Journal of
Medicinal Plants Research

  • Abbreviation: J. Med. Plants Res.
  • Language: English
  • ISSN: 1996-0875
  • DOI: 10.5897/JMPR
  • Start Year: 2007
  • Published Articles: 3846

Full Length Research Paper

Isolation, characterization and biological activity of organic extractives from Calodendrum capense (L.f.) Thunb.(Rutaceae)

Okwemba R. I.
  • Okwemba R. I.
  • Chemistry Department, School of Pure and Applied Chemistry, Kenyatta University, P. O. Box 43844, Nairobi, Kenya.
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Tarus P. K.
  • Tarus P. K.
  • Department of Chemistry and Biochemistry, University of Eldoret, P. O. Box 1125 - 30100, Eldoret, Kenya.
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Machocho A. K.
  • Machocho A. K.
  • Chemistry Department, School of Pure and Applied Chemistry, Kenyatta University, P. O. Box 43844, Nairobi, Kenya.
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Wanyonyi A. W.
  • Wanyonyi A. W.
  • Chemistry Department, School of Pure and Applied Chemistry, Kenyatta University, P. O. Box 43844, Nairobi, Kenya.
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Waweru, I. M.
  • Waweru, I. M.
  • Chemistry Department, School of Pure and Applied Chemistry, Kenyatta University, P. O. Box 43844, Nairobi, Kenya.
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Onyancha J. M.
  • Onyancha J. M.
  • Department of Pharmacy and Complementary/Alternative medicine, School of Medicine, Kenyatta University, P. O. Box 43844, Nairobi, Kenya.
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Onani M. O.
  • Onani M. O.
  • Department of Chemistry, Faculty of Natural Science, University of the Western Cape, Modderdam Road, Private bag X1, Belleville, South Africa.
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Amuka O.
  • Amuka O.
  • Department of Applied Sciences, Maseno University, Private Bag Maseno, Kenya.
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  •  Received: 27 December 2017
  •  Accepted: 03 April 2018
  •  Published: 05 April 2018

 ABSTRACT

A novel prenylfuroquinoline alkaloid capensenin (1), an alkaloid confusameline (2), two furanocoumarins psolaren (3) and bergapten (4), were isolated from hexane and dichloromethane crude extracts of stem bark, leaves and fruit pericarp of Calodendrum capense. Limonin (5) was also isolated from the stem bark, while limonin diosphenol (6) was isolated from the seeds. Capensenin (1) showed weak antimicrobial activity against Bacillus subtilis, while the leaves, stem bark and fruit pericarp crude extracts exhibited activity against Staphylococcus aureus, B. subtilis and P. citrinum. Hexane pericarp extract showed slight cytotoxicity to Vero cell E199 in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The structures of the compounds were elucidated by 1D and 2D nuclear magnetic resonance (NMR) spectroscopy, mass spectrometry and infra-red spectroscopy.

Key words: Capensenin, 4-hydroxyfuroquinoline, furanocoumarin, Calodendrum capense, alkaloid, Rutaceae.


 INTRODUCTION

Calodendrum capense (Linnaeus Filius) Thunberg belongs to the family Rutaceae. It is a semi-deciduous tree with tough timber used in house building, for tool handles and poles and as fuelwood (Dharani, 2011). The bark is used as an ingredient of skin ointments and is sold at traditional medicine markets in South Africa. Previous analytical studies carried out on seed kernels reported, antioxidant element copper, magnesium, manganese and zinc composition of the oil (Nawiri et al., 2012), performance of domestic cooking wick stove using fatty acid methyl ester of oil (Wagutu et al., 2010). Phytochemical studies revealed the composition of fatty acids in the isolated oil (Munavu, 1983) and isolation from the seeds of limonin, limonin diosphenol and rutaevin (Dreyer, 1967). A degraded limonoid, calodendrolide has been isolated from the root bark (Cassady and Lui, 1972). The C. capense metabolites, limonin and limonin diosphenol have been reported to exhibit biological activity in larvicidal activity in Aedes aegypti (Kiprop et al., 2005) and the wood extract showed mimicry of juvenile hormone activity in Oncopeltus fasiatus (Jacobson et al., 1975). In the chemotaxonomy of rutaceous plants, biologically active metabolites reported include; alkaloids with quinoline skeleton, coumarins, limonoids, essential oils and coumarins. Alkaloids and coumarins have not been reported and no phytochemical work has been done on the leaves and fruit pericarp of C. capense. As part of phytochemical investigation in screening for biological activity of crude extracts in medicinal plants and presence of these secondary metabolites, the isolation and characterization of two furoquinoline alkaloids, two furocoumarins and the common rutaceae limonoids, limonin and limonin diosphenol from organic extracts obtained from leaves, stem bark, fruit pericarp and seed kernels of C. capense were reported.


 MATERIALS AND METHODS

Instrumentation
 
Melting points of the isolated compounds were determined using Sanyo GallenKamp (UK) electronic melting point apparatus. Infrared spectra were obtained using NaCl pellets with acetone as solvent from Shimadzu Fourier Transform (FTIR-8400) Spectrophotometer. Nuclear Magnetic resonance NMR spectra were measured using Bruker Avance 400 Proton 1H NMR(400MHz); Carbon 13C NMR (100 MHz). Solvents used were deuterated chloroform CDCl3, methanol CD3OD and acetone (CD3)2CO. Chemical shifts were given in ppm values with trimethylsilane (TMS) used as the internal standard. Mass spectra were measured on electron impact mass spectra (EI-MS) using a Finnigan Gas chromatography Mass spectrometer GC-MS analyses. The samples were analysed on an Agilent GC-MS apparatus equipped with DB-5SIL MS (30 m × 0.25 mm i.d., 0.25 μm film thickness) fused-silica capillary column; helium (at 2 ml/min) was used as a carrier gas. For vacuum liquid chromatography (VLC), silica gel 60 (0.063-0.2 mm, Merck Chemicals Ltd, South Africa) was used. For column chromatography, silica gel 60 (70-230 mm, Merck Chemicals Ltd, South Africa) was used. For analytical thin layer chromatography TLC, aluminium plates coated with fluorescence indicator F254 (Alugram Sil G/UV 254, Macherey- Nagel, Germany) were used. Sephadex LH-20 was used as filter gel.
 
