NPC 2010 Vol. 5 No. 2 175 - 178 Natural Product Communications Antimosquito and Antimicrobial Clerodanoids and a Chlorobenzenoid from Tessmannia species Charles Kihampaa,c, Mayunga H.H. Nkunya b,*, Cosam C. Josephb, Stephen M. Magesad, Ahmed Hassanalic, Matthias Heydenreiche and Erich Kleinpetere a Department of Environmental Science and Management, Ardhi University, P. O. Box 35176, Dar es Salaam, Tanzania b Department of Chemistry, University of Dar es Salaam, P.O. Box 35061, Dar es Salaam, Tanzania c Behavioural and Chemical Ecology Department, International Centre for Insect Physiology and Ecology, P.O. Box 30772, Nairobi 00100, Kenya d Amani Medical Research Centre, National Institute for Medical Research, P.O. Box 81, Muheza, Tanga, Tanzania e Institut für Chemie, Universität Potsdam, Postfach 601553, D-14415 Potsdam, Germany nkunya@chem.udsm.ac.tz, mnkunya@tcu.go.tz Received: April 7th, 2009; Accepted: August 23rd, 2009 The clerodane diterpenoids trans-kolavenolic acid, 18-oxocleroda-3,13(E)-dien-15-oic acid, ent-(18-hydroxycarbonyl)-cleroda3,13(E)-dien-15-oate, 2-oxo-ent-cleroda-3,13(Z)-dien-15-oic acid and trans-2-oxo-ent-cleroda-13(Z)-en-15-oic acid, and the chlorobenzenoid O-(3-hydroxy-4-hydroxycarbonyl-5-pentylphenyl)-3-chloro-4-methoxy-6-pentyl-2-oxybenzoic acid were isolated from Tessmannia martiniana var pauloi and T. martiniana var matiniana. Structures were established based on interpretation of spectroscopic data. Some of the compounds exhibited significant antimosquito, antifungal and antibacterial activities. Keywords: Tessmannia martiniana var pauloi, T. martiniana var martiniana, Caesalpiniaceae, ent-clerodanoids, chlorobenzenoid, anti-mosquitoes, antimicrobials. In our previous paper [1] we reported the isolation, structural determination, insect repellency and antimicrobial activity of nor-halimanoid diterpenes and some other compounds from Tessmannia densiflora. In continuation with investigations of Tanzania Tessmannia species for their antimosquito, antimicrobial and other constituents we now analyzed the root and stem barks of T. martiniana var pauloi Harms and T. martiniana var martiniana Harms. In Tanzania the plant species grow in the coastal evergreen forest reserves of Pugu and Zaraninge, respectively. None of them has previously been investigated chemically for bioactive or any other constituents. We now report the isolation and antimosquito, antibacterial and antifungal activities of ent-clerodane diterpenoids and a chlorobenzenoid from these plant species. The larvicidal methanol extracts from the root bark of T. martiniana var pauloi on repeated chromatography yielded trans-kolavenolic acid (1) [2], 18-oxocleroda3,13(E)-dien-15-oic acid (2), which was previously OH OH OMe O O O H H H OH O H H O OH CHO 1 O COOH 2 OH 1 OH O 4 3 5 3 HOOC 5 7 5'' 3'' O 1'' 3' Cl 1' MeO COOH 7' MeO O R 5' 6 1''' 3''' 5''' OH O 7: R = H 8: R = Cl isolated as an antifeedant constituent of Detarium microcarpum [3], and ent-(18-hydroxy-carbonyl)cleroda-3,13(E)-dien-15-oate (3) [4]. Similarly, an active chloroform extract of the root bark of T. martiniana var martiniana, apart from 3, also gave 2-oxo-ent-cleroda-3,13(Z)-dien-15-oic acid (4) [5], and 2-oxo-ent-cleroda-13(Z)-dien-15-oic acid (5), while the 176 Natural Product Communications Vol. 5 (2) 2010 Table 1: 1H and 13C NMR spectral data for the chlorobenzenoid 6. H/C 1 2 3 4 5 6 7 1' 2' 3' 4' 5' 6' 7' 1" 2" 3" 4" 5" 1"' 2"' 3"' 4"' 5"' OMe COOH OH δH 6.75 6.63 6.44 3.01 1.68 1.34 1.34 0.91 3.01 1.68 1.34 1.34 0.88 3.99 11.30 11.70 J (Hz) d, 2.1 d, 2.4 s m m m m t, 7.5 m m m m t, 7.0 s br s br s δC 174.7 108.6 165.2 108.6 154.7 115.9 146.8 169.2 105.2 160.5 107.9 159.8 106.5 150.1 36.5 32.0 31.3 22.5 14.0 37.6 32.1 31.9 22.4 14.0 56.3 COSY HMBC H-6 