Abstract: Phytochemical investigation of the stems of Lecaniodiscus cupanioides Planch (Sapindaceae) afforded two triterpenoid saponins identified as 3-O- [α-L-arabinofuranosyl-(1→3)- α-L-rhamnopyranosyl- (1→2) - α -L-arabinopyranosyl-]-hederagenin (1) and 3-O- [α -L-arabinopyranosyl- (1→3)- α -L-rhamnopyranosyl (1→2)- α -L-arabinopyranosyl-]-hederagenin (2). The structures of the compounds were determined based on chemical investigations and comprehensive NMR spectroscopic studies including 1H, 13C, DEPT, COSY, HMQC, HMBC, MS and comparison with literature data. The compounds exhibited antifungal activity against C. albicans, C. neoformans and A. fumigatus.
INTRODUCTION
Almost every culture within the wide diversity of the world´s population and habitats uses local plants within its environs as medicines in one form or another. Chemical and biological investigations of folkloric medicinal plants with the reputation of curative potential have provided the world with many of the common clinical drugs and herbal remedies of today (Hamburger and Hostettmann, 1991). It has been reported that several compounds derived from plant species could be regarded as important drugs currently in use and that about 25% of the pharmaceuticals prescribed by doctors in the developed world have their antecedents in chemicals produced by flowering plants (Balandrin et al., 1985). There is also a growing interest in the acquisition of botanical enterprises by multinational pharmaceutical companies in recent years (Leaders, 1996).
Lecaniodiscus cupanioides Planch (Sapindaceae) is a tree sometimes planted as a shade-tree and as an ornamental. It appears as a weed in rice fields in Nigeria. The bark is used for cough and broncho-pneumonial infections (Burkill, 2000). Ethnobotanical information revealed its use as galactogen, laxative and febrifuge and has autonomic effects such as lacrimation and skeletal muscle relaxant activity in rats (Sandberg and Cronlund, 1977). It is also used as an aphrodisiac and cases of sexual asthenia (Ghana Herbal Pharmacopoeia, 1992). The aqueous root extract of this plant was reported to have central nervous system depressant activity (Yemitan and Adeyemi, 2005). Preliminary screening of extracts from marine organisms and plants for antifungal activities in our laboratory revealed that the stem extract of L. cupanioides had promising antifungal properties. Incidence of fungal infection is increasing worldwide and despite treatment, mortality remains very high. Presently, few antifungal agents are available and their use may be limited by harmful side effects (Lorthoraly et al., 1999; Andriole, 1999). The present study thus deals with the isolation and characterization of the saponins from L. cupanioides and their antifungal effects.
MATERIALS AND METHODS
1H and 13C-NMR Spectra were measured and reported in ppm by using the residual solvent peak as an internal standard. ESI-FTMS analysis was measured on a Bruker-Magnex BioAPEX 30ES ion cyclotron HR HPLC-FT spectrometer by direct injection into an electrospray interphase. Semi-preparative HPLC was carried out on a Waters 510 system with a gradient programmer.
Plant Material
The stems of L. cupanioides were collected at Sango, Ogun State,
Nigeria and authenticated by Mr. Odewo of the Forestry Research Institute
of Nigeria (FRIN) Ibadan, voucher specimen FHI.105, 353 was deposited
at the herbarium.
