Abstract
A new iridoid glucoside, 10-methoxy apodanthoside (1), and a new monoterpene glycoside, (3S,6S)-cis linalool-3,7-oxide O-β-D-glucopyranosyl-(1″→5′)-β-D-xylofuranoside (2), were isolated from V. edulis (Rubiaceae), along with eighteen known compounds (3–20), including monoterpenes, iridoid glycosides, and a lignin, which were encountered for the first time in the genus Vangueria,. The structural elucidation of the isolates was based on the analysis of spectroscopic (1D and 2D NMR) and HR-ESI-MS data. Detailed stereochemical studies of 1 and related iridoid glucosides (compounds 3, 4 and 8) were made by matching the calculated ECD peaks with the experimental ones. All isolates were tested for their antiprotozoal, antifungal, and antiplasmodial activities. Compounds 9, 15 and 16 showed good trypanocidal activities against Trypanosoma brucei brucei with IC50 values of 8.18, 9.02 and 7.80 μg/mL, respectively and IC90 values of >10, >10 and 9.76 μg/mL, respectively. Compound 16 showed a moderate activity against Candida glabrata with an IC50 value of 8.66 μg/mL. Compound 20 showed a weak antiplasmodial activity against chloroquine-sensitive (D6) and resistant (W2) Plasmodium falciparum with IC50 values of 3.29 (SI, >1.4) and 4.53 (SI, >1) μg/mL, respectively.
Keywords: Vangueria edulis, Rubiaceae, Iridoid glucosides, Monoterpene glycosides, ECD spectra, Trypanocidal
Vangueria edulis (Vahl) Vahl (Rubiaceae) is a perennial shrub or small tree which is widely distributed in the tropics, especially Madagascar and Africa [1]. It is grown for its tasty edible fruits and is known as Spanish tamarind [2]. Some Vangueria species have been shown to possess antimicrobial activity and are fed to cattle suffering from East Coast Fever (caused by the protozoan parasite Theileria parva), which cause an annual loss of over one million cattle. Also, they were used for the cure of parasitic worm infections due to their anthelmintic and antiplasmodial activities [3]. There are relatively few phytochemical studies on V. edulis, including our previous study on its leaves [4]. In continuation of our search for new active metabolites, the flowers and leaves of this species have been further investigated. As a result, two new compounds 1 and 2 were isolated along with 18 known compounds.
Compound 1 was isolated as an optically active oily residue. The positive HR-EIS-MS suggested a molecular formula of C18H24O11 by providing a molecular ion peak at m/z 439.1216 [M+Na]+ (calcd. 439.1216) and confirmed by the negative HR-EIS-MS ion at m/z 451.1028 [M+Cl]− (calcd. 451.1007). Its UV absorption (λmax 233.9 nm, log ε 4.1) indicated the presence of a conjugated enol-ether system [5]. The 1H NMR spectrum showed the characteristic signals of an iridoid skeleton in addition to two carbomethoxy groups at δH 3.794 and 3.805, which were correlated in the HMBC spectrum with two carbonyl groups at δC 174.7 and 168.9, respectively confirming the presence of two methyl esters in the compound. An anomeric doublet at δH 4.742 (J= 8.0 Hz), characteristic of a β-D-glucosyl unit, correlated in the HMBC spectrum to C-1, as in most iridoid glucosides [6]. Its 13C NMR spectrum confirmed the presence of these functionalities and was in accordance with the apodanthoside structural pattern [6], except for the presence of an additional methoxy group at C-10. The stereochemistry of compound 1 was determined on the basis of ROESY experiment, which showed diagnostic cross peaks between H-1/H-1′, H-5/H-9 and H-8/H-1, and confirmed by ECD analysis. The conformational space of the aglycone moiety was searched to check for the degree of flexibility of compound 1. The aglycone moiety showed considerable rigidity with 6 major non-redundant conformers adopting two ring geometries. This rigidity reduced the computational expenses required for geometry optimization and ECD calculations. The experimental ECD spectrum showed a peak of negative Cotton effect at 260 nm. The spectra of the possible stereoisomers of compound 1 were calculated to match with the experimental one, and (1S,4aS, 7S,7aS)1-(β-D-glucopyranosyloxy)-1,4a,7,7a tetrahydrocyclopenta[c]pyran-4,7-dicarboxylic acid 4,7-dimethyl ester was confirmed to be the stereochemical notation of compound 1 by comparing its calculated ECD with the experimental one. The dominant conformer of the assigned stereoisomer of compound 1 is the one whose calculated ECD spectrum showed the best matching to the experimental spectrum (Figure 2). Therefore, compound 1 was identified as 10-methyl apodanthoside, which is a new natural iridoid glucoside.
