PHYTOTHERAPY RESEARCH Phytother. Res. 16, 66–70 (2002) DOI: 10.1002/ptr.849
SHORT COMMUNICATION
Chemical Constituents and Biological Activities of Zanthoxylum usambarense Weidong He,1 Luc Van Puyvelde,1 Norbert De Kimpe,1* Luc Verbruggen,2 Kristel Anthonissen,2 Mark Van der Flaas,2 Jan Bosselaers,2 Simon G. Mathenge3 and Francis P. Mudida4 1 Department of Organic Chemistry, Faculty of Agricultural and Applied Biological Sciences, University of Gent, Coupure Links 653, B9000 Gent, Belgium 2 Plant Protection Research, Janssen Research Foundation, Turnhoutseweg 30, B-2340 Beerse, Belgium 3 Botany Department, University of Nairobi, P.O. Box 30197, Nairobi, Kenya 4 TRAMEDA, P.O. Box 66514, Nairobi, Kenya
Bioassay-guided fractionation of the dichloromethane extracts of the roots and the bark of Zanthoxylum usambarense led to the isolation of two physiologically active compounds, i.e. canthin-6-one 1 (fungicide) and pellitorine 4 (insecticide). Together with oxychelerythrine 2, norchelerythrine 3, ()-sesamin 5 and ()-piperitol-3,3-dimethylallyl ether 6, they were isolated for the first time from this plant. Copyright # 2002 John Wiley & Sons, Ltd. Keywords: Zanthoxylum usambarense Kokwaro; Rutaceae; insecticides; fungicides.
INTRODUCTION Zanthoxylum usambarense (Engl.) Kokwaro (Rutaceae), formerly known as Fagara becquetii G. Gilbert (Troupin, 1983) and Fagara usambarensis Engl. (Beentje, 1994), is a highly branched tree (2.5–15 m) of oriental, intertropical Africa. In Kenya, it is known under the name of Muguchwa, Muvu, Mulasi, Sagawaita, Ol-Oisugi or Loisugi. The wood is used in house building, for furniture and to make bows. In the traditional medicine the bark and roots are used as a cough remedy, while a decoction of the bark is drunk to treat rheumatism. In the native medicine, the bark and the roots serve as an emetic and are employed against malaria (Beentje, 1994; Kokwaro, 1976). Zanthoxylum usambarense was investigated previously. Kokwaro et al. (1983) isolated the coumarin derivative O-methylcedrelopsin in 1983. Recently Kato et al. studied the stems and the roots of Z. usambarense and isolated by ion-pair HPLC a number of alkaloids which were identified as ()-tembetarine, ()-magnoflorine, ( )- edulinine, ()-N-methylplatydesmine, ( )blongine, ( )-usambarine, usambanoline, ( )-cis- Nmethylcanadine, nitidine and chelerythrine without description of their biological activities (Kato et al., 1996). Upon biological screening, antifungal and insecticidal activities were found in the roots and the bark of Z. usambarense. This led to a close examination of the plant in order to identify the compounds responsible for these * Correspondence to: N. D. Kimpe, Department of Organic Chemistry, Faculty of Agricultural and Applied Biological Sciences, Ghent University, Coupure Links 653, B-9000 Ghent, Belgium. Email:
[email protected] Contract/grant sponsor: Janssen Research Foundation, Beerse, Belgium.
Copyright # 2002 John Wiley & Sons, Ltd.
activities. The results of the bioassay-guided phytochemical study of the roots and the bark are described below.