Plant material
 
The plant samples of C. capense were collected from City Park in Nairobi. Plant authentication was done by a plant taxonomist Mathias Mbale and a voucher RO/001 deposited in plant herbarium, National Museum of Kenya.
 
Extraction and isolation
 
The hexane (37.8 g) and dichloromethane DCM (37.8 g) fruit pericarp extracts were combined based on observation of the Rf of their thin layer chromatography (TLC) profiles and separated by vacuum liquid chromatography (VLC) using solvent systems of increasing polarity from n-hexane to ethylacetate EtOAc (Hexane:DCM; 100:0 to 0:100; DCM:EtOAc; 100:0 to 0:100). The VLC fractions 63-93 obtained with the solvent system with the ratios DCM:EtOAc (4:1) were combined and passed through a filter gel column (Sephadex LH 20) using solvent mixture (DCM:MeOH; 1:1). The fractions obtained were spotted and developed on TLC plates with solvent system (Hexane:EtOAc; 3:2), subjected to separation by preparative-TLC using the solvent system Hex:EtOAc to obtain 18.2 mg compound 1, 22.8 mg compound 2, 11.5 mg compounds 3 and 4. A mass of 3.1 kg of dried ground seed kernels used was defatted using hexane and 72 g of a white solid DCM extract was obtained. The white solid was separated by column chromatography using DCM: EtOAc (9:1) and 114 fractions were collected which showed a positive purple test with anisaldehyde reagent. Fractions 109-114 were combined and separated by preparative-TLC DCM:EtOAc; 9:1 to afford compound (5) 41 mg. The fraction103-108 was subjected to Preparative-TLC Hexane: EtOAc 3:2 gave compound (6) 22.8 mg which was UV active observed at 275 nm.
 
Antimicrobial in vitro assay
 
Antifungal test on crude extracts from C. capense was done against Candida albicans American Type Culture Collection ATCC 90028, Trichophyton mentagrophytes, Penicillium citrinium and Aspergillus niger using agar diffusion method (Elgayyar et al., 2001). Sterile Petri dishes were filled with sterilized medium of yeast, malt extracts and Potato Dextrose Agar (PDA) to a depth of 4 mm and seeded with the spores of fungi. Two hundred mg extract was dissolved in 1 ml of methanol and 10 µl of solution was loaded onto a 6 mm sterile filter paper, dried (2 mg/disc) and placed on the Petri dishes containing the medium which was sterilized using an autoclave set at 121oC for 15 min, allowed to cool and seeded with fungi. Fluconazole was used as standard, negative control plates had discs with sterile distilled water and methanol. The Petri dishes containing Aspergillus niger were incubated at room temperature (about 25°C) and the zones of inhibition were read after 72 h (Chabbra and Uiso, 1992). Antibacterial test on crude extracts from C. capense was done on Escherichia coli American Type Culture Collection ATCC 25923, Bacillus subtilis, and Staphylococcus aureus ATCC 25922, by Plate diffusion method (Elgayyar et al., 2001). Chloramphenicol was used as standard antibiotic. For each drug, 100 mg was dissolved in 1 ml of dimethylsulphoxide (DMSO). Twenty eight grams per liter of nutrient Agar in distilled water was sterilized by autoclaving at 121°C for 15 min. Fifteen milliliter agar was poured into the Petri dishes in a lamina flow machine under sterile conditions and 0.1 ml of the bacterial solution was added to it. Filter papers containing the drug test were put on the Petri dish and incubated at 37°C for 24 h. After which diameter of the zone of inhibition from the initial 6 mm was measured (Chhabra and Uiso, 1991). The active extracts from the antimicrobial screening were tested for minimum inhibitory concentrations (MIC). The MICs were determined using a two-fold serial dilution method in a peptone water solution for bacteria and potato dextrose agar (PDA) broth for yeast and fungi for active extracts to give a final extract concentration of between 1.95 and 8000 µg/ml. The negative control of the disc diffusion testing was done by the use of methanol that showed no inhibition, while the negative control was done by the use of standard antibiotic discs. The average zone of inhibition was calculated for three replicates. A clearing zone of 9 mm for bacteria and 10 mm or more for fungi was used as a criterion for designating significant antibacterial and antifungal activity (Faizi et al., 2003).
 
In vitro antiproliferative assay
 
This was carried out following a modified rapid calorimetric assay (Mosmann, 1983) using two cell lines; Vero-199 cells and Vero-E6 cells sourced from American Type Culture Collection (ATCC), by Kenya Medical Research Institute KEMRI. These cells were maintained in Eagle’s Minimum Essential Medium (MEM) supplemented with 10% foetal bovine serum (FBS) and 2 mM L-glutamine. On 96 well micro titer plates 5 × 103 cells/well suspension were seeded and incubated at 37°C/5% CO2 (Kamuhabwa et al., 2000). Samples were added to the cultured cells over a concentration range of 1000 to 0.14 µg/ml dimethylsulphoxide (DMSO). The plates were incubated for 48 h at 37°C and 5% CO2, after which 10 µL of MTT (Thiazoil Blue Tetrazolium Bromide) dye was added and incubated for another 4 h. Media was removed from all wells and 100 µl of DMSO was added. The plates were then read (colour absorbance) on an enzyme-linked immunosorbent assay ELISA scanning multiwell spectrophotometer (Multiskan Ex labs systems) at 562 and 690 nm as reference. Data was transferred onto a graphic programme (EXCEL) and expressed as percentage of the untreated controls. The 50% inhibition concentration (IC50) value was evaluated by linear regression analysis. Podophylotoxin (PPT) drug with an Initial concentration of 100 µg/ml DMSO was used as the control standard for the experiment.


