Planta Med 2004; 70(5): 437-440
DOI: 10.1055/s-2004-818972
Original Paper
Natural Product Chemistry
© Georg Thieme Verlag KG Stuttgart · New York

Antiplasmodial Agents from the Leaves of Glossocalyx brevipes

James A. Mbah1 , Pierre Tane1 , Bonaventure T. Ngadjui2 , Joseph D. Connolly3 , Christopher C. Okunji4 , Maurice M. Iwu4 , Brian M. Schuster4
  • 1Chemistry Department, Faculty of Science, University of Dschang, Dschang, Cameroon
  • 2Organic Chemistry Department, Faculty of Science, University of Yaoundé I, Yaoundé, Cameroon
  • 3Department of Chemistry, University of Glasgow, Glasgow, Scotland
  • 4Division of Experimental Therapeutics, Walter Reed Army Institute of Research, Washington, DC, USA
We gratefully acknowledge financial support from the ICBG ”Drug Development and Conservation in West and Central Africa” Grant No TW01023 - 01-AP2 from the Fogarty Center, NIH, and from the International Science Program (ISP), Grant No CAM:02, Uppsala University, Sweden
Further Information

Prof. Pierre Tane

Department of Chemistry

Faculty of Science

University of Dschang

Box 67

Dschang

Cameroon

Fax: +237-345-1202

Email: ptane@yahoo.com

Publication History

Received: August 5, 2003

Accepted: February 16, 2004

Publication Date:
04 May 2004 (online)

Table of Contents #

Abstract

A phytochemical study of the methylene chloride/methanol (1/1) extract of the leaves of Glossocalyx brevipes Benth. (Monimiaceae) afforded three new derivatives of homogentisic acid, methyl 2-(1′β-geranyl-5′β-hydroxy-2′-oxocyclohex-3′-enyl)acetate (1), 2-(1′β-geranyl-5′β-hydroxy-2′-oxocyclohex-3′-enyl)acetic acid (2), methyl 2-(1′β-geranyl-5′β-hydroxy-4′β-methoxy-2′-oxocyclohexyl)acetate (3), and two known alkaloids, aristololactam BII and liriodenine. Compounds 1 and 2 and liriodenine showed modest in vitro activity against Plasmodium falciparum.

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Introduction

Glossocalyx brevipes Benth (Monimiaceae) is a shrub with hairy stems and leaves, growing in the humid rain forests of West and Central Africa [1]. In Cameroonian folk medicine, the macerated leaves are added to antifever preparations. A previous phytochemical study reported the isolation of aporphine alkaloids, but these were not tested for antimalarial activity [2]. In a continuation of our search for new anti-plasmodial agents from plant sources [3], the CH2Cl2/CH3OH (1/1) extract of the leaves of G. brevipes, that revealed moderate antiplasmodial activity, was investigated and three homogentisic acid derivatives and two alkaloids were isolated.

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Materials and Methods

Melting points were measured on a Köfler hot stage 277 938 and are uncorrected. 1H-NMR (400.13 MHz) and 13C-NMR (100.6 MHz) were registered in CDCl3 with a Bruker DPX 400 spectrometer. Chemical shifts are reported in parts per million (ppm) and coupling constants in Hertz (Hz). A 1D-NOE experiment using PFGSE (pulsed field gradient selective excitation) was used to obtain NOEs. UV spectra were recorded in chloroform with a Shimadzu UV-3101 PC. IR spectra were registered with a JASCO FTIR-410 spectrometer. Mass spectra were obtained in the EI mode with a Joel JMS 700 spectrometer at 70 eV. Optical rotations were measured with a Perkin-Elmer 241 polarimeter at 20 °C. TLC plates (eluting with mixtures of methylene chloride/acetone or chloroform/methanol) were visualised with UV light (254 nm and 366 nm) and sprayed with 50 % H2SO4 and other reagents, followed by heating in an oven. SiO2 60 (70 - 230 mesh) was used for CC while SiO2 F254 was used for TLC plates.