Figure 2.
The experimental and calculated ECD spectra of compound 1. Both spectra showed a negative Cotton effect at 260 nm.
Compound 2 was isolated as a colorless oily residue. Its molecular formula was established as C21H36O11 from the positive HR-ESI-MS, which gave a molecular ion peak at m/z 487.2155 [M+Na]+ (calcd. 487.2154). The 1H NMR spectrum showed three singlets for primary methyl groups, methylene signals, and terminal vinyl group signals which suggested cis linalool-3,7-oxide as the aglycone part [7], attached to two sugar units with β configuration at δH 4.314 (J = 8.0 Hz) and δH 4.383 (J = 7.6 Hz). The 13C NMR spectrum, showing 21 carbon signals, including two anomeric carbons at δC 106.3 and 104.8, confirmed these functionalities. Acidic hydrolysis of compound 2 yielded β-D-glucose and β-D-xylose as the sugar components by comparison with authentic samples. The sugar unit connectivities were deduced from the HMBC correlations (Figure 3), in addition to agreement of sugar signals with those of xyloside, except for the downfield shift of C-5′ due to glycosylation [8]. The stereochemistry of compound 2 was established by comparison of its 1H and 13C NMR data with those previously reported for analogues [7]. Therefore, compound 2 was identified as (3S,6S)-cis linalool-3,7-oxide β-D-glucopyranosyl-(1″→5′)-β-D-xylofuranoside. To the best of our knowledge, compound 2 is a newly reported natural compound.
Figure 3.
Important HMBC (H→C) correlations of compound 2.
Compounds 3, 4 and 8 were identified as the iridoid glucosides: geniposidic acid [9], apodanthoside [6], and geniposide [10], respectively. Confirmation of the stereochemistry of these iridoids was made using ECD analysis for the first time. Similar to compound 1, compounds 3, 4 and 8 adopted two major ring geometries for the aglycone moieties. The calculated and experimental spectra of the defined stereoisomers are almost identical and show the same peak positions and amplitude. Compound 4 has 6 non-redundant conformers for the aglycone moiety, and the dominant conformer of this stereoisomer is detected by comparing the calculated ECD of all non-redundant conformers with the experimental ECD spectrum. A negative ECD band at 260 nm is observed in the spectral region, which is similar to that of its ester derivative, compound 1. A negative spectral band is found in experimental and calculated ECD spectra of compound 3 ~ 225 nm, representing a prominent negative Cotton effect. As in compounds 1 and 4, the aglycone ring adopts two different geometries represented by the dihedral angle (O2-C1-C9-C8) of 67° for one and 162° for the other. After geometry optimization and comparison of the calculated and experimental ECD spectra of each conformer set, we defined the dominant conformer of compound 3 to adopt the geometry of the lowest energy aglycone moiety. In order to confirm our results, the calculated ECD spectrum was compared with the experimental ones of the sample and authentic geniposidic acid. The experimental ECD spectrum of compound 8 revealed a negative band at 225 nm. The calculated ECD showed perfect matching with the experimental data, strongly confirming the stereochemical information. Because compound 8 is the ester derivative of compound 3, we compared its ECD spectra with that of authentic geniposidic acid for confirmation.
