research Centre of Pakistan Council of Scientific and Industrial Research Laboratories Complex, Karachi 75280, Pakistan 9 Department of Chemistry, Higher Teacher Training College, University of Yaoundé I, P.O. Box 47 Yaoundé, Cameroon 1 2 ABSTRACT ARTICLE HISTORY Ficus vallis-choudae Delile has been reported to exhibit antifungal, anticonvulsant, anti-inflammatory and antinociceptive activities. Herein, we report the first ever pharmacochemical studies on the figs of Ficus vallis-choudae Delile resulting in the isolation of a new ceramide named nkwenamide (1). In addition, seven known compounds including the binary mixture of β-amyrin palmitate (2) and lupeol palmitate (3), olean12-en-3-one (4), n-hexacosan-1-ol (5), β-sitosterol (6), and mixture of β-amyrin (7) and lupeol (8) were isolated. Their structures were elucidated using spectroscopic methods. The methanol extract from the figs of this plant exhibited urease, and α-glucosidase inhibitory activities and showed DPPH radical scavenging potency with IC50 values, 61.7, 73.7 and 87.4 µg/mL, respectively. It also showed a weak chemiluminescence activity as compared to ibuprofen. The mixture of 2 and 3 exhibited maximum urease inhibitory activity with IC50=23.9 µg/mL while the mixture of 7 and 8 showed the maximum α-glucosidase inhibition with an IC50 value of 44.0 µg/mL. All the isolates showed weak chemiluminescence activity. Received: 21 August 2017 Revised: 23 October 2017 Accepted: 01 November 2017 ePublished: 12 December 2017 KEYWORDS Ficus vallis-choudae Isolation Nkwenamide Bioactive compounds Antioxidant activity Biological activity © 2017 Islamic Azad University, Shahrood Branch Press, All rights reserved. 1. Introduction Medicinal plants have long been used in traditional medicines for the treatment of many illnesses. This situation has pushed researchers to screen a variety of plants for their biological activities and their chemical constituents (Mohammadhosseini, 2017; Mohammadhosseini et al., 2017). In this context, we carried out the study of the figs of Ficus vallis-choudae Delile, a Cameroonian medicinal plant of the family Moraceae. Ficus vallis-choudae D. is a tropical and subtropical shrub or tree of the Moraceae family, with white latex (Evans, 1996). It is distributed in Tropical Africa from Senegal to Cameroon, from Sudan to Ethiopia and Malawi (Vivien and Faure, 1996). The decoctions of leaves and young leafy stems are used as local drug for jaundice, nausea, bronchial and gastrointestinal disorders (Oliver, 1960). The figs are edible and are really appreciated by children (Vivien and Faure, 1996). Its bark extract has been reported to possess antifungal and anticonvulsant activities (Adekunle et al., 2005; Malami et al., 2010) as well as anti-inflammatory and antinociceptive effects (Lawan et al., 2008). Corresponding authors: Jean Jules Kezetas Bankeu and Bruno Ndjakou Lenta Tel: +237-677-955-630; Fax: +237-675-097-561 E-mail address: bk_jeanjules@yahoo.fr; lentabruno@yahoo.fr 236 Bankeu et al. / Trends in Phytochemical Research 1(4) 2017 235-242 Helicobacter pylori bacteria is recognized as class 1 carcinogen by the World Health Organization (WHO), consequently efforts are being focused worldwide for its eradication through the application of several therapies. Treatment of H. pylori using synthetic compounds is associated with several problems such as high pretreatment cost, pretreatment bacterial resistance and adverse side effects (Yesilada et al., 1999; Huang et al., 2017). Therefore, exploration of some safer urease inhibitors derived from medicinal plants is becoming important as an alternate therapy against H. pylori based infections (Yesilada et al., 1999; Huang et al., 2017). According to chemical literature survey, about 80-90% plants found in nature has great potential against a large number of naturally occurring antimicrobial agents and these agents are the best sources for