C-2, C-3, C-5, C-6 H-4 C-2, C-4, C-5, C-1" C-2', C-4', C-5', C-1''' H-2'' C-2, C-6, C-7, C-2'', C-3'' H-1'', H-3'' C-1'', C-4'' H-2'', H-4'' C-2'', C-4'' H-3'', H-5'' C-2'', C-4'', C-5'' H-4'' C-2'', C-3'', C-4'' H-2"' C-2', C-6', C-7', C-2"', C-3" H-1"', H-3"' C-1"', C-4"' H-2"', H-4"' C-2"', C-4"' H-3"', H-5"' C-2"', C-4"', C-5"' H-4"' C-2"', C-3"', C-4"' C-5' new chlorobenzenoid (6) was obtained from stem bark of T. martiniana var martiniana. Structure 6 for the new chlorobenzenoid was established on the basis of its 1H and 13C NMR spectral data (Table 1) and MS, all of which indicated that the compound consisted of two units derived from 7 and 8 that we recently obtained from T. densiflora [1]. The presence of a chlorine atom was deduced from the high resolution EIMS that showed peaks at m/z 477.1626 and 479.1635 corresponding to the [M–1]+ and [M+1]+ fragment ions, and 3:1 relative intensity ratio, which corresponds to the natural abundance of Cl isotopes {calculated for C25H30ClO7 = 477.1680 corresponding to [M-1]+}. The position of Cl was derived from the MS fragmentation pattern and upon analysis of HMBC interactions (Table 1), which also indicated the relative positions of all the protonated C substituents and hence confirming structure 6 for the isolated compound. When assayed for mosquito larvicidal properties the ent-clerodanoids 2–5 and a chlorobenzenoid 6 showed moderate activity after 24 h larvae exposure, their efficacy being enhanced with prolonged time of exposure to 48 and 72 h (Table 2). However, the compounds exhibited insignificant difference in activity compared with the crude extract after 24 h [6]. Compounds 2 and 3 (Table 2), as well as their crude extract, exhibited stronger activity than their refined precursor fractions. This suggested that the efficacy of the active principles 2 and 3 could have been masked by impurities in the semi-purified fractions. On the other hand, the stronger activity of the crude extract was presumed to result from synergistic effects not only involving 2 and 3, but also other compounds such as 1 Kihampa et al. and others, which occurred in small amounts and hence could not be isolated. The enhanced activity of the crude extract could also be attributed to decomposition products formed during the isolation process, as has been previously observed elsewhere [7]. However, the fact that the crude extract displayed very interesting insect growth regulatory and larvicidal effects [6] indicates that whether in crude extract, refined fractions or as the pure compounds, the constituents of T. martiniana var pauloi root barks are potential botanical mosquito larvicides. Compound 3, which was obtained in appreciable amounts, was also assayed for mosquito repellency, for which it exhibited very low activity, being less than 50% as active as DEET. Table 2 shows that of compounds 2 – 6 assayed for larvicidal activity, compound 3 was the most active, being nearly three times more active than its crude extract after 24 h larvae exposure (methanol extract of the root bark of T. martiniana var pauloi and chloroform extract of the stem bark T. martiniana var martiniana). Compound 5 was the least active, being four times less potent than its crude extract after 24 h larvae exposure. The higher larvicidal efficacy of compounds 2 – 4 compared with 5 could be attributed to the presence of an α,β-unsaturated carbonyl system in 2 – 4, whose enhanced contribution to bioactivity has previously been reported [7,8]. The cleradonoids 2 and 3 also showed antimicrobial activity at different levels against both Gram-positive and Gram-negative bacterial strains, as well as against the tested fungal species. Compound 2 was the least active; it exhibited activity only against the Grampositive bacterium