Other compounds isolated from the flowers were identified as the monoterpene, 6,7- dihydroxy linalool (5) [11], β-sitosterol glucoside (6) [12], the monoterpene glycosides: (3S,6R)-cis-linalool-3,6-oxide β-D-glucopyranoside (7) [7], and (3S, 6R) cis-linalool 3,6 oxide, O-β-D-xylopyranosyl-(1″→6′)-β-D-glucopyranoside (9) [7], D-mannitol (10) [13], p-hydroxy benzoic acid (11) [14], chlorogenic acid methyl ester (12) [15], quercetin 3-O-rutinoside-7-O-rhamnoside (13) [16], kaempferol-7-O-α-L-rhamnopyranoside (14) [17], quercetin-7-O-α-L-rhamnopyranoside (15) [16], and quercetin (16) [17]. From the leaves, 6 compounds were isolated and identified as scandoside methyl ester (17) [9], syringaresinol-4′-O-β-D-monoglucopyranoside (18) [18], kaempferol-3-O-rutinoside-7-O-rhamnoside (19) [19] and 4,5-dicaffeoyl quinic acid methyl ester (20) [20] in addition to compounds 12 and 14. All known isolated compounds, except 6 and 16, were isolated for the first time from the genus Vangueria. Monoterpenes, iridoids and lignan were encountered for the first time in Vangueria, which might be useful for the chemotaxonomic evaluation of the genus. It is also apprehensible to encounter iridoids in plants containing monoterpenes.
Compounds 9, 15 and 16 showed good trypanocidal activities against Trypanosoma brucei brucei with IC50 values of 8.18, 9.02 and 7.80 μg/mL, respectively and IC90 values of >10, >10 and 9.76 μg/mL, respectively compared with the positive control DFMO, difluoromethylornithine (IC50 and IC90 values of 2.30 and 5.20 μg/mL). Compound 16 showed a moderate activity against Candida glabrata with an IC50 value of 8.66 μg/mL compared with the positive control AMB, amphotericin B (IC50 0.34 μg/mL). Compound 20 showed a weak antiplasmodial activity against the chloroquine-sensitive (D6) and resistant (W2) Plasmodium falciparum with IC50 values of 3.29 (SI, >1.4) and 4.53 (SI, >1) μg/mL, respectively compared with the positive control chloroquine (IC50 values of <26.0 (SI, >9) and 116 (SI, >2.1) ng/mL, respectively).
Experimental
General
Optical rotations, Autopol IV polarimeter; IR, Bruker Tensor 27 instrument; UV, Cary-50 Bio spectrophotometer; CD, JASCO J-715 spectrometer; NMR, Bruker Avance DRX-500 instrument at 500 (1H) and 125 MHz (13C) and a Varian Mercury 400 MHz spectrometer at 400 (1H) and 100 (13C); HR-ESI-MS, Bruker BioApex-FTMS with electrospray ionization (ESI). Column chromatography (CC) was performed on silica gel 60 F254 (0.2 mm, Merck), Diaion HP-20, Sephadex™ LH-20, SPE columns 10 g RP (C-18) and MN-polyamide-SC-6.
Conformational analysis and geometry optimization
OMEGA 2.5.1.4 was used for conformational sampling [21–23]. The 3D structural construction and initial geometry refinement were performed using OMEGA’s MMFF94s force field variant with no electrostatic term for Coulomb interactions. All generated conformers were kept within a 10.0 kcal/mol energy window. A root mean square (RMS) Cartesian distance of less than 0.5 was used to remove redundant and duplicate conformers. For the ab initio optimization, we used B3LYP hybrid density functional theory (DFT) and 6–31G** basis set (B3LYP/6–31G**) in Gaussian 09 [24]. The geometry in the vacuum and in polarizable continuum DMSO solvent model was optimized.
ECD calculation
Time-dependent density functional theory (TDDFT) was used to calculate the ECD spectra. Electronic excitation energies, oscillator and rotational strengths were calculated using B3LYP/6–31G** at excited states of 40 [24]. The PCM methanol solvent model was used in all calculations.