the treatment of H. pylori (Yesilada et al., 1999). Intestinal α-glucosidase is a key enzyme for carbohydrate digestion, located at the epithelium of the small intestine. α-Glucosidase has been recognized as a therapeutic target for the modulation of postprandial hyperglycemia, which is the earliest metabolic abnormality that occurs in Type II diabetes (Yao et al., 2010). Natural products are still the most available source of α-glucosidase inhibitors. Therefore, screening of α-glucosidase inhibitors in medicinal plants has received much attention. Inhibition of reactive oxygen species (ROS) can be used for the regulation of the inflammatory responses in the innate immune system. Reactive oxygen species play a key role in several inflammatory disorders, such as cancer, atherosclerosis, and ischemic heart diseases (Rimess et al., 2016). Previous phytochemical studies of plants of Ficus genus have revealed the occurrence of ceramides, cerebrosides, steroids, pentacyclic triterpenes, flavonoids and phenolic compounds (Bankeu et al., 2010; Bankeu et al., 2011; Fongang et al., 2015). Even though a preliminary phytochemical analysis on the bark of F. vallis-choudae D. revealed the presence of flavonoids, glycosides, alkaloids, tannins and saponins (Lawan et al., 2008), no attempt has been made so far to isolate its compounds. Therefore, the purpose of present study was to isolate and characterize compounds from the methanolic extract of F. vallis-choudae D. figs and to assess their DPPH radical scavenging and chemiluminescence activities as well as their inhibitory effect on urease and α-glucosidase. 2. Experimental 2.1. Plant material The figs of F. vallis-choudae D., Moraceae, were collected in March, 2014 from Nkwen, Bamenda, North West Region of Cameroon and identified by two botanists from the Department of Biological Sciences, Faculty of Science, The University of Bamenda, Cameroon, and compared with voucher specimens formerly kept at the National Herbarium of Cameroon under the registration number of HNC Nº 5115 SRF/ Can (YA). 2.2. Chemicals For this study, the following reagents and technical and laboratory grade solvents (Fisher) were used: methanol was used for the extraction of the plant material; n-hexane, dichloromethane, ethyl acetate and methanol were used as pure or binary mixtures at different concentrations for purification of compounds. Column chromatography was performed on silica gel (230-400 mesh). Fractions were monitored by TLC using Merck pre-coated silica gel sheets (60 F254), and the identification of spots on the TLC plate was carried out by spraying ceric sulfate reagent solution and heating the plate at about 80 ºC. Depending on the solubility of the isolated compound, deuterated solvents including CDCl₃ and MeOD were used for ¹H and 13C NMR experiments. Phenol, sodium nitroprusside, dipotassium hydrogen phosphate trihydrate, EDTA, lithium chloride and thiourea, all purchased from Sigma were used for the urease assay. Potassium phosphate, p-nitrophenylα-glucopyranoside, dimethylsulfoxide, glycine and 1-deoxynojirimycin (Sigma) were used for α-glucosidase assay while 1,1′-diphenyl-2-picrylhydrazyl, ethanol and butylated hydroxyanisole (Sigma) were used for DPPH. Hanks balanced salt solution and luminol (Sigma) were used for chemiluminescence assay, as well. 2.3. Apparatus Melting points were obtained on a Büchi M-560 melting point apparatus. Optical rotations were measured with a JASCO DIP-360 polarimeter. UV spectra were recorded on a Hitachi UV 3200 spectrophotometer. A JASCO 320-A spectrophotometer was used for scanning IR spectroscopy using KBr pellets. 1D and 2D NMR spectra of the isolates were run on three Bruker spectrometers operating at 75, 100, 150, 400, 500 and 600 MHz, respectively where chemical shifts (δ) were expressed in ppm with reference to the TMS. EIMS spectra were obtained on Varian MAT 311A mass spectrometer operating at 300 ºC. FAB-MS spectra were measured on a JEOL JMS-HX-110 mass Spectrometer. These spectrometers use a magnetic sector and an electric sector analyzer. 