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Plant material

The leaves of Glossocalyx brevipes Benth (Monimiaceae) were collected in Kumba, South West Province of Cameroon, in September 1997 and authentication was carried out at the Limbe Botanical Garden. A voucher specimen (UD 337) is deposited at the herbarium of the Botany Department, University of Dschang.

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Extraction and isolation

The air-dried and powdered leaves (5 kg) of G. brevipes were macerated in a mixture of methylene chloride/methanol (1/1) for seven days. Filtration and concentration on a rotavapor afforded 1200 g of extract. Part of this extract (300 g) was treated with 0.1 M sulfamic acid (20 mL) followed by extraction with methylene chloride to yield the neutral fraction (230 g). The aqueous phase was basified to pH 7 with 5 % aqueous ammonia and the crude alkaloid material (6 g) was obtained by extraction with methylene chloride. Vacuum liquid chromatography of the neutral fraction (150 g) on silica gel using a gradient of EtOAc in n-hexane (5 : 95, 10 : 90, 20 : 80, 40 : 60, 60 : 40 and 100 : 0) gave sixty fractions (500 mL each) which were concentrated and combined on the basis of TLC profiles. Chlorophyll was removed from fractions 29 - 35 (obtained with 20 % EtOAc); 44 - 48 (obtained with 40 % EtOAc); and 54 - 57 (with 60 % EtOAc) by gel permeation through Sephadex LH-20 (50 g) (CH2Cl2/MeOH [8 : 2] 100 mL for each elution). The residue obtained from fractions 29 - 35 (6 g) was purified on a silica gel (125 g) column eluted with hexane/EtOAc (9 : 1; 150 mL for each fraction) to yield 1 (300 mg) and 3 (200 mg). Compound 2 (208 mg) was obtained from the residue of fractions 44 - 48 (7 g) and aristololactam BII (8 mg) [5] from fractions 54 - 57 (4 g) under further purification by gel permeation through Sephadex LH-20 (40 g), eluted with CH2Cl2 (50 mL for each elution) and hexane/CH2Cl2 (7 : 3; 50 mL for each elution), respectively. Liriodenine (20 mg) [6] was isolated from the alkaloid portion of the initial extract by gel permeation via Sephadex LH-20 (40 g) (CH2Cl2/MeOH [7 : 3], 50 mL for each elution) to remove the chlorophyll, followed by chromatography over basic alumina (type III) using gradient of CH2Cl2-hexane (0 : 100, 10 : 90, 20 : 80, 40 : 60, 60 : 40, 100 : 0; 250 mL for each fraction) and then silica gel (100 g) (CH2Cl2/acetone [9 : 1]; 150 mL for each elution).

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Isolated compounds

Methyl 2-(1′β-geranyl-5′β-hydroxy-2′-oxocyclohex-3′-en-yl)acetate (1): Colourless oil; [α]D 22: -2.9° (c, 0.1, CHCl3); analysis found: C 71.22 %, H 8.81 %; C19H28O4 requires: C 71.24 %, H 8.80 %; UV (CHCl3): λmax = 246 nm (ε 1200); IR (KBr): νmax = 3520, 2918, 2254, 1732, 1679, 1438, 1382, 1200, 1175, 1093, 921, 757 cm-1; EIMS (probe, 70 eV): m/z (rel. int.) = 320 [M]+ (7), 219 (44), 173 (55), 168 (98), 134 (98), 107 (65), 93 (85), 69 (100); 1H-NMR data (Table [1]); 13C-NMR data (Table [2]).

2-(1′β-Geranyl-5′β-hydroxy-2′-oxocyclohex-3′-enyl)acetic acid (2): Colourless oil; [α]D 22: -2.9° (c, 0.1, CHCl3); analysis found: C 70.56 %, H 8.55 %; C18H26O4 requires: C 70.54 %, H 8.52 %; UV (CHCl3): λmax = 246 nm (ε 1200); IR (KBr): νmax = 3604, 3155, 2969, 2918, 2254, 1710, 1678, 1448, 1383, 1217, 1093, 1023, 922, 708 cm-1; EIMS (probe; 70 eV): m/z (rel. int.) = 306 [M]+ (6), 288 (6), 219 (15), 173 (20), 135 (36), 134 (30), 93 (32), 69 (100); 1H-NMR data (Table [1]); 13C-NMR data (Table [2]).