Plant material
Flowers and leaves of Vangueria edulis were collected from El-Zohria Research Garden, Cairo, Egypt in May 2013. The plant material was identified by Dr Mo’men M. Mahmoud, Professor of Taxonomy, Assiut University, Egypt. A voucher specimen (No. 35) has been deposited at the herbarium of the Pharmacognosy Department, Assiut University, Assiut, Egypt.
Antiprotozoal screening
Leishmania donovani promastigote, L. donovani amastigote, L. donovani amastigote/THP1 cells and Trypanosoma brucei brucei strains were used for antiprotozoal screening. The in vitro antileishmanial and antitrypanosomal assays were made on cell cultures of L. donovani promastigotes, axenic amastigotes, THP1-amastigotes, and Trypanosoma brucei trypomastigotes by Alamar Blue assays, as described earlier [25]. DFMO was used as a positive control.
Antifungal screening
Candida albicans ATCC 90028, C. glabrata ATCC 90030, C. krusei ATCC 6258, Cryptococcus neoformans ATCC 90113, and Aspergillus fumigatus ATCC 204305 were used for antifungal testing [26]. IC50 values were calculated using the XLfit curve fitting software. Amphotericin B was used as a positive control.
Antiplasmodial screening
All isolates were tested against chloroquine-sensitive (D6, Sierra Leone) and resistant (W2, Indo China) strains of P. falciparum by measuring plasmodial LDH activity, as described earlier [27], in addition to the VERO mammalian cell line as an indicator of general cytotoxicity. The selectivity indices (SI), and the ratio of VERO IC50 to either D6 or W2 IC50, were calculated. IC50 values were calculated using the XLfit software. Chloroquine was used as a positive control.
Acid hydrolysis of compound 2
Compound 2 (1.5 mg) was refluxed with 2 mL 1 N HCl (aq.) at 80°C for 1 h. After cooling, the reaction mixture was extracted with EtOAc. The EtOAc was removed under reduced pressure. The H2O layer was subjected to co-TLC analysis using CHCl3-MeOH-H2O (6:4:1), and two sugars were identified after comparison with authentic samples.
Extraction and isolation
Air-dried powdered flowers (300 g) were exhaustively extracted by maceration with MeOH (3 L × 3) at room temperature for 3 days. VLC on silica gel (900 g) was used for fractionation of the methanolic extract (30 g), which was eluted with n-hexane, EtOAc and MeOH (2.0 L, each) to give F1-F3. F3 (15.2 g) was subjected to Diaion-HP20 CC which was eluted with H2O then MeOH. The MeOH sub-fraction (6.8 g) was subjected to MN-polyamide-SC-6 (225 g) CC which was eluted with water then a gradient of decreased polarities with water-methanol systems to deliver 5 sub-fractions (F3a to F3e). F3a (2.0 g) was subjected to silica gel (100 g) CC, which was eluted initially with DCM-MeOH (95:5) then DCM-MeOH gradient elution, resulting in 5 sub-fractions (F3a-1 to F3a-5). F3a-1 (476 mg) was purified by Sephadex LH-20 (50 g) CC, eluting with DCM-MeOH (1:1) to afford 1 (20 mg). F3a-3 (263.5 mg) was subjected to Sephadex LH-20 (30.0 g) CC to afford 3 sub-fractions (1–9), (10–16) and (17–23). Further purification of F3a-3-(17–23) (40.0 mg) using SPE-C18 (2 g) eluted with H2O then a H2O-MeOH gradient afforded 3 (8.6 mg). F3a-4 (223 mg) was subjected to Sephadex LH-20 (30.0 g) CC eluted with MeOH to afford 3 sub-fractions (1–5), (6–10) and (11–14). F3a-4-(6–10) was