Extraction and Isolation
The stems of L. cupanioides were chopped into small bits and
dried using an electric oven at 40 °C for 3 days. The dried stems
were ground in a roller mill. About 1 kg of powdered plant material was
extracted exhaustively using Soxhlet apparatus with 96% ethanol (4 L)
for 48 h. The filtrate was concentrated under reduced pressure using rotatory
evaporator until a semi-solid residue (5.1%w/w) was obtained. The extract
was subjected to vacuum liquid chromatography over Si gel column, eluted
successively with n-hexane-CHCl3-MeOH in a step gradient by
using different ratios to give eleven fractions (V1-V11). Fraction V8
(4.3 g) obtained on elution with CHCl3-MeOH (7:3) was chromatographed
over a Si gel column (4.0x70 cm) (EtOAc-Acetone-AcOH-H2O, 6:2:1:1)
isocratically to give eleven subfractions (S1-S11). Fractions S8 (0.31
g) and S9 (0.06 g) gave semi-pure 1 and fractions S10 (0.07 g) and S11
(0.09 g) gave semi-pure 2 which were purified by preparative TLC (Merck
60Å Si gel, 20x20 cm, 1000 μm), developed with EtOAc-Acetone-AcOH-H2O
(6:2:1:1). Final purification was by reverse phase HPLC (Luna 5 μM,
C8 100Å, 250x21.20 mm) using CH3CN-H2O (40:60)
as an eluent (flow rate of 5 mL min-1 gradiently and UV detection
at 225 nm) yielded 15 mg of 1 (tR = 40 min) and 12 mg of 2
(tR = 40 min).
Compound 1
Colourless crystals; [α]D25: +11.2 °
(c 0.5, EtOH); ESI-HRMS m/z 882.3 ([M-H]-); 750 [M-arabinose-H]-;
604 [M-(arabinose+rhamnose)-H]-; 471.3[M-(2arabinose+ rhamnose)
-H]- (calc. for [C46H74O16
-1], 882.49767400).
1H NMR (400.13 MHZ, C6D5N) δ 0.79 (3H, s, H-24), 0.80 (3H, s, H-26), 0.86 (3H, s, H-29), 0.88 (3H, s, H-30), 0.99 (3H, s, H-25), 1.11 (3H, s, H-27), 5.33 (1H, br s, H-12), 4.16 (1H, m, H-3α), 4.94 (1H, d, J = 7.6 Hz, H-1´), 6.25 (1H, d, J = 7.8 Hz, H-1´´), 6.05 (1H, d, J = 7.9 Hz, H-1´´´).
13C NMR data (100 MHZ, C6D5N) δ 38.4 (C-1, t), 25.7 (C-2, t), 80.7 (C-3, d), 43.0 (C-4, s), 47.3 (C-5, d), 17.6 (C-6, t), 32.3 (C-7, t), 39.1 (C-8, s), 47.6 (C-9, d), 36.3 (C-10, s), 23.0 (C-11, t), 122.0 (C-12, d), 144.2 (C-13, s), 41.5 (C-14, s), 27.7 (C-15, t), 23.2 (C-16, t), 46.0 (C-17 s), 41.1 (C-18, d), 45.8 (C-19, t), 30.3 (C-20, s), 33.6 (C-21, t), 32.6 (C-22, t), 63.5 (C-23, s), 13.5 (C-24, q), 15.5 (C-25, q), 16.8 (C-26, q), 25.5 (C-27, q), 179.0 (C-28, s), 32.7 (C-29, q), 23.1 (C-30, q), 104.1 (C-1´, d), 74.7 (C-2´, d), 74.6 (C-3´, d), 69.1 (C-4´, d), 65.6 (C-5´, t), 100.5 (C-1´´, d), 71.2 (C-2´´, d), 78.7 (C-3´´, d), 71.8 (C-4´´, d), 68.8 (C-5´´, d), 18.0 (C-6´´, q), 110.3 (C-1´´´, d), 81.8 (C-2´´´, d), 78.2 (C-3´´´, d), 87.5 (C-4´´, d), 62.1 (C-5´´, t).
Compound 2
Colourless crystals; [α]D25: +15.4 °
(c 0.5, EtOH); ESI-HRMS m/z 882.2 ([M-H]-), - (calc.
for [C46H74O16 -1], 882.49767400).
1H NMR (400.13 MHZ, C6D5N): δ 1.16 (3H, br s, H-24), 0.95 (3H, s, H-25), 1.05 (3H, s, H-26), 1.57 (3H, s, H-6´´), 1.25 (3H, s, H-27), 1.00 (3H, s, H-30), 0.95 (1H, s, H-29). 4.90 (1H, d, J = 7.8 Hz, H-1´), 6.30 (1H, d, J = 7.8 Hz, H-1´´), 6.15 (1H, d, J = 7.6 Hz, H-1´´´).