Methyl 2-(1′β-geranyl-5′β-hydroxy-4′β-methoxy-2′-oxocyclohexyl) acetate (3): Colourless oil; [α]D 22: -59° (c, 0.103, CHCl3); analysis found: C 68.15 %, H 9.15 %; C20H32O5 requires: C 68.17 %, H 9.18 %; UV (CHCl3): λmax = 290 nm (ε 15); IR (KBr): νmax = 3571, 2932, 2254, 1729, 1706, 1438, 1352, 1201, 1102, 983, 922, 757, 710 cm-1; EIMS (probe; 70 eV): m/z (rel. int.) = 352 [M]+ (11), 283 (18), 251 (18), 216 (40), 166 (52), 136 (28), 107 (25), 93 (45), 69 (100); 1H-NMR data (Table [1]); 13C-NMR data (Table [2]).

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Biological assays

Antiplasmodial assay: The in vitro antiplasmodial assays were performed by using a modification of the semi-automated microdilution technique described earlier [4]. Two Plasmodium falciparum malarial parasite clones designated Indochina (W-2) and Sierra Leone (D-6) were utilised in susceptibility tests. The W-2 clone is resistant to chloroquine, pyrimethamine, sulfadoxine and quinine while the D-6 clone is resistant to mefloquine. The tested compounds were dissolved in DMSO and serially diluted using malarial growth medium. Drug-induced reduction in uptake of tritiated hypoxantine was used as index of inhibition of parasite growth.

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Results and Discussion

Compound 1 was obtained as a colourless oil. Its IR spectrum showed absorptions at 3520 (OH), 1732 (ester) and 1680 cm-1 (C = O). The mass spectrum (EIMS) displayed a molecular ion peak at m/z = 320 and the presence of nineteen carbon signals in the 13C-NMR spectrum enabled us to deduce the molecular formula as C19H28O4. The 1H-NMR (400 MHz) spectrum showed olefinic protons at δ = 5.99 (1H, dd, J = 10.2, 1.8 Hz, H-3′), 6.89 (1H, ddd, J = 10.2, 2.8, 1.4 Hz, H-4′) (α,β-unsaturated ketone) and 5.05 (2H, m, H-2′′ and H-6′′). A proton on an oxygenated sp 3 carbon appeared at δ = 4.68 (1H, m, H-5′) and coupled to a hydroxy proton at δ = 2.21 (d, J = 5.7 Hz). Three vinyl methyl groups were observed at δ = 1.67 (3H, s, Me-8′′), 1.60 (3H, s, Me-9′′), 1.59 (3H, s, Me-10′′) and a methoxycarbonyl group at 3.67 (Table [1]). The vinyl methyls together with the two trisubstituted double bonds suggested the presence of a geranyl group. The 13C-NMR (100.6 MHz) spectrum showed an enone carbonyl at δ = 201.1 (C-2′), an ester carbon function at δ = 171.8 (C-1) and a secondary oxygenated carbon at δ = 64.8 (C-4′). The signals of the geranyl group appeared at 35.6 (C-1′′), 119.0 (C-2′′), 131.5 (C-3′′), 40.4 (C-4′′), 26.9 (C-5′′), 124.5 (C-6′′), 139.9 (C-7′′), 26.1 (C-8′′), 18.1 (C-9′′) and 16.7 (10′′) (Table [2]). The DEPT and HMQC spectra enabled us to conclude that 1 had five quaternary, five tertiary, five secondary and four primary carbon atoms. The COSY spectrum readily revealed a 1H-1H spin system involving the enone protons H-3′ and H-4′, the carbinol proton H-5′ and the C-6 methylene protons. There are long-range couplings between H-3′ and H-5′ (allylic) and H-4′ and H-6′ (pseudo-equatorial, α) (4 J H,H). The COSY also showed the isolated AB system H2 - 2 and the first methylene group H2 - 1′′ of the geranyl group. HMBC spectral correlations such as H-2 to C-1, C-1′, C-2′, C-6′ and C-1′′; H-3′ to C-4′, C-2′, C-1′ and C-5′; H-1′′ to C-1′, C-2′′, C-3′′, C-2′; and H-5′ to C-4′, C-3′, C-6′ enabled us to assign structure 1, methyl 2-(1′β-geranyl-5′β-hydroxy-2′-oxocyclohex-3"-enyl)acetate, to this compound, which is a new homogentisic acid derivative. Similar derivatives bearing a farnesyl residue have been reported from Otoba parvifolia [7].