B. subtilis and the filamentous fungus Aspergillus niger at a level lower than that shown by the standard antibiotic and antifungal agent Ampicillin and Fluconazole, respectively. Compound 3 showed activity against the three bacterial species P. aeruginosa, S. aureus and B. subtilis and for the l atter, the activity being comparable to that of the standard antibiotic Ampicillin. Compounds 1, 4 – 6 could not be tested for antimicrobial activity due to paucity of the isolated amounts. These results further demonstrate the versatility of the family Caesalpiniaceae in accumulating bioactive metabolites of interest to biomedical research. Experimental General experimental procedures: CC: silica gel 60 (0.063-0.200 mm, Merck); TLC: silica gel 60 F254 (Merck) precoated plastic plates; visualization: UV-Vis and anisaldehyde spray [9]; IR: CHCl3 or KBr; specific Diterpenoids and a chlorobenzenoid from Tessmannia martiniana Natural Product Communications Vol. 5 (2) 2010 177 Table 2: Activity (% mortality) of 2 – 6 and crude extracts against Anopheles gambiae larvae. Concentration (ppm) Cp T (h) 2 3 4 5 6 TMRM* LC50 (ppm) 95% CL 24 48 15.62 nd nd 31.25 nd nd 62.5 33.3 + 3.3 53.3 + 8.8 125 46.7 + 3.3 63.3 + 3.3 250 80 + 5.7 96.7 + 3.3 500 90 + 5.7 96.7 + 3.3 72 nd nd 70 + 5.7 90 + 5.7 100 + 0 100 + 0 24 16.7+3.3 30 +5.7 50 + 0 83.3+3.3 100 + 0 100 + 0 ∼ 11 (nd) 48 (29-73) 48 46.7+3.3 70 +5.7 90 + 5.7 100 + 0 100 + 0 100 + 0 19 (7-29) 72 83.3+3.3 86.7+3.3 100 + 0 100 + 0 100 + 0 100 + 0 24 nd nd 20 + 5.7 33.3+3.3 46.7 +3.3 80 + 5.7 ∼ 1 (nd) 212 (114-520) ∼ 55 (nd) 125 (57-204) ∼ 57 (nd) 48 nd nd 50 + 5.7 70 + 0 73.3 +3.3 100 + 0 72 nd nd 80 + 5.7 100 + 0 100 + 0 100 + 0 ∼ 22 (nd) 24 nd nd 10 + 1 16.7 +6.7 20 + 5.7 36.7 +3.3 ∼ 737 (nd) 48 nd nd 16.7+8.8 33.3+8.8 36.7 + 12 66.7 +3.3 72 nd nd 20 + 5.7 43.3 +12 36.7 + 12 80 + 5.7 ∼ 345 (nd) 256 (149-708) 24 16.7+3.3 50 + 0 53.3+3.3 60 + 5.7 70 + 5.7 100 + 0 62 (30-111) 48 50 + 5.7 83.3 +3.3 100 + 0 80 + 5.7 100 + 0 100 + 0 15 (2-26) 72 60 + 5.7 100 + 0 100 + 0 100 + 0 100 + 0 100 + 0 24 - - - - - - ∼ 3 (nd) 114 (44-186) TMMRC* 24 - - - - - - 204 (133-340) TMMSC* 24 - - - - - - 256 (149-708) Cp = compound; CL = Confidence limits; nd = not determined; TMRM = T. martiniana var pauloi root bark crude methanol extract; TMMRC = T. martiniana var martiniana root bark crude chloroform extract; TMMSC = T. martiniana var martiniana stem bark crude chloroform extract; * quoted from ref. [6]. rotation: CHCl3; 1D NMR: 300 or 500 MHz (1H), and 75 or 125 MHz (13C); 2D NMR (HMQC, HMBC, COSY, NOESY) at 500 MHz (1H); chemical shift in ppm referenced to internal standard TMS (δ = 0) for 1 H and CDCl3 (δ = 77.0 ppm) for 13C NMR; MS: DSQII (Axel Semrau GmbH), GC-TOF (micromass) and Q-TOF micro (micromass) equipment. (MeOH/CHCl3, 1:1 v/v) yielded compounds 1, 2 and 3 (from T. martiniana var pauloi MeOH extract); 3, 4 and 5 (from T. martiniana var martiniana root bark chloroform extract) and 6 from T. martiniana var martiniana stem bark chloroform extract, while constituents of several other active fractions decomposed during the isolation process. Plant materials: The root and stem barks of Tessmannia martiniana var pauloi and T. martiniana var martiniana were collected in March 2006 from Pugu and Zaraninge Forest Reserves, respectively in Kisarawe and Bagamoyo Districts in Tanzania. The plant species were authenticated at the Herbarium of the Department of Botany at the University of Dar es Salaam, Tanzania, where voucher specimens are preserved under reference numbers FMM 1321 and 3374 respectively. O-(3-Hydroxy-4-hydroxycarbonyl-5-pentylphenyl)3-chloro-4-methoxy-6-pentyl-2-oxybenzoic acid (6) MP: 149°C. Yield: 264 mg (0.018%). Anisaldehyde: red. 1 H and 13C NMR: Table 1. MS, m/z (% rel. int.) 479 ([M+1]+, 