MATERIAL AND METHODS General experimental procedures. The 1H, 13C, DEPT, COSY and HETCOR NMR spectra were obtained with a JNM-EX270 FT NMR system (JOEL, Japan). Mass spectra were measured with a MAT-112S (70eV) mass spectrometer (Varian, USA). Optical rotations were determined on an AA-10 automatic polarimeter (Optical Active Ltd, England). IR spectra were measured with a Nicolet Impact 410 FT-IR spectrometer (Thermo Optec, USA). Melting points were determined on a Bu¨chi 535 apparatus (Bu¨chi, Switzerland). Preparative counter current chromatography (CCC) was carried out at a revolution speed of 1000 rpm with a CCC-1000 high speed countercurrent chromatograph (Pharma-Tech Research Corporation, USA), equipped with a SSI 300 pump, a Pharmacia LKB Uvicord S II detector (254 and 280 nm) and a Retriever II fraction collector. Flash liquid chromatography (FLC) was executed with differently sized columns, packed with silica gel G60 (15–40 mm) or Lichroprep RP-18 (40–63 mm) (Merck, Germany). All precoated TLC plates for both analytical and preparative use, and the solvents used for isolation and purification, were from Merck, Germany. Sephadex LH-20 was obtained from Pharmacia Biotech (The Netherlands). Plant material. The roots and bark of the stem of Zanthoxylum usambarense were collected at the Karura forest near Nairobi (Kenya) at an altitude of 1700 m on 26 March 1994. The plant was identified by S. G. Mathenge, F. P. Mudida and A. Kamadi. A voucher herbarium species was deposited at the Herbarium of the DepartReceived 26 January 2000 Accepted 8 November 2000
ACTIVITIES OF ZANTHOXYLUM USAMBARENSE
ment of Botany of the University of Gent. The roots and bark were air-dried and powdered mechanically. Extraction and isolation. The powdered roots (118.9 g) were extracted (five times) in a percolator with 1.5 L of dichloromethane. The extract was filtered and concentrated under reduced pressure at 40 °C yielding a dark yellow dichloromethane residue (7.16 g). 6.42 g of the dichloromethane extract, absorbed on silica gel and as a slurry in n-hexane, was applied to the top of an FLC column (f 7.0 cm), containing 600 g silica gel, eluted with a n-hexane–ethyl acetate–methanol gradient (from 100/0/0 through 0/100/0 to 0/0/100). Fractions of 100 mL were collected. After monitoring by thin-layer chromatography (TLC), 27 fractions were obtained (TLC conditions: Merck silica-gel 60 F254, 20 20 cm, 0.25 mm; solvent: n-hexane–ethyl acetate 75/25, 40/60, ethyl acetate–methanol 75/25; detection at 254 nm and 365 nm after spraying with 5% SbCl3 in 10% HCl, 105 °C for 10 min). The fractions 10 (382.7 mg), 11 (418.5 mg) and 12 (168.7 mg) showing antifungal activities were submitted separately to RP-18 FLC purification using water–methanol–dichloromethane gradient elution. The fractions (338.3 mg) eluted with water– methanol 30/70, showed strong antifungal activity, were crystallized from acetone to obtain canthin-6-one 1 (183.9 mg). The fractions (135.3 mg), eluted with water–methanol 10/90, were redissolved in acetone to afford a white fine powder, oxychelerythrine 2 (69.5 mg). 140 mg of fraction 4 was fractionated by CCC with a nhexane–ethyl acetate–methanol–water (10/5/5/1.5) system: column volume, 350 mL; elution mode, head to tail, upper phase of the solvent as stationary phase, lower phase as mobile phase; revolution speed, 1080 rpm with a pressure about 100 psi; flow rate, 1 mL/min; equilibrium volume, 44 mL; fraction collection, in 10 mL; total run 260 min; push-out with a flow rate of 3 mL/min with collection in 12 mL; sample loop, 10 mL; detection at 254 nm and 280 nm. The elutes 8–12 (38.9 mg) were Copyright # 2002 John Wiley & Sons, Ltd.
67
purified by preparative TLC (silica gel 60 F254, 20 20 mm, 2mm) using ethyl acetate–methanol 95/5 to yield norchelerythrine 3 (2.3 mg, R f 0.85). The powdered bark (105.8 g) was extracted (six times) with 1.5 L of dichloromethane. After concentration of the dichloromethane extract under reduced pressure, 8.54 g of the residue was obtained. The dichloromethane extract (8.31 g) was applied to FLC on a column containing 1000 g of silica-gel eluted with a n-hexane–ethyl acetate– methanol gradient (from 100/0/0 through 0/100/0 to 0/0/ 100). The fractions (1.06 g), eluted with hexane–ethyl acetate 50/50 to 40/60, showing insecticidal activities, were then injected to a CCC system to give 19 fractions after combination into pools according to their similar TLC patterns (CCC conditions: solvent, n-hexane–ethyl acetate–methanol–water (15/5/5/1.5); column volume, 950 mL; elution mode, head to tail, upper phase of the solvent as stationary phase, lower phase as mobile phase; revolution speed, 1060 rpm with a pressure about 80 psi; flow rate, 4 mL/min; equilibrium volume, 137 mL; fraction collection, in 12 mL; total run 450 min; pushout with a flow rate of 8 mL/min and collection in 16 mL; sample loop, 20 mL; detection, 254 nm and 280 nm). Fraction 6 (26.1 mg), showing insecticidal activity, was purified by gel permeation chromatography (GPC) on a Sephadex LH-20 column (100 20 mm) by eluting with 270 mL of methanol. Re-crystallization of the 75–105 mL-elute using methanol afforded pellitorine 4 (11.7 mg). Fraction 7 (36.6 mg) was dissolved in methanol and kept at 20 °C to afford slightly yellow needles, ()-sesamin 5 (20.5 mg). Fraction 8 (168.2 mg) was submitted to preparative TLC (silica gel) developed (four times) with the upper phase of n-hexane–ethyl acetate–methanol–water (15/5/5/1.5) to afford ()-piperitol-3,3-dimethylallyl ether 6 (112.5 mg). Compound 1 was found in fungicidal active fractions. 6H-indolo[3,2,1-de][1,5]naphthyridin-6-one (canthin6-one): 1. C14H8N2O, yellow needles, mp 156.6 °– Phytother. Res. 16, 66–70 (2002)