 RESULTS AND DISCUSSION

Antimicrobial assay in vitro
 
The organic crude extracts of leaves, stem bark and fruit pericarp exhibited weak antimicrobial activity against S. aureus, B. subtilis and P. citrinum. There was no activity observed against Candida albicans, Trichophyton mentagrophytes, and Aspergillus niger. Crude extracts of leaves and fruit pericarp of C. capense showed moderate inhibition triplicates Appendix 1 from which mean inhibition zones in Table 1 between 11.0 to 12.7 mm were obtained, this shows potential use of the plant part in treatment as antimicrobial diseases. In a similar investigation of ethanol stem extracts of Cressa cretica used in traditional medicine as an expectorant and antibilious, it showed a zone of inhibition of 32.2 mm against P. citrinum (Mandeel and Taha, 2005). Ethylacetate crude extracts of C. capense leaves showed moderate MIC of 1250 µg/ml and fruit pericarp hexane extract showed weak MIC of 2500 µg/ml; the extracts show potential antifungal use. Grewia asitica leaves used on pustular eruptions showed MIC of 1500 µg/ml when methanolic extracts of the leaves were used (Sangita et al., 2009).  The hexane stem bark and leaves extract and ethylacetate stem bark extract showed inhibition zone triplicates in Appendix 2 moderate activity with mean inhibition zone of 14 mm against S. aureus with chloroamphenicol as positive control drug showing 22 mm (Table 2). This shows potential of these extracts in antibacterial application. The results are in close agreement with those carried out on Acalypha wilkesiana leaves used in the treatment of gastrointestinal disorders; ethanol extract showed of 11.5 mm with positive control drug ciprofloxacin showing an inhibition zone 27.3 mm (Akinyemi et al., 2006).
 
 
 
 
The hexane leaves crude extract and ethylacetate stem bark showed moderate activity on S. aureus with MIC of 1250 µg/ml (Table 2). Acalypha fruticosa used in treatment of skin infection and diarrhea showed MIC of 512 µg/ml against S. aureus for methanolic leaves extract (Sama Fonkeng et al., 2015). Sequential leaves and fruit pericarp extracts of C. capense in Table 3 and 4 showed moderate activity against B. subtilis with mean inhibition zones between 9.0 and 13.0 mm with standard drug showing 24 mm, the triplicate inhibition zones are shown in Appendix 3 and 4 In comparison to a similar study carried on Cinnamomum tamala methanol and ethylacetate extracts showed close agreement between 11.7 and 12.5 mm in inhibition zone against B. subtilis and 34.2 mm using tetracycline as positive control drug (Goyal et al., 2009). Hexane leaves and ethylacetate fruit pericarp extract showed moderate activity with MIC of 1250 µg/ml against B. subtilis while the other sequential extracts of leaves and fruit pericarp had weak activity with MIC of 2500 µg/ml. In comparison, C. tamala methanol extracts showed MIC value of 4096 µg/ml (Goyal et al., 2009).
 
 
 
 
 
 
Antiproliferative assay in vitro
 
 
Cytotoxicity screening of plant extracts is a preliminary aspect of safety evaluation for crude extract and isolated compounds ensuring that bioactivity is not due to general toxic effect of the extracts or compounds. The 96 well plate replicate results for podophylotoxin and fruit pericarp in Appendix 5 and 6 were analyzed and IC50 values determined from which the mean IC50 values were determined and the variation of cell viability against represented graphically in Figures 1 and 3 respectively. The regression curves were represented in Figures 2 and 4. The hexane fruit pericarp extract and podophylotoxin both showed weak cytotoxicity IC50 of 81.49 ± 0.689 µg/ml and 65.13 ± 0.0.599 µg/ml respectively (Tables 5 and 6). Significant cytotoxicity is considered when the IC50 value is ≤20 µg/ml; however their MIC values against B. subtilis differ significantly 2500 g/ml for hexane pericarp and 50 µg/ml for podophylotoxin. The selectivity index (SI) is calculated as a ratio of IC50 value of Vero cells to MICs (Vicente et al., 2009) and is a measure of tolerability of cells in vitro to extracts or compounds. The selectivity indices greater than 1.0 indicate safety of the drug on the host as compared to the pathogen, for SI less than 1.0, high amount of extractible will be required to be applied in eradication of the pathogen. When selectivity index is ≥10 the compound is considered suitable for further investigations (Oliveira et al., 2014). The SI values presented in Table 7 is slightly low for the hexane fruit pericarp extract 0.033 µg/ml compared to the standard drug podophylotoxin, a medical cream applied topically to treat genital warts which is 1.243 µg/ml against B. subtilis, this means that lower amount of standard drug can be used in eradication of the pathogen since larger amounts could be toxic. In a previous investigation using plant extracts, the selectivity indices ranged between 0.02 and 0.68 µg/ml against S. aureus with one plant extract showing a value of 2.87 µg/ml, while doxorubicin used as positive control showed cytotoxicity of 8.3 ± 1.76 µg/ml (Elisha et al., 2017). The antimicrobial activities of the extracts on P. citrinium, S. aureus and B. subtilis, was therefore not due to toxic effect of the extracts.

 

 

 

 

Structure elucidation of purified compounds
 
The purification of the hexane and DCM crude extracts obtained from seed kernels, leaves, stem bark and fruit pericarp of C. capense yielded two furoquinoline alkaloids, capensenin (1), confusameline (2); two furocoumarins, psolaren (3), bergapten (4) and limonoids, limonin (5) and limonin diosphenol (6).
 