further purified with Sephadex LH-20 to afford 4 (4 mg). F3b (1.5 g) was subjected to silica gel (60 g) CC eluted initially with EtOAc-DCM-MeOH-H2O (15:8:4:1) then (20:10:11:5) to afford F3b-1 to F3b-6. F3b-1 (107.0 mg) was subjected to Sephadex LH-20 (15 g) CC eluted with MeOH to afford F3b-1-1, F3b-1-2, and compound 5 (28 mg). F3b-2 (40 mg) was subjected to Sephadex LH-20 (10 g) CC using MeOH as eluent to afford 6 (2.8 mg). F3b-3 (75 mg) was subjected to silica gel (6 g) CC eluting initially with DCM-MeOH (95:5) then (90:10), (85:15) and (80:20) to afford 7 (10 mg). F3b-4 (130 mg) was subjected to SPE-C18 (10 g) CC starting with H2O then H2O-MeOH gradient elution to afford 8 (6 mg). F3b-5 (460 mg) was subjected to SPE-C18 (20 g) CC starting with H2O then H2O-MeOH gradient elution to afford F3b-5-1 to F3B-5-5. F3b-5-2 (35 mg) was purified with Sephadex LH-20 (10 g), eluted with MeOH to afford 9 (5 mg). F3b-6 (380 mg) was subjected to Sephadex LH-20 (40 g) CC to afford 10 (20 mg). F3c (838.5 mg) was subjected to silica gel (40 g) CC, which was eluted initially with DCM-MeOH (95:5) then (80:20) to give F3c-1 to F3c-3. F3c-2 (230 mg) was purified by Sephadex LH-20 (25 g) CC eluting with MeOH to afford 2 (5 mg) and 11 (4 mg). F3d (348 mg) was subjected to silica gel (15 g) CC eluting initially with DCM-MeOH (98:2) then (85:15) to afford 12 (25 mg). F3e (542 mg) was subjected to Sephadex LH-20 (55 g) CC to obtain 2 main fractions F3d-1 (160 mg) and F3d-2 (153 mg), each contains two main spots on TLC examination. Both of them were subjected separately to Sephadex LH-20 (16 g) CC, eluting with MeOH to afford 13 (10 mg) and 14 (1.7 mg) from sub-fraction F3d-1, and 15 (2.8 mg) and 16 (6.3 mg) from sub-fraction F3d-2.
The EtOAc fraction of the leaves (14 g), obtained by the same method as in flowers fractionation, was subjected to diaion-HP-20 (30 g) CC with elution with distilled water then MeOH. The methanol fraction (7.7 g) was subjected to MN-polyamide-SC-6 CC (250 g) CC, which was eluted with H2O 100%, H2O-MeOH (90:10), (80:20), (70:30) and (60:40) to deliver F-1 to F-5. F-1 (2.3 g) was subjected to silica gel (92 g) CC which was eluted initially with DCM-MeOH (95:5) then DCM-MeOH (90:10), (85:15) and (80:20) to give F-1-A to F-1-D. F-1-C (181 mg) was subjected to Sephadex LH-20 (20 g) CC using MeOH as eluent to afford 17 (5 mg). F-2 (900 mg) was fractionated by Sephadex LH-20 (90 g) using MeOH as eluent to afford 3 sub-fractions (F-2-A to F-2-C). F-2-B (200 mg) was separated by silica gel (10 g) CC eluting initially with DCM-MeOH (95:5), (90:10) and (80:20) to afford 18 (5.6 mg). F-3 (1.2 g) was subjected to silica gel (50 g) CC eluting with DCM-MeOH (98:2), (95:5) and (85-15) to afford 12 (35 mg). F-4 (950 mg) was fractionated by Sephadex LH-20 (100 g) CC, eluting with MeOH, to obtain 2 fractions, F-4-A (500 mg) and F-4-B (214 mg), and 14 (4.2 mg). F-4-B (214 mg) was subjected to silica gel (10 mg) CC eluted initially with DCM-MeOH (95:5) then with a gradient polarity increased to (75:25) to afford 19 (15 g). F-5 (872 mg) was fractionated by Sephadex LH-20 (90 g) CC eluted with MeOH to give F-5-A to F-5-C. F-5-B (360 mg) was subjected to silica gel (15 g) CC which was eluted with DCM-MeOH (98:2), (95:5) and (90:10) to afford 20 (9 mg).