68
W. HE ET AL.
Table 1. Activity against fungi at 1000 ppm of the crude extracts of Zanthoxylum usambarense Fungus Coriolus versicolor Coniophora puteana Chaetomium globosum Aureobasidium pullulans Penicillium islandicum Cladosporium resinae Aspergillus niger Aspergillus ¯avus Trichoderma viride a b
CH2Cl2 extract of the roots
CH2Cl2 extract of bark
xb x x x x x x x x
x x x x x x x
Ref.*a x x x x x x x x x
Propiconazole as reference sample and DMSO as blank control. Fully active at 1000 ppm.
157.0 °C. IR (KBr): v 1670 (amide), 1630–1680 (aromatic double bonds) cm 1. 1H NMR data: see the literature (Oehl et al., 1976; Anderson et al., 1983). 13C NMR (67.5 MHz, CDCl3): d 116.32 (=CH-1), 117.22 (=CH-8), 122.61 (=CH-11), 124.30 (=Cquat N=, C-11c), 125.59 (=CH-9), 128.86 (=CH-5), 130.19 (=Cquat-11a), 130.82 (=CH-10), 131.95 (=Cquat-N=, C-7a), 136.14 (=Cquat-11b), 139.35 (=Cquat=N-, C-3a), 139.55 (=CH4), 145.80 (=CH-2) and 159.46 (C=O). MS: m/z 220 (M, 100), 192 (52), 166 (6), 165 (9), 164 (10), 139 (11), 114 (6) and 96 (10). 7,8-Dimethoxy-5-methyl-2,3-methylenedioxybenzo[c]phenanthridin-6(5H)-one (oxychelerythrine): 2. C21H17NO5, white fine solid, mp 191.6 °–192.5 °C. IR (KBr): v 1655 (NC=O), 1060 (C-O), 1493, 1300 and 2820-2930 (-NCH3) cm 1. 1H NMR data: see the literature (Hanaoka et al., 1986). 13C NMR (67.5 MHz, CDCl3): d 40.77 (NCH3), 56.55 and 61.73 (OCH3-7 and -8), 101.45 (OCH2O), 102.43 (=CH-4), 104.60 (=CH-1), 117.75 (=CH- 9), 117.75 and 118.40 (=CH-10, =CH-11), 123.24 (=CH-12), 135.56 (=Cquat-4b), 147.01 and 147.44 (=Cquat-7, =Cquat-8), 150.06 and 152.63 (=Cquat-2, =Cquat3), 162.57 (C=O), and five quaternary carbons: 117.12, 119.70, 120.95, 128.86 and 131.63 (=Cquat-4a, -6a, -10a, 10b and -12a). MS: m/z 363 (M, 100), 348 (54), 334 (46), 305 (32), 320 (27), 181 (20), 174 (35) and 43 (19). 7,8-Dimethoxy-2,3-methylenedioxybenzo[c]phenanthridine (norchelerythrine): 3. C20H15NO4, slightly yellow solid, mp 210.0 °–210.8 °C. 1H NMR (270 MHz, CDCl3): d 4.06 and 4.13 (3H each, 2xs, OCH3-7 and -8), 6.14 (2H, s, OCH2O), 7.27 (1H, s, H-1), 8.73 (1H, s, H-4), 7.60 (1H, d, AX, J=9.24Hz, H-9), 7.86 (1H, d, AX, J=8.24Hz, H-12), 8.36 (1H, d, AX, J=8.24Hz, H-11), 8.37 (2H, d, AX, J=9.24Hz, H-10), 9.75 (1H, s, H-6). 13C NMR (67.5 MHz, CDCl3): d 56.80 and 61.94 (OCH3-7 and -8), 101.33 (OCH2O), 102.15 (=CH-4), 104.38 (=CH-1), 118.22 (=CH-9), 118.31 (=CH-10), 118.67 (=CH-11), 127.06 (=CH-12), 148.48 (N=CH-6). Ten of quaternary carbons were not observed due to lack of sample. MS: m/z 333 (M, 100), 318 (8), 290 (20), 275 (15), 232 (5), 167 (6) and 144 (5). N-(2-methylpropyl)-2,4-decadienamide (pellitorine): 4. C14H25NO, colourless fine needles, mp 87.6 °– 88.2 °C. IR (NaCl): v 3260 (NH), 2920, 1650 (C=C), 1630 (-CO-NH-), 1610 (C=), 1535, 1460, 990 Copyright # 2002 John Wiley & Sons, Ltd.