Compound 1
 
White needles, m.p. 85.6 - 87.0°C; IR λmax.cm-1 (NaCl) 3417 (-OH), 3132 (furan), 2920 (CH2), 1624(C=C), 1585 (C=C) and 1097 (=COC). The EI-MS (m/z; % int.) 269 (18.6) [M]+ molecular formula C16H15NO3, 268 (100), 200 (39.9), 172 (14.4), and 68 (17.6); 1H-NMR ((CD3)2CO); δ 7.93 (1H, d, J = 9.6 Hz, H-6), δ 7.84 (1H, d, J = 2.2 Hz, H-2), δ 7.49 (1H, s, H-8), δ 6.89 (1H, d, J = 2.2 Hz, H-3), δ 6.23 (1H, d, J = 9.6 Hz, H-5), δ 5.46 (1H, t, H-2'), δ 4.86 (2H, d, J = 7.12, H-1'), δ 1.58 (3H, H-4') and δ 1.58 (3H, H-5'); 13C-NMR ((CD3)2CO) δ 160.5 (1a), δ 149.5 (C-4), δ 148.5 (C-2), δ 145.5 (C-6), δ 144.9 (C-8a), δ 139.8 (C-3'), δ 132.0 (C-7), δ 126.9 (C-3a), δ 120.9 (C-2'), δ 117.8 (C-4a), δ 115.2 (C-5), δ 114.8 (C-8), δ 107.5 (C-3), δ 70.4 (C-1'), δ 25.8 (C-5') and 18.1 (C-4'). Compound (1) was isolated from fruit pericarp as white needles (Hexane:EtOAc; 3:2). The IR spectrum showed peaks at 3417 cm-1 for hydroxyl group (-OH), 3132 cm-1for furan, 2920 cm-1 exomethylene (CH2),1624 cm-1 for olefinic (C=C), 1585 cm-1 for conjugated (C=C) and 1097 cm-1for (=COC). The developed TLC plate showed orange colour when sprayed with Dragendorff reagent as positive test for presence of alkaloid compounds. The EI-MS fragmentation pattern revealed a base peak at m/z 269 Appendix 7 to 11 representing the molecular ion [C16H15NO3]+ corresponding to molecular formula C16H15NO3. A prominent peak was observed at m/z268 (100%),[C16H14NO3]+resulting from the loss of a labile proton from the hydroxyl group at C-4.The subsequent loss of prenyl carbocation m/z 68 [C5H8]+,from the side chain gave rise to the fragment ion m/z 200[C11H6NO3]+in agreement with reported fragmentation of linear and angular furoquinoline alkaloids, where a large substituent group attached to the furoquinoline nucleus such as prenyloxy side-chain [C5H8]+is lost leaving a carbonyl group at C-7 position as reported in 7-O-dimethyallyl-γ-fagarine (O’Donnell et al., 2006). The peak at m/z 172 [C10H6NO2]+ resulted due to loss of carbon monoxide molecule (Figures 5 and 6).
 
 
 
 
 
 
 
 
 
 
 
The 13C-NMR spectrum of compound (1), Appendix 9 and 10 exhibited 16 carbon signals resolved by DEPT spectrum as; two methyl, one methylene, a total of six methine; one olefinic methine δ 120.9 (C-2'), two methine signals associated with the furan ring δ 148.5 (C-2) and δ 107.5 (C-3), three aromatic methine and seven quarternary carbon signals comprising of highly deshielded oxygenated carbon at δ 160.5 (9a), δ 149.5 (C-4), and δ 132.0 (C-7). The multiplicities were assigned from DEPT spectrum and confirmed unambiguously by HSQC spectrum. The 1H- NMR spectrum exhibited a total of eight proton signals, five of which were characteristic of a furoquinoline alkaloid; two doublets at δ 7.84 (1H, d, J = 2.2 Hz, H-2) and δ 6.89 (1H, d, J = 2.2 Hz, H-3) showed significant cross peaks from the COSY and NOESY spectrum characteristic of a furan ring. Three aromatic ABX type proton signals; a singlet at δ 7.49 (1H, s, H-8), two ortho-coupled aromatic doublets at δ 6.23 (1H, d, J = 9.6 Hz, H-5) and δ 7.93 (1H, d, J = 9.6 Hz, H-6) with significant 1H-1H COSY and NOESY correlation. The position of furan protons in the furoquinoline nucleus was confirmed by significant three bond HMBC (3J), between H-2 and δ 117.8 (C-3a), H-3 and δ 149.5 (C-4), also two bond HMBC (2J) between H-2 and C-3a. The position of aromatic proton at H-5 was confirmed by 2J correlation with δ 126.9 (C-4a), while H-6 showed 3Jcorrelation with δ 126.9 (C-4a) and δ 114.8 (C-8). The position of H-8 showed 2Jcorrelation with δ 144.9 (C-8a). Three proton signals displayed characteristics of a prenyloxy moiety; an oxymethylene doublet integrating into two protons which resonated at δ 4.86 (2H, d, J = 7.12, H-1'), an olefinic proton triplet δ 5.46 (1H, t, H-2') and a proton signal integrating to six proton representing methyl protons δ 1.58 (3H, H-4') and (3H, H-5'). The prenyloxy moiety showed coupling of protons with significant cross peaks from COSY and NOESY spectra between H-2′ and H-1′, hence confirmed the assignment of the triplet at H-2′ with characteristic allylic 4J correlation. The orientation of protons in the prenyloxy moiety was confirmed from HMBC due to correlation peaks between; methyl protons H-5' resonating at δ1.58 and δ 18.1 (C-4'), δ 139.8 (C-3'), δ120.9 (C-2'). The methylene proton, δ 4.86 (H-1') showed, HMBC correlation with δ 120.9 (C-2') and δ 139.8 (C-3'). The connectivity of the prenyloxy side chain to the furoquinoline ring at C-7 was confirmed by HMBC correlation between methylene proton δ 4.86 (H-1') with δ 132.0 (C-7) and the chemical shifts Table 8 were in close agreement with those of prenyloxy in tecleabine (Al-Rehaily et al., 2003; Tarus et al., 2005). On the basis of the findings above the structure of compound (1) was established as 4-hydroxy-7-prenyloxyfuroquinoline. This is the first time isolation of the compound from a natural source and from C. capense. Capensenin represents a pivotal metabolite in furoquinoline biosynthesis as many authors report 4-hydroxy-2-quinolone undergoes C-3 prenylation, the hydroxyl group at C-4 is alkylated, commonly to form a 4-methoxy group before furan ring formation. The lack of such alkylation in capensenin (1) shows the divergent furoquinoline biosynthetic pathways in Rutaceae.