The relative stereochemistry of 1 was determined using NOE experiments. Irradiation of H-5′, which must be pseudo-axial in view of its 8.3 Hz coupling with H-6′ at 2.01 ppm, afforded NOEs towards H-6′ equatorial (2.40 ppm) and H-2 (2.64 ppm). Therefore H-5′ and the acetic acid side chain lie on the same face of the molecule. The E configuration of the Δ1 ′′ geranyl double bond follows from the observation of an NOE between the C-1′′ protons and the C10′′ methyl protons. Thus the relative stereochemistry is as shown in 1.

Compound 2 was obtained as a colourless oil whose IR spectrum showed absorptions at 3604, 3155 (OH), 1710 (COOH), and 1678 cm-1 (C = O). The mass spectrum afforded a molecular ion peak at m/z = 306 compatible with the molecular formula C18H26O4. The spectroscopic data were in all respects similar to those of 1 except for the absence of the methoxy signal at 3.67 ppm in the 1H-NMR (Table [1]) and 52.1 ppm in the 13C-NMR spectra (Table [2]). Thus 2 is 2-(1′β-geranyl-5′β-hydroxy-2′-oxocyclohex-3′-enyl) acetic acid, another new homogentisic acid derivative.[]

Compound 3 was obtained as a colourless oil whose IR spectrum showed absorptions at 3571 (OH), 1729 (ester), and 1706 cm-1 (C = O). The mass spectrum afforded a molecular ion peak at m/z = 352, compatible with the molecular formula C20H32O5. The 1H-NMR (400 MHz) spectrum (Table [1]) showed a geranyl moiety as in 1 and two methoxy groups at δ = 3.43 and 3.71 (Table [1]). The 13C-NMR (100.6 MHz) spectrum (Table [2]) confirmed the presence of a geranyl moiety and, in addition, showed signals at δ = 211.5 (C-2′), 173.0 (C-1), 79.8 (C-4′), 67.1 (C-5′), 56.7 (OMe) and 51.9 (COOMe). DEPT and HMQC experiments showed that the compound had five quaternary, five tertiary, six secondary and five primary carbon atoms. The COSY and HMBC data enabled us to attribute structure 3, methyl 2-(1′β-geranyl-5′β-hydroxy-4′β-methoxy-2′-oxocyclohexyl) acetate, to this compound, a third new homogentisic acid derivative. The COSY spectrum readily revealed the coupled system around the cyclohexanone ring while the HMBC spectrum displayed correlations from MeO-4′ to C-4′ and from H-4′ to C-2′, C-3′, C-5′ and C-6′.

The relative stereochemistry of 3 was determined by NOE experiments and consideration of coupling constants. Irradiation of H-4′, which must be axial, showed NOEs towards H-3′α, H-5′α, H-6′α, while irradiation of H2 - 1′′ gave NOEs to Me-10′′, confirming the E configuration of the double bond. Also H2 - 1′′ had NOEs to H-3′β, H-6′β and H2 - 2. These results indicate that the cyclohexanone ring of 3 essentially has a chair configuration with the methoxy and acetic acid side chain in equatorial and the hydroxyl and geranyl groups in axial orientation.