13C NMR data (100 MHZ, C6D5N) δ 39.2 (C-1, t), 26.5 (C-2, t), 81.4 (C-3, d), 43.8 (C-4, s), 47.9 (C-5,d), 18.4 (C-6, t), 33.1 (C-7, t), 39.9 (C-8, s), 48.3 (C-9, d), 37.0 (C-10, s), 23.8 (C-11, t), 122.8 (C-12, d), 144.9 (C-13, s), 42.3 (C-14, s), 28.5 (C-15, t), 24.4 (C-16, t), 46.8 (C-17 s), 39.9 (C-18, d), 46.5 (C-19, t), 31.1 (C-20, s), 34.4 (C-21, t), 33.3 (C-22, t), 64.2 (C-23, s), 14.3 (C-24, q), 16.2 (C-25, q), 17.6 (C-26, q), 26.3 (C-27, q), 180.4 (C-28, s), 33.4 (C-29, q), 23.9 (C-30, q), 104.8 (C-1´, d), 75.2 (C-2´, d), 74.7 (C-3´, d), 69.7 (C-4´, d), 66.4 (C-5´, t), 101.5 (C-1´´, d), 72.2 (C-2´´, d), 83.1 (C-3´´, d), 73.2 (C-4´´, d), 69.9 (C-5´´, d), 18.6 (C-6´´, q), 107.7 (C-1´´´, d), 73.3 (C-2´´´, d), 79.9 (C-3´´´, d), 69.9 (C-4´´´, d), 67.3 (C-5´´´, t).
Acid Hydrolysis of 1 and 2
Each isolate (10 mg) was heated in a mixture of 20% HCl (6.5 mL) and
CH3OH (3.5 mL) under reflux for 7 h. The reaction mixture was
concentrated under reduced pressure to remove methanol and diluted with
water (2 mL) and extracted with chloroform (10 mL x3). The H2O
layer was neutralized with Na2CO3, filtered and
concentrated under reduced pressure and residue which contained sugars
were subjected to paper chromatography analysis with standard sugars glucose,
rhamnose, arabinose and xylose. (BuOH-HOAc-H2O (4:1:5) was
used as solvent and detection was by aniline/phthalate spray.
Antifungal Assay
The organisms used in this study were Candida albicans (ATCC
90028), Candida neoformans (ATCC 90113) and Aspergillus fumigatus
(ATCC 90906). The antifungal assays were evaluated by agar diffusion method.
The antifungal activity was determined by measuring the diameter of zone
of inhibition (mm). The determination of MIC was performed using a two
fold dilution technique as previously described (Peterson et al.,
1992). The MIC, μg mL-1, was recorded as the lowest concentration
that prevented visible growth (Hamann et al., 1993). The antifungal
agent amphotericin B was included as positive control in each assay.
RESULTS AND DISCUSSION
Evidence from 13C NMR chemical shifts showed that both 1 and 2, possess the same triterpenoid skeleton, differing only in the sugar regions. A comparison of the 13C NMR signals due to the compounds with those of reported saponins revealed that they are monodesmosides of 3-O-glycoside and the aglycones as hederagenins (Li et al., 1990).
Mineral acid hydrolysis of 1 yielded arabinose and rhamnose as the sugar components. The negative ion HRESI mass spectrum of 1 revealed the molecular ion peaks appearing at m/z 882.3 [M-H]- corresponding to the molecular formula C46H74O16 with fragment peaks appeared at m/z 750.9, 604.0 and 471.3 and indicating the loss of arabinose, arabinose + rhamnose and 2xarabinose+ rhamnose. This sequence indicated that a terminal arabinose moiety is linked to an inner rhamnose that is linked to inner arabinose and this, in turn, is attached to the C-3 of hederagenin (Mahato et al., 1991). This fragmentation pattern confirms interglycosidic linkages in all the sugars of 1. Comparison of 13C NMR spectra of the sugar portions with corresponding methylglycosides revealed that the sugar moieties are of α-L-arabinofuranose, α-L-arabinopyranose and α-L-rhamnopyranose (Gorin and Mazurek, 1975).