 

 
Compound 2
 
White crystals, m.p. 105.4 – 107.7°C; EI-MS (m/z) 216 [M+H]+ molecular formula C12H9NO3, 201, 173, 145; 1H-NMR ((CD3)2CO) δ 8.24 (1H, d, J = 9.7 Hz, H-5), δ 7.89 (1H, d, J = 2.4 Hz, H-2), δ 7.35 (1H, d, J = 2.4 Hz, H-3), δ 7.34 (1H, d, J = 0.84 Hz, H-8), δ 6.28 (1H, d, J = 9.9 Hz, H-6) and δ 4.26 (3H, s, -OCH3); 13C-NMR ((CD3)2CO) δ 160.8 (C-7), δ 159.3 (C-1a), δ 153.8 (C-8a), δ 150.8 (C-4), δ 146.2 (C-2), δ 139.9 (C-5), δ 113.5 (C-3a), δ 113.3 (C-6), δ 139.9 (C-5), δ 106.3 (C-3), δ 93.9 (C-8) and δ 70.4 (4-OMe). Compound (2) was isolated as a white crystalline solid. The 1H NMR spectrum Appendix 12a and b showed six protons characteristic of a linear furoquinoline alkaloid; two proton doublets with signals δ 7.89 (1H, J = 2.4, H-2) and δ 7.35 (1H, J = 2.4, H-3) which showed correlation according to COSY and NOESY spectrum. A prominent proton signal at δ 4.26 (3H, -OCH3) integrating to three proton singlets characteristic of a methoxy group was observed. The aromatic protons were observed at δ 8.24 (1H, d, J = 9.7, H-5) diagnostic of the type of substitution in the benzenoid ring, the ortho-proton δ 6.28 (1H, d, J = 9.9, H-6) showing correlation as observed in the COSY spectrum and NOESY spectrum. The proton at para-position resonated at δ 7.34 (1H, d, J = 0.84, H-8). The 13C NMR spectrum in Appendix 13 and 14 exhibited 12 signals resolved by DEPT spectrum as five methine, one methyl and six quarternary carbons. The chemical shifts were characteristic of a furoquinoline alkaloid. The positions of the 12 carbon atoms were confirmed from HSQC spectrum and HMBC spectrum. There was HMBC correlation of H-2 to C-3a (δ 113.5) and C-9a (δ 159.3). There was HMBC spectra revealed correlation of H-2 to C-3a (δ 113.5) and C-9a (δ 159.3). The proton H-3 shows HMBC correlation with C-2 (δ 146.2), C-3a and C-9a hence confirming C-3a and C-9a to be the bridge carbon atoms between the furan ring and the heterocyclic ring. There was HMBC correlation between the methoxy hydrogen atoms and with a highly desheilded oxygenated C-4 (δ 150.8).
 
 
 
 
 
 
 
The aromatic proton H-5 showed HMBC correlation with C-4, C-8 (δ 153.8), and the highly deshielded oxygenated centre C-7 (δ 160.8). There was observed correlation between H-6 to C-4a and C-7 from the HMBC spectrum. The proton H-8 exhibited correlation with carbon atoms C-4a, C6, C-7 and C8a from the HMBC spectrum. The electron impact mass spectrum Appendix 15 of compound 2 showed a molecular ion peak at m/z 216 [M+1]+ corresponding to molecular formula C12H9NO3. Other prominent peaks observed in EI-MS were at m/z (rel.int); 215 (M+, 100), 200 (M+-CH3, 42.1), 172 (200 –CO, 25.6), 144 (172 –CO, 12.8). The peak at m/z 201 [M+1]+ corresponded to the fragment ion [C11H6NO3]+ due to the loss of a methyl group from the molecular ion. The peak at m/z 173 [M+1]+ corresponded to [C10H6NO2]+ and arose due to the loss of carbon monoxide and a further loss of carbon monoxide molecule gave rise to a peak at m/z 145 [M+1]+ which corresponded to the fragment ion [C9H6NO]+. The proposed fragmentation pattern of compound 2 was rationalized by mass spectrometric studies carried out on substituted furoquinoline alkaloids, where those with methoxy substituent at position C-4 and C-8 lose the methyl group leaving behind a carbonyl at these positions, followed by the subsequent loss of carbon monoxide molecules (Glugston and Maclean, 1965; O’Donnell et al., 2006). 1H-NMR and MS values were in close agreement with literature values for confusameline (Kang and Woo, 2010).
 
 
 