(C=C) cm 1. 1H NMR data: see the literature (Semple, 1995). 13C NMR data: see the literature (Yosuda et al., 1981). MS: m/z 223 (26, M), 155 (63), 100 (47), 96 (59), 83 (59), 69 (36), 57 (100), 40 (88). ()-2,6-Bis(3,4-methylenedioxyphenyl)-3,7-dioxabicyclo[3.3.0]octane (()-sesamin): 5. C20H18O6, slightly yellow needles, mp 122.4 °–122.8 °C. [a]D20 = 40.3 ° (c 0.30, CHCl3). IR (NaCl): n 2840–2890, 1600–1720 (arom), 1480, 1420, 1240 and 1040 cm 1. 1H and 13C NMR data: see the literature (Pelter et al., 1982). MS: m/z 354 (M, 68), 323(18), 161(34), 149(100) and 135 (40). ()-Piperitol-3,3-dimethylallyl ether: 6. C25H28O6, white fine solid, mp 58.1 °–59.0 °C. [a]D20 = 32.8 ° (c 0.88, CHCl3). IR (NaCl): n 2950, 2986, 1610, 1590, 1515, 1445, 1250, 1140, 1080, 1000, 935, 855 and 810 cm 1. 1 H NMR data: see the literature (Vaquette et al., 1979). 13 C NMR data: see the literature (Arruda et al., 1994). MS: m/z 424 (M, 4), 356 (95), 325 (16), 203 (15), 163 (22), 161 (30), 151 (67), 149 (100), 135 (45) and 69 (35). Fungicidal activity. Compounds 1, 2, 4, 5 and 6 were dissolved in DMSO and added to malt agar in 24 well microtitre plates in order to yield a 100/50/25/12.5/6.2/ 3.1 mM concentration series for each compound. Each microtitre plate well was subsequently inoculated with one of the nine fungi listed in Table 2 and incubated at 22 °C and 75% relative air humidity. Fungal growth compared with untreated controls was evaluated after 3 days. Insecticidal activity. Compounds 1, 2, 4, 5 and 6 were dissolved in DMSO in order to yield a 20/5/1.25/0.31/ 0.08/0.02 mM concentration series for each compound. 25 mL quantities of these solutions were applied to the bottom of 2 cm2 circular wells of 24 well microtitre plates. Solvent evaporation on a hot plate resulted in compound application rates equivalent to 5000/1250/ 313/78/20/5 g/ha respectively. After evaporation, three adult houseflies (1 or 2 days old) were added to the microtitre plate wells and incubated at 30 °C, 60% RH under a 16 h/8 h light/dark cycle. The experiment was repeated four times. Evaluation of activity, based on the mortality of the flies, was performed after 24 h. Permethrin was used as reference sample while DMSO was used as a blank control. Phytother. Res. 16, 66–70 (2002)
ACTIVITIES OF ZANTHOXYLUM USAMBARENSE
69
Table 2. MIC-values (minimum inhibitory concentration in mM) of five compounds, isolated from Zanthoxylum usambarense Fungus Coriolus versicolor Coniophora puteana Chaetomium globosum Aureobasidium pullulans Penicillium islandicum Cladosporium resinae Aspergillus niger Aspergillus ¯avus Trichoderma viride a
1
2
4
5
6
Ref.a
25 25 25 100 100 25 25 50 100
>100 >100 >100 >100 >100 >100 >100 >100 >100
>100 >100 >100 >100 >100 >100 >100 >100 >100
>100 >100 >100 >100 >100 >100 >100 >100 >100
>100 >100 >100 >100 >100 >100 >100 >100 >100