2.4. Procedures 2.4.1. Extraction and isolation of compounds The figs of F. vallis-choudae D. (3.8 kg) were harvested from a planted tree, chopped and airdried under shade and ground in a locally made mill Bankeu et al. / Trends in Phytochemical Research 1(4) 2017 235-242 and then extracted with MeOH (methanol) (15 L) (72 hours, repeated three times) at room temperature (24 ºC). The extract was then concentrated to dryness under vacuum at low temperature (40 ºC) to give 129 g of grey crude extract. Part of the extract (128 g) was subjected to medium pressure liquid column (Buchner funnel: 13 cm diameter, 10 cm height) chromatography over silica gel (200 g) (Merck, 230-400 mesh) eluting with mixtures of n-hexane/EtOAc (ethyl acetate) and EtOAc/MeOH of increasing polarities. One hundred and fifty subfractions, each containing 500 mL, were collected and combined according to their TLC profiles on pre-coated silica gel 60 F254 plates developed with n-hexane/EtOAc and CHCl3/MeOH mixture to give 3 fractions (F1-3). Fraction F1 (78 g) was subjected to column chromatography (CC) over silica gel (Merck 230-400 mesh), eluted with n-hexane/EtOAc mixture starting from 100% n-hexane to 50% of the mixture. This resulted in the isolation of a (1:1) mixture of β-amyrin palmitate (2) and lupeol palmitate (3) (5.05 g) (1:99 n-hexane/EtOAc), olean-12-en-3-one (4) (500 mg) (2:98 n-hexane/EtOAc) and n-hexacosan-1-ol (5) (3.0 mg) (1:99 n-hexane/EtOAc). Fraction 2 (22 g) was also subjected to successive CC using the same quality of silica gel and eluted with a mixture of EtOAc and n-hexane (varying from 1:9 n-hexane/EtOAc to 100% EtOAc) to give: β-sitosterol (6) (14 mg) (10:90 n-hexane/ EtOAc), mixture of β-amyrin (7) and lupeol (8) (250 mg) (15:85 n-hexane/EtOAc). Fraction 3 (18 g) was eluted with the same mixture of solvents with different polarity to yield nkwenamide (1) (5.5 mg) (98:2 CHCl3/MeOH). 2.4.2. Methanolysis of 1 Compound 1 (2 mg) was heated with 5% HCl in MeOH (1 mL) at 70 ºC for 12 h in a sealed small-volume vial. The reaction was monitored by TLC analysis. On completion of the reaction, the solution was extracted with n-hexane. The n-hexane layer (0.8 mg) was then separated and concentrated for further analysis using GC-MS, to yield methyl 2-hydroxydocosanoate (1a) (m/z 370 [M]+) (Bakhat et al., 2014). 2.4.3. Bioassays 2.4.3.1. Urease inhibition assay Urease activity was determined by measuring ammonia production using the indophenol method described by Weatherburn (1967) with little modification (Pervez et al., 2016). Reaction mixtures with 25 μL of enzyme (Jack bean Urease purchased from Sigma) solution and 55 μL of buffers containing 100 mM urea were incubated with 5 μL of test samples (extract and compounds 1-8) (1 mM concentration) at 30 ºC for 15 min in 96-well plates. Briefly, 45 μL each of phenol reagent (1% w/v phenol and 0.005% w/v 237 sodium nitroprusside) and 70 μL of alkali reagent (0.5% w/v NaOH and 0.1% active chloride NaOCl) were added to each well. The absorbance at 630 nm was measured after 50 min, using a microplate reader (Molecular Device, USA). All reactions were performed in triplicate in a final volume of 200 μL. The results (change in absorbance per min) were processed by using Soft Max Pro software (Molecular Device, USA). All the assays were performed at pH 8.2 (0.01 M K₂HPO₄•3H₂O, 1 mM EDTA and 0.01 M LiCl). Percentage inhibitions were calculated from the formula (Eqn. 1): Urease inhibition(%)= 100-(ODtest / ODcontrol)×100 (Eqn. 1) Where OD stands for optical density. Thiourea was used as the standard inhibitor of urease. 