 
Compound 3
 
 
White solid IR λmax.(NaCl)cm-1; 1724 (C=O), 1627, 1535 (C=C); the EI-MS (m/z; % int.) 186 (92) [M]+ molecular formula C11H6O3, 158 (100)[C10H6O2]+, 130 (34)[C9H6O]+, 102 (50)[C8H6]+, 76 (25)[C6H4]+, 63 (16)[C5H3]+ and 51 (42)[C4H3]+; 1H-NMR ((CD3)2CO) δ 7.96 (1H, d, J = 9.6 Hz, H-4), δ 7.74 (1H, d, J = 2.4 Hz, H-2'), δ 7.40 (1H, s, H-8), δ 7.19 (1H, d, J = 0.68 Hz, H-5)δ 6.89 (1H, d, J = 2.4 Hz, H-3'), δ 6.23 (1H, d, J = 9.6 Hz, H-3); 13C-NMR((CD3)2CO) δ 159.0 (C-2), δ 153.0 (C-1a) , δ 148.3 (C-7), δ 146.2 (C-3′), δ 145.5 (C-4), δ 127.0 (C-6), δ 121.5 (C-5), δ 115.3 (C-4a), δ 113.3 (C-3), δ 107.5 (C-2′), δ 100.0 (C-8). Analysis of the mass spectrum Appendix 18 showed a base peak of m/z 186 corresponding to molecular formula C11H6O3. The 1H NMR spectrum Appendix 16 revealed five proton signals; two doublets at δ 6.23 (1H, d, 9.6, H-3), δ 7.96 (1H, d, 9.6, H-4) which showed correlation on analysis of correlation spectroscopy (COSY) spectrum characteristic of an α, β-unsaturated ketone of a pyrone ring, as well as two aromatic signals at δ 7.19 (1H, d, 0.68, H-5) and 7.40 (1H, s, H-8) indicating the presence of a disubstituted aromatic ring. The carbon-13 nuclear magnetic resonance (13C NMR) spectrum Appendix 17 showed nine carbon signals characteristic of a coumarin. The connectivity of hydrogen atoms to carbon atoms were unambiguously assigned by analysis of the heteronuclear single quantum correlation (HSQC) spectrum. The six methine carbon signals were resolved from analysis of distortionless enhancement by polarization transfer (DEPT) spectrum assigned asC-2' (δ 107.5), C-3' (δ 146.2), C-3 (δ 113.3), C-4 (δ 145.3), C-5 (δ 106.3) and C-8 (δ 100.0). The proton signal δ 7.96 H-4 showed correlation peak with C-2 (δ 159.0) and C-8a (δ 153.0) on analysis of HMBC spectrum which confirmed the location of C-4 in the pyrone ring in close proximity to the highly desheilded carbonyl C-2. The proton signal δ 7.19 H-5 showed correlation peaks Figure 8 with C-4 (δ 145.3) and C-8 (δ 100.0) while the proton at δ 7.40 H-8 showed HMBC correlation peak with C-5 (δ 106.3) and C-8a (δ 153.0), thereby confirming the position of C-7 in the benzenoid ring in close proximity with C-5. Compound 3 structure shown in Figure 7 was elucidated as psolaren.
 
 
 
 
 
 
Compound 4
 
White solid IR λmax.(NaCl); 1724 cm-1 (C=O), 1627, 1535 cm-1 (C=C);The EI-MS (m/z; % int.) 216 (100) [M]+ molecular formula C12H8O5, 201 (32.4)[C11H5O4]+, 173 (75.0)[C10H5O3]+, 145 (36.8)[C9H5O2]+, 117 (7.35)[C8H5O]+, 89 (72.1) [C7H5]+, 63 (58.8)[C5H3]+and 51 (20.8)[C4H3]+; 1H-NMR ((CD3)2CO) δ 8.09 (1H, d, J = 9.8 Hz, H-4), δ 7.74 (1H, d, J = 2.4 Hz, H-2'), δ 7.02 (1H, s, H-8), δ 6.89 (1H, d, J = 2.4 Hz, H-3'), δ 6.12 (1H, d, J = 9.8 Hz, H-3), δ 4.30 (3H, s-OCH3); 13C-NMR((CD3)2CO) δ 160.80 (C-2), δ 157 (C-7) , δ 154 (C-4a), δ 151 (C-5), δ 146.2 (C-2'), δ 139.9 (C-4), δ 115.3 (C-6), δ 113.5 (C-3), δ 107.5 (C-3’), δ 107.0 (C-1a), δ 93.8 (C-8), δ 60.8 (5-OMe). The compound 4 was isolated as a white solid (Hexane: EtOAc; 3:2). It showed fluorescence (yellow) under UV 365 nm and gave a blue colour on spraying with anisaldehyde locating agent and warming to 120°C. The infra-red (IR) spectrum exhibited absorption band at 1724 cm-1 (C=O) and 1627, 1535 cm-1 (C=C). The 1H NMR spectrum Appendix 16 exhibited six proton signals characteristic of a furanocoumarin. Two proton doublets resonated at δ 6.12 (1H, d, 9.8, H-3) and 8.09 (1H, d, 9.9, H-4) which showed correlation in the correlation spectroscopy (COSY) spectrum characteristic of α, β-unsaturated ketone of a pyrone ring in the coumarin nucleus. Two doublets resonated at δ 7.74 (1H, d, 2.4, H-2’) and δ 6.89 (1H, d, 1.6, H-3’) is characteristic of a furan ring proton. An aromatic proton singlet was observed at 7.02 (1H, s, H-8) and a proton signal at δ 4.30 (3H, s) integrating into 3 hydrogen atoms characteristic of a methoxy group. The 13C NMR spectrum Appendix 17 exhibited 12 signals resolved by distortionless enhancement by polarization transfer (DEPT) spectrum as; five methine, six quaternary and one methyl carbon signal. The connectivity between hydrogen and carbon atoms was determined unambiguously from the heteronuclear single quantum correlation (HSQC) spectrum. The proton δ 8.09 H-4 showed correlation to C-4a (δ 154) and C-5 (δ 151) on analysis of heteronuclear multiple bond correlation (HMBC) spectrum hence confirming the location of C-4 (δ 139.9) in the pyrone ring and C-4a (δ 154.0) as a bridge atom between the pyrone and benzenoid ring. The proton δ 7.02 H-8 showed HMBC correlation Figure 8 with C-4a and C-7 (δ 157) the latter being the bridge atom between benzenoid and furan ring hence supporting the location of the proton at C-8. The highly desheilded signal was assigned as C-2 (δ 160.80) due to the presence of carbonyl group, the furan ring carbon atoms were assigned as C-2’ (146.2) and C-3’ (107.5). The desheilded C-5 (δ 151.0) was assigned as an oxygenated centre, the position of attachment of methoxy group. The MS of compound 4 Appendix 19 showed a base peak of m/z of 216 corresponding to molecular formula C12H8O5, hence compound 4 structure shown in Figure 7 was confirmed to be 5-methoxypsolaren commonly known as bergapten (Yu et al., 2010;Chi Chunyan et al., 2009). This is the first time isolation of the compound from C. capense. Bergapten and psoralen are found in bergamot essential oil and many other citrus essential oils, and is the chemical in bergamot oil that causes phototoxicity (Frérot and Decorzanr, 2004; Saita et al., 2004).
 