| Structure of compound (1) |
The 13C NMR spectrum of 1 demonstrated 46 carbon resonances in partial agreement with the molecular formula C46H74O16 indicating three sugar moieties, two pentoses and one hexose while the remaining 30 signals assigned to the aglycone. The 13C NMR spectrum showed anomeric carbon signals at δC 111.0, 104.9 and 101.3 which were consistent with the presence of trisaccharide chain. The DEPT spectrum displayed 7 methyl, 13 methylene, 18 methine and 8 quarternary carbon atoms. The 1H and 13C NMR and HMBC data indicated the presence of a trisubstituted double bond δH 5.33 ppm (1H, br s, H-12) and δC 144.2 and 122.0 ppm, characteristic of a Δ12 double bond in an oleanane skeleton (Ahmad et al., 1993; Silverstein and Webster, 1997). The appearance of signal at δC 179.0 is due to the presence of CO2H group and seven methyl groups: δH 0.99 ppm (3H, s, H-24)/δC 13.5 ppm (C-24), δH 0.80 ppm (3H, s, H-25)/δC 15.5 ppm (C-25), δH 0.88 ppm (3H, s, H-26)/δC 16.8 ppm (C-26), δH 1.46 ppm (3H, s, H-5´´)/δC 18.10 ppm (C-5´´), δH 0.88 ppm (3H, s, H-30)/δC 23.1 ppm (C-30), δH 1.11 ppm (3H, s, H-27)/δC 25.5 ppm (C-27) and δH 0.80 ppm (3H, s, H-29)/δC 32.7 ppm (C-29).
The points of attachment of the sugar units in 1 were determined from the 13C NMR chemical shifts (Table 1). The C-3 of the aglycone resonated at δC 80.7, thus showing +7.0 ppm desheilding as compared to hederagenin (Li et al., 1990). This is an indication that the sugar moieties are attached at this carbon. The upfield shift of C-2 signal by 1.90 ppm and the 3J-HMBC correlation of H-3 (δ 4.16) to C-1´ (δ 104.1) also confirmed the above proposed site of glycosidation. The presence of hydroxy group attached to C-23 is evidenced by signal in the downfield region of the 13C NMR spectrum, δC 63.5 ppm. A comparison of the chemical shift of C-2´ of arabinose (C-2´, δ 74.7) with that of methyl arabinose (C-2, δ 71.8) allowed the assignment of a 1→2 linkage between arabinose and rhamnose. The down field 13C NMR chemical shift of C-3´´ of rhamnose at 78.7 and small upfield shift of C-4´´ of rhamnose at 71.8 indicated 1→3 linkage between rhamnose and the terminal arabinose. The nature of the interglycosidic linkage was further confirmed by long-range connectivity information obtained from HMBC spectrum which showed 3J-HMBC interaction of protons at δ 4.45 (H-2´) and δ 4.64 (H-3´´) with anomeric carbons at δ 100.5 (C-1´´) and δ 110.3 (C-1´´´) in agreement with presence of (1→2) and (1→3) linkages between arabinose and rhamnose and rhamose and the terminal arabinose, respectively. From these results the structure of 1 was elucidated as 3-O- [α-L-arabinofuranosyl- (1→3)-α-L-rhamnopyranosyl- (1→2)-α-L-arabinopyranosyl-]-hederagenin.