2.4.3.2. α-Glucosidase inhibition assay The enzyme inhibition assay is based on the breakdown of substrate to produce a coloured product, followed by measuring the absorbance over a period of time (Kurihara et al., 1994). In brief, α-glucosidase (Sigma, type III, from yeast) was dissolved in buffer A (0.1 mol/L potassium phosphate, 3.2 mmol/LMgCl₂, pH=6.8) (0.1 units/mL). p-Nitrophenyl-α-Dglucopyranoside dissolved in buffer A at 6 mmol/L was used as substrates. 102 µL buffer B (0.5 mol/L potassium phosphate, 16 mmol/L-MgCl₂, pH=6.8), 120 µL sample solution (extract or compound 0.6 mg/mL in dimethyl sulfoxide: DMSO), 282 µL deionized water and 200 µL substrate were mixed. This mixture was incubated in a water-bath at 37 ºC for 5 min and then 200 µL enzyme solution was added and mixed. The enzyme reaction was carried out at 37 ºC for 30 min and then 1.2 mL of 0.4 mol/L glycine buffer (pH=10.4) was added to terminate the reaction. Enzymatic activity was quantified by measuring the absorbance at 410 nm. The crude extract showed various colours due to pigments, so the background absorption of every sample was considered. 1-Deoxynojirimycin hydrochloride (DNJ) was used as standard inhibitor of α-glucosidase. The percent inhibition was calculated using the following equation (Eqn. 2): Inhibition % = Absorbance of control − Absorbance of test sample × 100 Absorbance of control (Eqn. 2) 2.4.3.3. DPPH radical scavenging activity The free radical scavenging activity was measured using 1,1'-diphenyl-2-picryl-hydrazyl (DPPH) (Gulcin et al., 2005). An alcoholic solution of DPPH (0.3 mM) was prepared in ethanol. Five microlitres of each sample of different concentrations (62.5 μg-500 μg) were mixed with 95 µL of DPPH solution in ethanol. The mixture was dispersed in 96 well plate and incubated in dark at 37 ºC for 30 min. The absorbance at 515 nm was measured by 238 Bankeu et al. / Trends in Phytochemical Research 1(4) 2017 235-242 microtitre plate reader (Spectramax plus 384 Molecular Device, USA) and percent radical scavenging activity was determined in comparison with the methanol treated control (Eqn. 3). Butylated hydroxyanisole (BHA) was used as standard. Ac − As DPPH scavenging effect (%) = × 100 Ac (Eqn. 3) Where Ac and As, respectively account for the absorbances of control (DMSO treated) and sample. 2.4.3.4. Chemiluminescence assay Luminol enhanced chemiluminescence assays were performed to study the effect of compounds on reactive oxygen species (ROS) from phagocytes (Yamamura et al., 1992). Briefly, 25 µL of diluted whole human blood [1:50 dilution in sterile Hanks balanced salt solution (HBSS++)] was incubated with 25 µL of serially diluted compounds with concentration ranges between 10, 50, 100 and 250 µg/mL. Control wells received HBSS++ and cells but no compounds. Serum-opsonized zymosan-A (SOZ) 25 µL, followed by 25 µL luminol (7×10-5 M) along with HBSS++, was added to each well to obtain a 100 µL volume/well. Tests were performed in white 96-well plates, which were incubated at 37 ºC for 30 min in the thermostated chamber of the luminometer. Results were measured as relative light unit (RLU) reading, with peak and total integral values set with repeated scans at 60-second intervals and 1-second point measuring time. 2.4.3.5. Statistical analysis The resulting data are shown as mean ± SD of Table 1 ¹H (400 MHz) and 13C (100 MHz) NMR data for compound 1 in CD3OD+CDCl3 (δ in ppm, J in Hz, TMS as internal standard). Atom δC Mult. 1 60.9 CH2 2 3 4 51.4 75.5 72.2 CH CH CH 5 34.2 CH2 6-13 14 15 16 17 18 19 20 1ʹ 2ʹ 29.2-29.5 31.8 129.7 130.6 31.8 25.1 22.5 13.9 175.7 71.8 CH2 CH2 CH CH CH2 CH2 CH2 CH3 C CH 3ʹ 32.5 CH2 4ʹ 5ʹ-20ʹ 21ʹ 22ʹ 25.7 29.2-29.5 22.5 13.9 CH2 CH2 CH2 CH3 δH (J in Hz) 3.70, dd, (4.5, 11.5) 3.63, dd, (4.5, 11.5) 3.98-3.99, m 3.43, overlapped dd 3.44-3.45, m 1.69-1.72, m 1.43-1.47, m 1.14-1.20, brs 1.85-1.87, m 5.29-5.30, m 5.29-5.30, m 1.85-1.87, m 1.30-1.32, 2H, m 1.14-1.20, 2H, m 0.77, t, (7.0) HMBC 3.94, dd, (3.0, 8.0) 1.60-1.56, m 1.30-1.32, m 1.14-1.20, brs 1.14-1.20, brs 1.14-1.20, brs 0.77, t, (7.0) 1ʹ 3 1, 3 4 3 15, 14, 14, 15, 16 17 17 16 three independent assays. One way analysis of variance (ANOVA) was carried out for the determination of difference between groups (GraphPad Prism 5.0, USA). P>0.05 was considered as significant. 