The known alkaloids were identified as aristololactam BII (m. p. 256 - 257 °C; C17H13O3N) [5] and liriodenine (m. p. 275 - 276 °C; C17H9O3N) [6] by analysis of their spectral data.

The results of the antiplasmodial tests of the compounds isolated from G. brevipes are summarised in Table [3] below. Methyl 2-(1′β-geranyl-5′β-hydroxy-2′-oxocyclohex-3′-enyl)acetate (1) showed the strongest activity while 2-(1′β-geranyl-5′β-hydroxy-2′-oxocyclohex-3′-en-yl)acetic acid (2) and liriodenine [6] demonstrated moderate activity. The isolation of these active principles supports the use of G. brevipes in anti-fever preparations:

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Table 1 1H-NMR (400 MHz) data (δ, mult. J) in CDCl3 for 1 - 3
Position 1 2 3
2 2.64, d, 14.9 2.67, d, 15.0 2.64, d, 14.8
2.38, d, 14.9 2.38, d, 15.0 2.38, d, 14.8
3′ 5.99, d, 10.1 5.99, d, 10.1 2.86, dd, 14.8, 10.8
- - 2.68, dd, 14.8, 4.5
4′ 6.89, dd, 10.1, 2.0 6.89, dd, 10.1, 2. 3.69, ddd, 10.1, 4.5, 2.7
5′ 4.68, brd, 2.2 4.69, m 4.32, m
6′ 2.33, dd, 13.9, 5.3 2.33, dd, 13.9, 5.3 2.16, dd, 14.8, 4.3
2.01, dd, 13.9, 8.3 2.01, dd, 13.9, 8.3 2.03, dd, 14.8, 8.5
1′′ 2.50, dd, 14.4, 6.5 2.50, dd, 14.4, 6.5 2.50, dd, 14.5, 6.5
2.31, dd, 14.1, 8.6 2.32, dd, 14.4, 8.3 2.33, dd, 14.5, 8.6
2′′ 5.10, dd, 8.6, 6.5 5.10, dd, 8.3, 6.5 5.10, dd, 8.5, 6.5
4′′ 2.10, m 2.10, m 2.10, m
2.07, m 2.06, m 2.07, m
5′′ 2.21, m 2.21, m 2.21, m
2.04, m 2.05, m 2.05, m
6′′ 5.10, m 5.10, m 5.10, m
8′′ 1.67, s 1.67, s 1.68, s
9′′ 1.60, s 1.60, s 1.60, s
10′′ 1.59, s 1.60, s 1.60, s
COOMe 3.67, s - 3.71, s
OMe - - 3.43, s
Table 2 13C-NMR (100.6 MHz) data in CDCl3 for 1 - 3
Position 1 2 3
1 171.8 176.0 173.0
2 40.7 40.6 40.0
1′ 48.0 47.8 49.4
2′ 201.1 201.1 211.5
3′ 128.7 128.7 39.5
4′ 150.6 150.5 79.6
5′ 64.8 64.7 67.1
6′ 39.2 39.0 35.7
1′′ 35.6 35.7 36.8
2′′ 119.0 118.8 118.7
3′′ 131.5 131.9 131.5
4′′ 40.4 40.4 40.3
5′′ 26.9 26.8 26.9
6′′ 124.5 124.5 124.5
7′′ 139.9 139.8 139.5
8′′ 26.1 26.1 26.1
9′′ 18.1 18.1 18.1
10′′ 16.7 16.7 16.7
COOMe 52.1 - 51.9
OMe - - 56.7
Table 3 Antiplasmodial activities of compounds from Glossocalyx brevipes.
Compound P. falciparum clones (IC50, ng/mL) D-6 W-2
Methyl 2-(1′β-geranyl-5′β-hydroxy-
2′-oxocyclohex-3"-enyl)acetate (1)
702.59 2 125.78
2-(1′β-geranyl-5′β-hydroxy-2′-
oxocyclohex-3′-enyl)acetic acid (2)
1 462.00 2 552.94
Methyl 2-(1′β-geranyl-5′β-hydroxy-4′β-methoxy-2′-oxocyclohexyl)acetate (3) NTa NTa
Aristololactam BII >5 000 >5 000
Liriodenine 1 326.30 2 373.71
Mefloquine (as standard) 11.67 4.78
Chloroquine (as standard) 4.77 145.30
a NT = not tested.
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References