 
 
 
Compound 5
 
 
White crystals, m.p. 295.0 - 297.5°C; IR λmaxcm-1.(NaCl) 3143 (furan), 2958 (CH2), 1751 (lactonic carbonyl),1277(ether); The EI-MS [M+H]+ (m/z) 356, 281, 207, 149, 96 and 58; 1H-NMR ((CD3)2CO) δ 7.48 (1H, m, H-23), δ 7.38 (1H, m, H-21), δ 6.33 (1H, m, H-22), δ 5.43 (1H, s, H-17), δ 4.77 (1H, d, J = 14.4 Hz, H-19a), δ 4.43 (1H, d, J = 14.4 Hz, H-19b), δ 4.02 (1H, m, H-1), δ 4.02 (1H, s, H-15), δ 2.98 (1H, dd, J = 18.0 Hz, 3.6 Hz, 2b), δ 2.84 (1H, dd, J = 14.4 Hz, 14.4 Hz, H-6b), δ 2.69 (1H, dd, J = 18.0 Hz, 3.6 Hz, H-2a), δ 2.56 (1H, dd, J = H-9), δ 2.47 (1H, dd, J = 14.4 Hz, 3.6 Hz, H-6a), δ 2.20 (1H, dd, J = H-5), δ 1.75 (1H, m, H-11), δ 1.47 (1H, m, H-12), δ 1.26 (3H, s, H-25a), δ 1.16 (3H, s, H-18), δ 1.06 (3H, s, H-24). 13C-NMR ((CD3)2CO) δ 208.2 (C-7), δ 170.1 (C-3), δ 167.6 (C-16), δ 144.1 (C-21), δ 142.5 (C-23), δ 121.7 (C-20), δ 110.9 (C-22), δ 80.6 (C-4), δ 80.1 (C-1), δ 78.6 (C-17), δ 67.3 (C-14), δ 65.9 (C-19), δ 60.2 (C-5), δ 54.9 (C-15), δ 51.9 (C-8), δ 48.5 (C-9), δ 46.8 (C-10), δ 38.9 (C-13), δ 37.2 (C-6), δ 36.5 (C-2), δ 30.9 (C-12), δ 21.8 (C-18), δ 20.6 (C-25a), δ 19.9 (C-25b), δ 19.1 (C-11), δ 17.9 (C-24). The compound 5 was isolated as a white crystalline solid with dichloromethane/methanol DCM/MeOH solvent system. The 1H NMR spectrum Appendix 20 revealed a total of 17 proton signals resolved by DEPT as; eight methine, five methylene and four methyl group protons. The ring A’, had methylene protons resonating at δ 4.77 (1H, d, 14.4, H-19a), δ 4.43 (1H, d, 14.4, H-19b), δ 2.69 (1H, dd, 18.0, 3.6, H-2a) and δ 2.98 (1H, dd, 18.0, 3.6, H-2b) which showed correlation in the correlation spectroscopy (COSY) spectrum with the methine proton δ 4.02 (1H, m, H-1).

 

 
 
In ring B, methylene protons were observed at δ 2.84 (1H, dd, 14.4, 14.4, H-6b), and 2.47 (1H, dd, 14.4, 3.6, H-6a); the latter exhibited correlation with δ 2.20 (1H, dd, H-5) on analysis of COSY spectrum. The methine proton at δ 2.56 (1H, dd, H-9) showed cross peak with one methylene proton at δ 1.75 (1H, m, H-11) on analysis of COSY spectrum. The methylene proton at δ 1.75 (1H, m, H-11) showed COSY cross peak with proton at δ 1.76 (1H, m, H-12) and in ring D, a methine proton resonates at δ 5.43 (1H, s, H-17). The furanyl ring protons resonated at δ 7.38 (1H, m, H-21), δ 6.33 (1H, m, H-22) and 7.48 (1H, m, H-23). There was significant COSY correlation between H-22 and H-23. The 13C NMR in Appendix 21 revealed 26 carbon signals, characteristic of a tetranortriterpenoid resolved by distortionless enhancement by polarization transfer (DEPT) spectrum as; 9 quaternary carbons, three of which were highly desheilded oxygenated centers at C-3 (δ 170.1), C-7 (δ 208.2), and C-16 (δ 167.6), five being bridge atoms in the pentacyclic ring at C-8 (δ 51.9), C-10 (δ 46.8), C-13 (δ 38.9), C-14 (δ 67.3), C-20 (δ 121.7) and one at C-4 (δ 80.6). There were 8 methine carbons at C-1 (δ 80.1), C-5 (δ 60.2), C-9 (δ 48.5), C-15 (δ 54.9), and C-17 (δ 78.6) and on the furanyl ring C-21 (δ 144.1), C-22 (δ 110.9) and C-23 (δ 142.5). The 5 methylene carbons were assigned to; C-2 (δ 36.5), C-6 (δ 37.2), C-11 (δ 19.1), C-12 (δ 30.9) and C-19 (δ 65.9). The 4 methyl carbons were assigned to C-18 (δ 21.8), C-24 (δ 17.9), C-25a (δ 20.6) and C-25b (δ 19.9). The connectivity of proton to carbon atoms were derived unambiguously from the heteronuclear single quantum correlation (HSQC) spectrum. The proton H-5 exhibited cross peaks with methyl C-25a (δC 80.6) and quaternary C-10 (δ 46.8) from the heteronuclear multiple bond correlation (HMBC) spectrum. 
 