2.5. MS and NMR data of the isolates Nkwenamide (1): (R)-2-hydroxy-N-((2S,3S,4R,E)-1,3,4trihydroxyicos-15-en-2-yl) docosanamide, colorless gummy solid. [α]D24 +51.2 (c 0.05, MeOH); UV λmax (MeOH) nm: 208 (6.5). IR (KBr) cm-1: 3510-3338 (O-H/ N-H), 2920 (C-H), 1657, 1538 (HN-C=O), 1636 (C=C), 722 (aliphatic Cs); ¹H (500 MHz, CD₃OD+CDCl₃) and 13 C NMR (125 MHz, MeOD+CDCl₃): see Table 1; EI-MS: m/z (%)=681 (1.9) [M]+, 663 (38) [M-H₂O]+, 647 (29), 645 (17) [M-2H2O]+, 408 (44), 384 (100), 339 (85), 283 (10), 281 (10), 223 (4), 111 (27), 97 (48), 83 (68), 57 (86), 43 (73); HR-FAB-MS: m/z 682.6350 (calcd 682.6344 for C42H84NO5 [M+H]+). β-Amyrin palmitate (2): colourless solid; EI-MS m/z (rel. int. %): 664 (M)+ (16.6), 649 (M-CH₃)+ (5.9), 409 (22.9), 218 (100.0), 203 (70.1), 189 (67.3); 13C-NMR (75 MHz, CDCl₃): δC 38.3 (C-1); 26.9 (C-2); 80.6 (C-3); 37.8 (C-4); 55.4 (C-5); 18.3 (C-6); 32.6 (C-7); 39.8 (C-8); 47.6 (C-9); 36.9 (C-10); 23.6 (C-11); 121.7 (C-12); 145.2 (C-13); 41.7 (C-14); 26.1 (C-15); 26.1 (C-16); 32.5 (C-17); 47.2 (C-18); 46.8 (C-19); 31.1 (C-20); 34.9 (C-21); 37.1 (C-22); 28.1 (C-23); 16.8 (C-24); 15.6 (C-25); 16.8 (C-26); 26.0 (C-27); 28.4 (C-28); 33.3 (C-29); 23.7 (C-30), 173.7 (C1ʹ), 31.9 (C-2ʹ), 25.2 (C-3ʹ), 29.2-29.8 (C-4ʹ - C-14ʹ), 22.7 (C-15ʹ), 14.1 (C-16ʹ). Lupeol palmitate (3): colourless solid; EI-MS m/z (rel. int. %): 664 (M)+ (16.6), 649 (M-CH₃)+ (5.9), 409 (22.9), 218 (100.0), 203 (70.1), 189 (67.3); 13C-NMR (75 MHz, CDCl₃): δC 38.4 (C-1); 27.4 (C-2); 80.6 (C-3); 37.8 (C-4); 55.4 (C-5); 18.2 (C-6); 34.7 (C-7); 40.9 (C-8); 50.4 (C-9); 37.1 (C-10); 21.0 (C-11); 25.1 (C-12); 38.1 (C-13); 42.8 (C-14); 27.4 (C-15); 35.6 (C-16); 43.0 (C-17); 48.3 (C-18); 48.0 (C-19); 151.0 (C-20); 29.8 (C-21); 40.0 (C-22); 28.0 (C-23); 16.0 (C-24); 16.6 (C-25); 16.2 (C-26); 14.5 (C-27); 18.0 (C-28); 109.3 (C-29); 19.3 (C-30), 173.7 (C-1ʹ), 31.9 (C-2ʹ), 25.2 (C-3ʹ), 29.2-29.8 (C-4ʹ - C-14ʹ), 22.7 (C-15ʹ), 14.1 (C-16ʹ). Olean-12-en-3-one (4): colourless polymorph solid; mp 166-167, IR (KBr) νmax 1701 cm-1; EI-MS m/z (rel. int. %): 424 (M)+ (9.1), 409 (M-CH₃)+ (7.0), 218 (100.0), 205 (16.0),189 (15.7); 13C-NMR (150 MHz, CDCl₃): δC 39.2 (C1); 34.2 (C-2); 217.7 (C-3); 47.4 (C-4); 55.3 (C-5); 19.6 (C6); 32.1 (C-7); 39.7 (C-8); 46.8 (C-9); 36.6 (C-10); 23.6 (C11); 121.4 (C-12); 145.2 (C-13); 41.8 (C-14); 26.1 (C-15); 26.9 (C-16); 32.5 (C-17); 47.2 (C-18); 46.7 (C-19); 31.0 (C-20); 34.7 (C-21); 37.1 (C-22); 26.4 (C-23); 21.5 (C-24); 15.2 (C-25); 25.8 (C-26); 16.7 (C-27); 28.4 (C-28); 33.3 (C-29); 23.6 (C-30). n-Hexacosan-1-ol (5): waxy colourless solid; EI-MS m/z (rel. int. %): 364 (M-H₂O)+ (3.6), 336 (5.2), 153 (23.0), 139 (28.6), 125 (42.5), 111 (83.1), 97 (100.0), 83 (88.3), 71 (48.1), 57 (61.3); ¹H-NMR (500 MHz, CDCl₃): δH 5.15 (1H, Bankeu et al. / Trends in Phytochemical Research 1(4) 2017 235-242 brs, OH), 3.87 (2H, t, J=6.5 Hz, H-1), 1.77-1.72 (2H, m, H-2), 1.53-1.47 (2H, m, H-3), 1.30-1.24 (44H, brs, H-4 to H-25), 0.85 (3H, t, J=6.5 Hz, H-26). β-Sitosterol (6): colourless solid; EI-MS m/z (rel. int. %): 414 (M)+ (6.1), 399 (M-CH₃)+ (12.3), 397 (97), 396 (100.0), 381 (16.5), 255 (19.2), 147 (26.2); ¹H-NMR (600 MHz, CDCl₃): δH 3.53-3.47 (1H, m, H-3), 5.33 (1H, dd, J=1.8, 4.8 Hz, H-6), 0.66 (3H, s, H-18), 0.99 (3H, s, H-19), 0.90 (3H, d, J=6.6 Hz, H-21), 0.79 (3H, d, J=6.6 Hz, H-26), 0.82 (3H, d, J=6.6 Hz, H-27), 0.86 (3H, t, J=7.2 Hz, H-29). β-Amyrin (7): colourless solid; EI-MS m/z (rel. int. %): 426 (M)+ (26.2), 411 (M-CH₃)+ (8.8), 218 (100.0), 203 (67. s1),189 (31.8); 13C-NMR (100 MHz, CDCl₃): δC 38.6 (C-1); 27.2 (C-2); 79.0 (C-3); 38.8 (C-4); 55.2 (C-5); 18.4 (C-6); 32.7 (C-7); 39.8 (C-8); 47.6 (C-9); 37.0 (C-10); 23.5 (C-11); 121.7 (C-12); 145.2 (C-13); 41.7 (C-14); 26.2 (C-15); 26.0 (C-16); 32.5 (C-17); 47.2 (C-18); 46.8 (C-19); 31.1 (C-20); 34.7 (C-21); 37.1 (C-22); 28.1 (C-23); 15.5 (C-24); 15.6 (C-25); 16.8 (C-26); 26.9 (C-27); 28.4 (C-28); 33.3 (C-29); 23.7 (C-30). Lupeol (8): colourless solid; EI-MS m/z (rel. int. %): 426 (M)+ (26.2), 411 (M-CH₃)+ (8.8), 218 (100.0), 203 (67.1),189 (31.8); 13C-NMR (100 MHz, CDCl₃): δC 38.7 (C-1); 27.5 (C-2); 79.0 (C-3); 38.9 (C-4); 55.3 (C-5); 18.3 (C-6); 34.3 (C-7); 40.8 (C-8); 50.4 (C-9); 37.2 (C-10); 20.9 (C-11); 25.2 (C-12); 38.1 (C-13); 42.8 (C-14); 27.4 (C-15); 35.6 (C-16); 43.0 (C-17); 48.3 (C-18); 48.0 (C-19); 151.0 (C-20); 29.9 (C-21); 40.0 (C-22); 28.0 (C-23); 15.4 (C-24); 16.1 (C-25); 16.0 (C-26); 14.5 (C-27); 18.0 (C-28); 109.3 (C-29); 19.3 (C-30). (4), n-hexacosan-1-ol (5), β-sitosterol (6), β-amyrin (7) and lupeol (8), respectively. The previously reported compounds 4, 5 and 6 were identified by comparison of their physical and spectral data with literature (Nakane et al., 2002; Ahmad et al., 2012; Chaturvedula and Prakash, 2012). The triterpenes 2/3 and 7/8 (Fig. 1) obtained as inseparable binary mixtures were identified by shifting of peaks in 13C NMR spectra and subsequent comparison with literature (Mahato and Kundu, 1994; Barreiros et al., 2002; Lakshmi et al., 2014). 3.1. Structure elucidation Compound 1 was isolated as a gummy colorless solid, mp 88-90 ºC, [α]D24 +51.2 (c 0.05, MeOH). The molecular formula, C42H83NO5, implying two degrees of unsaturation, was deduced from the detailed analysis of one- and two-dimensional NMR data, the EI-MS fragmentation pattern and the positive mode HR-FABMS which showed a pseudomolecular ion peak [M+H]+ at m/z 682.6350 (calcd 682.6344 for C42H84NO5). The UV spectrum in MeOH exhibited absorption bands at λmax 208 and 226 nm, suggesting a ceramide skeleton (Bakhat et al., 2014). The IR spectrum showed absorption bands for amide, hydroxy (3200-3500 cm-1), and secondary amide (1657 cm-1) functionalities (Bakhat et al., 2014). The ¹H NMR spectrum (Table 1) exhibited resonances for two olefinic protons at δH 5.30-5.29 (2H, m, H-15 and H-16), two oxymethylene protons at δH 3.70 (1H, dd, J=11.5, 4.5, Hz, H-1a) and 3.63 (1H, dd, J=4.5, 11.5 Hz, H-1b) and two methyl protons at δH 0.78 (6H, t, J=7.0 Hz, H-20 and H-22ʹ), a downfield methine proton between δH 3.99-3.98 (1H, m, H-2), three oxymethine protons at δH 3.94 (1H, dd, J=3.0, 8.0 Hz, H-2ʹ), 3.43 (1H, overlapped dd, H-3), 3.45-3.44 (1H, m, H-4) four methylene groups between δH 1.31-1.91, and the rest of the methylene protons between δH 1.53-1.20 (br, s, 28×CH2). These signals confirmed the basic skeleton of 3. Results and Discussion The MeOH extract of the figs of F. vallis-choudae D. was fractionated and subjected to repeated column chromatography on silica gel to afford eight compounds including nkwenamide (1), β-amyrin palmitate (2), lupeol palmitate (3), olean-12-en-3-one OH ′ ′ ′ ′ ′ O 1′ ′ 3′ ′ 5′ ′ 7′ ′ 9′ ′11 ′ 13 ′15′ ′17 ′19′ ′ 21 22 2 8 4 20 14 16 18 10 12 6 NH OH 16 HO 1 11 13 17 19 20 3 7 9 4 5 6 8 12 14 15 2 10 18 1 OH 30 30 29 29 12 H 25 3 R 1 10 H 5 18 17 8 5 18 H 22 H H 27 R H O H 3 R= Palmitoyl 8 R= H 2 R= Palmitoyl 7 R= H 19 17 18 20 23 H 21 26 27 H H HO 6 24 25 H 1 Fig. 1. Structures of the isolates. 1 26 CH3(CH2)24CH2OH 19 21 H 22 28 H 6 24 23 239 H 4 240 Bankeu et al. / Trends in Phytochemical Research 1(4) 2017 235-242 OH OH O O NH OH NH HO OH HO 1 OH 5% HCl/MeOH OH Fig. 3. Important COSY ( OH ) and HMBC ( ) correlations of 1. O 1a OMe + NH2 OH HO OH 1b Fig. 2. Methanolysis of 1. 