  • 1 Fouilloy R. Flore du Cameroun. Muséum National d’Histoire Naturelle Paris; 1974 Vol. 18: pp 30-1
  • 2 Montgomery T C, Freyer A J, Guinandeau H, Shama M, Fagbule M O, Olatunji G, Gbile Z. (+)-N-Methyllaurotetanine β-N-oxide from Glossocalyx brevipes .  Journal of Natural Products. 1985;  48 833-4
  • 3 Tchuendem M -HK, Mbah J A, Tsopmo A, Ayafor J F, Sterner O, Okunji C O, Iwu M M, Schuster B M. Reneilmol, A new antiplasmodial isodaucane and other sesquiterpenoids from Reneilmia cincinnata .  Phytochemistry. 1999;  52 1095-9
  • 4 Desjardins R E, Canfield C J, Haynes J D, Chulay J D. Quantitative assessment of antimalarial activity in vitro by a semiautomated microdilution technique.  Antimicrob Agents Chemother. 1979;  16 710-8
  • 5 Priestap H A. Seven aristolactams from Aristolochia argentina .  Phytochemistry. 1985;  24 849-52
  • 6 Bick I RC, Douglas C K. Yellow alkaloids of Atherosperma moschatum Labill. Tetrahedron Letters 1964: 1629-33
  • 7 Ferreira A G, Fernandes J B, Vieira P C, Gottlieb O R, Gottlieb H E. Further farnesylhomogentisic acid derivatives from Otoba parvifolia .  Phytochemistry. 1995;  40 1723-28

Prof. Pierre Tane

Department of Chemistry

Faculty of Science

University of Dschang

Box 67

Dschang

Cameroon

Fax: +237-345-1202

Email: ptane@yahoo.com

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References

  • 1 Fouilloy R. Flore du Cameroun. Muséum National d’Histoire Naturelle Paris; 1974 Vol. 18: pp 30-1
  • 2 Montgomery T C, Freyer A J, Guinandeau H, Shama M, Fagbule M O, Olatunji G, Gbile Z. (+)-N-Methyllaurotetanine β-N-oxide from Glossocalyx brevipes .  Journal of Natural Products. 1985;  48 833-4
  • 3 Tchuendem M -HK, Mbah J A, Tsopmo A, Ayafor J F, Sterner O, Okunji C O, Iwu M M, Schuster B M. Reneilmol, A new antiplasmodial isodaucane and other sesquiterpenoids from Reneilmia cincinnata .  Phytochemistry. 1999;  52 1095-9
  • 4 Desjardins R E, Canfield C J, Haynes J D, Chulay J D. Quantitative assessment of antimalarial activity in vitro by a semiautomated microdilution technique.  Antimicrob Agents Chemother. 1979;  16 710-8
  • 5 Priestap H A. Seven aristolactams from Aristolochia argentina .  Phytochemistry. 1985;  24 849-52
  • 6 Bick I RC, Douglas C K. Yellow alkaloids of Atherosperma moschatum Labill. Tetrahedron Letters 1964: 1629-33
  • 7 Ferreira A G, Fernandes J B, Vieira P C, Gottlieb O R, Gottlieb H E. Further farnesylhomogentisic acid derivatives from Otoba parvifolia .  Phytochemistry. 1995;  40 1723-28

Prof. Pierre Tane

Department of Chemistry

Faculty of Science

University of Dschang

Box 67

Dschang

Cameroon

Fax: +237-345-1202

Email: ptane@yahoo.com

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