10-Methoxy apodanthoside (1)
Oily residue.
[α]D20: +14.0 (c 0.05, MeOH).
IR (KBr) νmax: 3379, 2924, 1707, 1636, 1010, 1035 cm−1.
UV λmax (log ε) nm (MeOH): 233.9 (4.1), 204.0 (4.0).
1H and 13C NMR (CD3OD): Table 1.
Table 1.
1H and 13C NMR spectroscopic data for compounds 1 and 2 (400 and 100 MHz) in CD3OD.
| Position | 1 | 2 | ||
|---|---|---|---|---|
|
| ||||
| δH, J (Hz) | δC | δH, J (Hz) | δC | |
| 1 | 5.268, d, 6.0 | 97.1 | 4.947, dd, 11.2, 18.0 | 111.5 |
| 3 | 7.571, d, 1.2 | 153.2 | 5.933, dd, 11.2, 18.0 | 147.4 |
| 4 | - | 110.9 | - | 75.0 |
| 5 | 3.817, m | 41.0 | 2.142, m, 1.599, m | 33.6 |
| 6 | 6.131, dt, 2.0, 5.6 | 137.0 | 1.942, m, 1.740, m | 25.8 |
| 7 | 5.821, dt, 2.0, 5.6 | 128.6 | 3.419, m | 85.7 |
| 8 | 3.728, m | 52.7 | - | 77.2 |
| 9 | 2.921, m | 44.6 | 1.226, s | 30.0 |
| 10 | - | 174.7 | 1.169, s | 22.0 |
| 10-OMe | 3.794, s | 53.1 | - | - |
| 11 | - | 168.9 | - | - |
| 11-OMe | 3.805, s | 51.8 | - | - |
| 1′ | 4.742, d, 8.0 | 100.5 | 4.309, d, 8.0 | 106.3 |
| 2′ | 3.319, t, 8.0 | 74.6 | 3.113, m | 75.3 |
| 3′ | 3.473, t, 8.4 | 77.9 | 3.425, m | 76.9 |
| 4′ | 3.400, m | 71.4 | 3.315, m | 71.4 |
| 5′ | 3.389, m | 78.2 | 4.088, dd, 2.4, 12.0 3.744, m |
69.8 |
| 6′ | 3.949, dd, 11.6, 2.0 3.763, dd, 12.0, 4.0 |
62.6 | - | - |
| 1″ | 4.383, d, 7.6 | 104.8 | ||
| 2″ | 3.186, m | 75.1 | ||
| 3″ | 3.279, m | 77.7 | ||
| 4″ | 3.247, m | 71.6 | ||
| 5″ | 3.298, m | 78.1 | ||
| 6″ | 3.640, m 3.840, m |
62.8 | ||
HR-ESI-MS: m/z 439.1216 [M+Na]+ (calcd. 439.1216) and m/z 451.1028 [M+Cl]− (calcd. 451.1007).
(3S,6S)-Cis linalool-3,7-oxide β-D-glucopyranosyl-(1″→5′)-β-D-xylofuranoside (2)
Colorless oily residue.
[α]D20: +14.9 (c 0.05, MeOH).
1H and 13C NMR (CD3OD): Table 1.
HR-ESI-MS: m/z 487.2155 [M+Na]+ (calcd. 487.2154).
Figure 1.
Chemical structures of compounds 1 and 2.
Acknowledgments
We are grateful to the Egyptian Government, the National Center for Natural Products Research, the University of Mississippi, USA and NIH COBRE grant P20GM104932 for financial support. We are also thankful to Dr Baharthi Avula for HR-ESI-MS, Dr B. Tekwani for antiprotozoal assay, Dr S. Khan for antiplasmodial assay, and Dr M. Jacob for antifungal screening.
Footnotes
Supplementary data: Supplementary data (conformers of compound 1, conformers and ECD spectra of compounds 3, 4, and 8, NMR spectroscopic data and HR-ESI-MS of compounds 1 and 2).
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