1 to be a sphingolipid (Bakhat et al., 2014). The broad band decoupled 13C NMR spectrum (Table 1) displayed carbon signals, which were sorted by DEPT and HSQC techniques into a quaternary carbon of an amide carbonyl at δC 175.7 (C-1ʹ), two olefinic methine carbons at δC 129.7 (C-15) and 130.6 (C-16), four methine carbons at δC 51.4 (C-2), 75.5 (C-3), 72.2 (C-4) and δ 71.8 (C-2ʹ) and an oxymethylene carbon at δC 60.9 (C-1). It also exhibited signals for aliphatic methylenic carbons in the range of δC 22.5-34.2, while the two terminal methyl carbons were observed at δC 13.9. Analysis of the ¹H-¹H COSY (Fig. 2), HSQC and HMBC (Fig. 2) spectra led to the assignment of proton and carbon signals for 1. The ¹H-¹H COSY spectrum exhibited the correlation between the oxygenated methylene hydrogens at δH 3.70 (H-1a) and 3.63 (H-1b) with the azomethine hydrogen between δH 3.99-3.98 (H-2) which in turn was connected to the oxymethine proton H-3 at δH 3.43. The proton H-3 at δH 3.43 also correlated with the oxymethine proton H-4 at δH 3.443.45 confirming the position of C-3 and C-4 for the two hydroxyl groups, respectively (Fig. 2). The position of the third hydroxyl group was located at C-2ʹ, based on the HMBC correlation observed between the proton H-2ʹ at δH 3.94 and the amide carbonyl carbon (δC 175.7). The geometry of the double bond was assigned as trans based on the chemical shift of C-14 (31.8) and C-17 (31.8). Typically, the signals of a carbon next to a trans double bond appear at δ≈32, while those of a cis double bond appear at δ≈27 (Bankeu et al., 2010). The stereochemistry at different stereocenters was further confirmed through NOESY spectrum, which showed correlation of the azomethine hydrogen at δH 3.983.99 (H-2) with H-4 at δH 3.44-3.45 and H-2′ at δH 3.94 similar to that of pakistamide C which has already been established (Bakhat et al., 2014). The number of carbons in the fatty acid chain of the sphingolipid was determined to be 22 based on the prominent ion peak observed on the EI-MS spectrum of 1 at m/z 339 (85%) corresponding to the cleavage between the nitrogen atom and the carbonyl carbon (Bakhat et al., 2014). This was further confirmed by the methanolysis of compound which yielded methyl 2-hydroxydocosanoate (m/z 370 [M]+) as the fatty acid methyl ester; therefore, the long-chain base length was composed of 20 carbons with double bond located in the base chain. In the same way, the position of the olefinic double bond was determined at C-15 due to appearance of prominent ion peaks in EI-MS at m/z 57 (86) and 83 (68) (Fig. 3) relating to the fragment ion C₄H₉+ and C₆H11+, respectively from the long-chain base branch. On the basis of these evidences the structure of nkwenamide (1) was determined as (R)-2′-hydroxyN-((2S,3S,4R,E)-1,3,4-trihydroxyicos-15-en-2-yl) docosanamide. 3.2. Methanolysis of 1 The methanolysis of compound 1 led to the formation of methyl 2-hydroxydocosanoate (1a) (m/z 370 [M]+) as the fatty acid methyl ester indicating that the long-chain base (1b) length was composed of 20 carbons with the double bond located in the base chain. 3.3. Biological activities 3.3.1. Urease enzyme inhibition The methanol extract of F. vallis-choudae D. and some isolated compounds showed considerable antiurease activity with IC50 values ranging from 23.9 to 61.7 µg/mL (Table 2). The binary mixture of 2 and 3 was the most potent among all the isolated compounds and as compared to the reference, thiourea with an IC50 value of 21.7 µg/mL (Table 2). This finding is in line with the traditional use of F. vallis-choudae D. In fact, leaves and Table 2 DPPH radical scavenging, urease inhibition and glucosidase inhibition activities of some isolates and the MeOH extract. Compounds 2 and 3 4 7 and 8 MeOH extract BHA Thiourea DNJ DPPH radical scavenging activity Urease inhibition activity Glucosidase inhibition activity IC50 (μg/mL) IC50 (μg/mL) IC50 (μg/mL) Nil Nil < 500 87.4 ± 0.10a 44.2 ± 0.09b - 23.9 ± 0.32a 32.7 ± 0.11b 43.6 ± 0.85c 61.7 ± 0.32d 21.7 ± 0.32a - 87.4 ± 0.85a 55.3 ± 0.22b 44.0 ± 0.31c 73.7 ± 0.43d 3.42 ± 1.7e Values are shown as mean ± SD of three independent assays. Superscript letters within the same column indicate significant differences (P<0.05). Bankeu et al. / Trends in Phytochemical Research 1(4) 2017 235-242 young leafy stems are used to treat gastrointestinal disorder (Oliver, 1960; Yesilada et al., 1999). This activity could be justified by the presence of urease inhibitors, an enzyme found in H. pylori. 