 
 
 
The proton H-19b showed HMBC correlation to C-10 (δ 46.8) indicating the β-orientation of the proton. H-6a showed long range HMBC correlation with C-5 (δ 60.2), while H-6b showed correlation with C-10 (δ 46.8). There were cross peaks observed between H-9 and H-11 in COSY spectra. In ring C, a cross peak correlation occurs between H-11 and H-12 in COSY spectra. There was long range connectivity from HMBC between H-18 and C-12. In ring D, the 14, 15 epoxide moiety was determined by long range HMBC correlation between H-17 (δH 5.43) and C-14 (δC 67.31), H-15 and C-14. The proton H-15 also showed correlation with C-8. There was HMBC correlation between H-17 proton and the furanyl quaternary carbon C-20 (δC 121.7). In the furanyl moiety H-21 proton signal at δH 7.38 showed HSQC connectivity with C-21 and HMBC correlation with C-20). The H-22 proton (δH 6.33) showed HMBC correlation with C-20 and C-23 (δC 142.5). There was significant COSY cross peaks between H-22 and H-23. The observed m/z prominent peaks in EI-MS [M+H]+ for spectrum of compound 5 Appendix 22 were 58, 96, 149, 207, 281 and 356 whereby only the fragments [C2O2+H]+ 58 and the furanyl [C4H3OCO+H]+ 96 could be rationalized. The spectral data, both 1H and 13C NMR of compound (5) and were in close agreement with literature data of limonin (Breksa et al., 2006; Teranishi et al., 1999; Hasegawa et al., 1986). The compound 5 structure shown in Figure 9 was proposed to be limonin and this is the first report of isolation from stem bark of C. capense.
 
 
 
 
Compound 6
 
 
White crystals m.p. 282.0 - 283.4°C; 1H-NMR ((CD3)2CO) δ 7.34 (1H, m, H-23), δ 7.33 (1H, m, H-21), δ 6.33 (1H, m, H-22), δ 4.01 (1H, m, H-1), δ 4.57 (1H, d, J = 3.2 Hz, H-19a), δ 4.56 (H, d, J = 3.2 Hz, H-19b), 4.05 (1H, s, H-15), δ 5.37 (1H, s, H-17), δ 2.93 (1H, dd, J =17.5, 2.4 Hz, H-2b), δ 2.88 (2H, dd, J =17.5, 2.4 Hz, H-2a), δ 2.62 (1H, dd, H-9), δ (2H, d, H-19a) δ (2H, d, H-19b), δ 1.89 (1H, m, H-11b), δ 1.75 (1H, m, H-12), δ 1.56 (H, m, H-11a), δ 1.49 (3H, s, H-18), δ 1.43 (3H, s, H-25a), δ 1.41 (3H, s, H-25b), δ 1.09 (3H, s, H-24); 13C-NMR ((CD3)2CO) δ 195.2 (C-7), δ 169.1 (C-3), δ 166.4 (C-16), δ 143.3 (C-21), δ 141.1 (C-23), δ 140.1 (C-5), δ 139.5 (C-6), δ 119.7 (C-20), δ 109.6 (C-22), δ 81.8 (C-4), δ 79.1 (C-17), δ 77.6 (C-1), δ 68.6 (C-19), δ 65.2 (C-14), δ 52.1 (C-15), δ 48.3 (C-10), δ 46.8 (C-8), δ 46.3 (C-9), δ 37.3 (C-13), δ 34.8 (C-2), δ 31.6 (C-12), δ 25.6 (C-25b), δ 25.2 (C-25a), δ 20.6 (C-24), δ 20.1 (C-11) and δ 18.1 (C-18). In compound 6, the proton and carbon chemical shifts Appendix 23 and 24 showed close correlation with those of compound 5. However observed differences were due to the presence of unsaturation between C-5 and C-6 with hydroxyl group at C-6, hence absence of proton H-5 and H-6, in addition the carbon atoms were highly deshielded. The proton H-19b showed heteronuclear multiple bond correlation (HMBC) to C-10 (48.3) and C-9 (46.3). The proton H-9 showed HMBC correlation with C-8 (46.8) and C-11 (20.4).

 

 
The methyl proton H-25b showed HMBC correlation with C-4 (81.8) and C-25a (25.2). H-2a showed HMBC correlation with C-3 (169.1) and C-10 (48.3). H-2b showed HMBC correlation with C-3 (169.1) indicating the close proximity between C-2 and C-3. The proton H-25a showed HMBC correlation with C-4 (81.8) and C-25b (25.6). The proton H-25b showed HMBC correlation with C-4 (81.8), C-5 (140.1) and C-6 (139.5), hence confirming orientation of C-25b. The proton H-15 showed HMBC correlation with C-14 (65.2) and C-16 (166.4) hence confirmed its location in the D-ring. The methyl proton H-18 showed correlation with C-14 (65.2) HMBC spectrum. The furanyl moiety showed HMBC correlation was observed between; H-21 to C-20 (119.7), H-22 to C-20 (119.7), C-21 (143.3) and C-23 (141.1) and H-23 to C-22 (109.6) and C-21 (143.3). The 1H and 13C NMR spectral data of compound 6 were comparable with literature data of limonin diosphenol 6 (Chang-qi et al., 2006; Nakatani et al., 1987).Thus compound 6 structure shown in Figure 10 was confirmed to be limonin diosphenol.
 
 


 CONCLUSION

The reported bioactivities of the crude extracts and compounds isolated shows the profound reported use of rutaceous plants in traditional and alternative medicine; the reported use of the stem bark in traditional medicine is justified by presence of alkaloids, coumarins and copius amounts of limonoids. This is the first reported isolation of alkaloids; capensinin (1), confusameline (2), coumarins; psolaren (3), bergapten (4) from leaves, fruit pericarp and stem bark and limonin (5) from stem bark of C. capense.


 RECOMMENDATIONS

Further studies should be carried out on the fruit pericarp of C. capense.


 CONFLICT OF INTERESTS

The authors have not declared any conflict of interests.

 


 ACKNOWLEDGEMENTS

The authors express their gratitude to Dr Erick Korir for the running of NMR and MS spectra in the University of kwaZulu-Natal, South Africa; Dr Christine Bii (Kenya Medical Research Institute) for providing facilities for antimicrobial assay and Mr. Nicholas O. Adipo (KEMRI) for assisting in MTT assay. Martin O. Onani wishes to thank National Research Foundation of South Africa and University of Western Cape.



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