Compound 2 afforded hederagenin as the aglycone and arabinose and rhamnose as sugar components on acid hydrolysis. A comparison of the 13C NMR spectra of the sugar portions with corresponding methylglycosides suggested that the sugar moieties are of 2 units of α-L-arabinopyranose and the other α-L-rhamnopyranose (Gorin and Mazurek, 1975) indicating a different sugar composition from 1. The negative ion HRESI mass spectrum of 2 exhibited the molecular ion peak at m/z 882.2 [M-H]- corresponding to the empirical molecular formula C46H74O16. The 1H and 13C NMR chemical shifts were compared with the literature reports for triterpenoidal sapogenins/saponins (Li et al., 1990), which confirmed the identity of the aglycone as hederagenin. The olefinic resonances at δC 144.9 and 122.8 corresponding to quaternary and methine behaviour, revealed the presence of unsaturation at C-12 in an oleanane skeleton (Tori et al., 1974). The appearance of a signal at δC 180.4 is ascribed to the carbon of CO2H group. The DEPT spectrum of 2 displayed 7 methyl, 13 methylene, 18 methine and 8 quarternary carbon atoms. There are seven methyl groups δH 1.16 ppm (3H, s, H-24)/δC 14.3 ppm (C-24), δH 0.95 ppm (3H, s, H-25) /δC 16.2 ppm (C-25), δH 1.05 ppm (3H, s, H-26)/δC 17.6 ppm (C-26), δH 1.57 ppm (3H, s, H-6´´) /δC 18.60 ppm (C-6´´), δH 1.25 ppm (3H, s, H-27)/δC 26.3 ppm (C-27), δH 1.00 ppm (3H, s, H-30) /δC 23.9 ppm (C-30) and δH 0.95 ppm (3H, s, H-29)/δC 33.4 ppm (C-29). The presence of hydroxy group attached to C-23 was evidenced by signal in the downfield region of the 13C NMR spectrum, δC 64.2 ppm. The anomeric carbon signals resonated at δ 107.7, 104.8 and 101.5 indicating the presence of three sugar moieties. Mass spectral fragmentation pattern and results of acid hydrolysis tend to suggest identical structure for 1 and 2.
| Structure of compound (2) |
| Table 1: | 13C (125 MHZ, C6H5N) NMR shifts for compounds 1-2 |
The sugar linkages in 2 were determined using the glycosidation rule (Tori et al., 1974; Seo et al., 1978; Mahato et al., 1991). The 13C NMR spectrum of 2 (Table 1) showed significant displacement of signals for C-3 (+7.7 ppm) of the aglycone, for C-2´ (+3.4 ppm) of the arabinopyranosyl moiety and for C-3´´ for inner rhamnopyranosyl moiety (+11.8 ppm) in comparison to the reported values for hederagenin (Tori et al., 1974) and methyl pyranoside due to glycosidation at these positions. The nature of the glycosidic linkage was further confirmed by the HMBC spectrum, which showed 2J-HMBC and 3J-HMBC interaction of protons at δ 4.47 (H-2´) and δ 4.63 (H-3), respectively with anomeric carbon C-1´ (δ 104.1) and 3J-HMBC interaction of proton at δ 4.47 (H-2´) with C-1´´ (δ 100.5). The C-3´´ proton at δ 4.64 also showed 2J-HMBC and 3J-HMBC to C-2´´ (δ 81.8) and C-1´´´ (δ 110.3), respectively. However in the 13C NMR spectrum, different resonance signals were obtained for the terminal α-L-arabinopyranosyl. It was observed that in 2 the signal of C-5´´´ due to arabinose was displaced downfield by 5.2 ppm and signal C-4´´´ was displaced upfield by 15.6 ppm. This disclosed that the terminal arabinose has C-5´´´ attached directly to oxygen to form a pyranose and not a furanose as it was the case with 1. Consequently, 2 was elucidated as 3-O- [α-L-arabinopyranosyl- (1→3)-α-L-rhamnopyranosyl (1→2)-α-L-arabinopyranosyl-]-hederagenin. Although the two compounds isolated were known compounds (Encarnacion et al., 1981) but the method of isolation and analysis of structures were different.
Compounds 1 and 2 were evaluated for antifungal activity in the agar well- diffusion assay. Compound 1 exhibited antifungal activity against C. albican, C. neoformans and A. fumigatus with IC50 of 4.5 μg mL-1, 15.0 μg mL-1 and MIC 10.0 μg mL-1, respectively and 2 exhibited similar activities with IC50 of 8.5 μg mL-1, 10.0 μg mL-1 and MIC >25.0 μg mL-1, respectively.
ACKNOWLEDGMENT
The authors are grateful to United State Government for the Fulbright fellowship awarded Dr. S.A. Adesegun to perform this research.