3.3.2. α-Glucosidase enzyme inhibition In the present study, we investigated the in vitro α-glucosidase inhibitory activity by using some compounds isolated from figs of F. vallis-choudae D. The tested compounds produced a weak α-glucosidase enzyme inhibition with IC50 values between 44.0 and 87.4 µg/mL while the control DNJ had an IC50 value of 3.42 µg/mL with the mixture of 7 and 8 being the most potent. Β-amyrin isolated from Memecylon umbellatum showed significant inhibition of α-glucosidase (Sridevi et al., 2015) as well as lupeol showed 13.1% inhibition of this enzyme at 20 μg/mL (Kakarla et al., 2016). 3.3.3. DPPH radical scavenging assay In this assay, only the extract showed a potent antioxidant activity against DPPH radical scavenging with an IC50 value of 87.4 µg/mL while all the tested isolates were inactive (Table 2). The synergetic action of secondary metabolites of the methanol extract could justify the poor activity of resulting compounds. As far as we are concerned, this is the first report of the antioxidant activity of F. vallis-choudae D. However, the antioxidant activity of Ficus species such as F. carica have been reported previously (Ali et al., 2012). 3.3.4. Chemiluminescence assay A cellular and a cell-free luminol-enhanced chemiluminescence assays were used to evaluate the antioxidant and ROS scavenger effects of compounds isolated from figs of F. vallis-choudae D. All the compounds exerted a weak inhibition, olean-12-en3-one (4) and nkwenamide (1) were more active than other isolates as shown in Table 3. These results were in accordance with previously Table 3 Effect of isolates on human whole blood assayed by luminolamplified chemiluminescence. Compounds IC50 values (μg/mL) 1 2 and 3 4 5 6 7 and 8 MeOH extract Ibuprofen 122.67 ± 3.67a 243.71 ± 6.33b 102.91 ± 2.37a 139.35 ± 2.45a 225.45 ± 5.06b 173.47 ± 3.37c 270.22 ± 4.79d 14.42 ± 0.35e Values are shown as mean ± SD of three independent assays. Superscript letters within the same column indicate significant differences (P<0.05). 241 reported pharmacological activities of the methanol stem bark extract F. vallis-choudae D. such as antiinflammatory and antinociceptive (Lawan et al., 2008). In fact, the inhibition of the production of ROS is in connection with the control of oxidative stress and inflammation. In addition, some traditional uses such as the use of leaves and young leafy stems for gastrointestinal troubles (Oliver, 1960; Yesilada et al., 1999) can probably be explained since the extract and some isolates inhibited urease enzyme found in H. pylori which is involved in such disorder. 4. Concluding remarks The phytochemical study of the figs of Ficus vallischoudae D. led to the isolation of a new ceramide, nkwenamide (1) and seven known compounds. Though methanol extract displayed urease, and α-glucosidase inhibitory activities and antioxidant potency, majority of isolated compounds only inhibited the activity of urease and α-glucosidase and the reactive oxygen species. The results obtained in this study support the use of Ficus vallis-choudae D. in Cameroonian pharmacopeia. Conflict of interest The authors declare that there is no conflict of interest. Acknowledgments The authors are very grateful to The World Academy of Sciences (TWAS) and the International Center for Chemical and Biological Sciences (ICCBS), University of Karachi, Pakistan for their financial and technical support through the ICCBS-TWAS Postdoctoral Fellowship number 3240280476 granted to BKJJ. We are also grateful to Dr Njouonkou André Ledoux and Mr Tacham Walter Ndam for the identification of the plant material. References Adekunle, A.A., Familoni, O.B., Okoli, S.O., 2005. 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