15), 477 ([M-1]+, 45), 223 (55), 205 (100) and 179 (25). HRMS, m/z 477.1626 and 479.1635 ([M-1]+ and [M+1]+, 3:1 ratio corresponding to the natural abundance of Cl isotopes). Extraction and isolation: The air-dried and pulverized T. martiniana var martiniana root and stem barks were (1.0 and 1.5 Kg respectively) extracted sequentially with CHCl3 and MeOH (2 x 48 h for each solvent). The air-dried and pulverized T. martiniana var pauloi root bark (560 g) was extracted only with MeOH (2 x 48 h) due to paucity of the available plant materials. All the extracts were kept at -20°C until the isolation process was undertaken. The T. martiniana var pauloi MeOH extract (20 g), T. martiniana var martiniana root bark CHCl3 extract (25 g) and the T. martiniana var martiniana stem bark CHCl3 extract (25 g) that showed larvicidal activity, on bioassay guided fractionation by vacuum liquid chromatography (VLC), and then repeated column chromatography on silica gel (light pet./EtOAc gradient elution), and Sephadex® LH-20 Biological assay: Larvicidal, antibacterial and antifungal assays were carried out as reported in the literature [10-12]. In the larvicidal assays 20 late 3rd or young 4th instar larvae of Anopheles gambiae s.s were used per beaker with 3 beakers per concentration (the water temperature being 25 ± 1°C) and for each test 3 beakers containing distilled water and test larvae, but without sample, were used as controls. Observation on mortality and deformities of the larvae was recorded after every 24 h of continuous exposure and expressed as percent mortality [11]. The lethal concentration at which 50% of the test larvae were killed (LC50) was determined using POLO PLUS computer package. The disc diffusion method was used in the antibacterial assay. Staphylococcus aureus and Bacillus subtilis were 178 Natural Product Communications Vol. 5 (2) 2010 used as the Gram-positive bacteria, Escherichia coli, Pseudomonas aeruginosa, Streptococus faecalis, Klebsiella pneumoniea and Salmonella typhimurium as the Gram-negative bacteria strains, and Ampicillin (10 μg/mL) and Gentamycin (10 μg/mL) were used as the standard antibiotics. Aspergillus fumigatus, A. niger and Candida albicans were used in the antifungal tests and Fluconazole (20 μg/mL) as the standard antifungal agent. The assays were conducted in triplicate at 10 mg/mL concentration of each tested compound. Kihampa et al. Acknowledgements - Thanks to the Germany Academic Exchange Services (DAAD) for a study grant for the research work that was also undertaken at the International Centre for Insect Physiology and Ecology (ICIPE) in Nairobi, Kenya, and Amani Medical Research Centre in Muheza, Tanzania. Financial support through a Sida/SAREC grant to the Faculty of Science at the University of Dar es Salaam is gratefully acknowledged. We thank Mr Frank M. Mbago of the Herbarium, Department of Botany at the University of Dar es Salaam for location and identification of the investigated plant species. References [1] Kihampa C, Nkunya MHH, Joseph CC, Magesa S, Hassanali A, Heydenreich M, Kleinpeter E. (2009) Anti-mosquito and antimicrobial nor–halimanoids, isocoumarins and an anilinoid from Tessmannia densiflora. Phytochemistry, 70, 1233-1238. [2] Misra R, Pandey RC, Dev S. (1979) Higher isoprene-IX, diterpenoids from the oleoresin of Hardwickia pinnata, Part 2: Kolavic, kolavenic, kolavenolic and kolavonic acids. Tetrahedron, 35, 979-984. [3] Lajide L, Escoubas P, Mizutani J. (1995) Termite antifeedant activity in Detarium microcarpum. Phytochemistry, 40, 1101-1104. [4] Nyasse B, Ngantchou I, Tchana EM, Sonke B, Denier C, Fontane C. (2004) Inhibition of both Trypanosoma brucei bloodstream forms and related glycolytic enzymes by a new kolavic acid derivative from Entada abyssinica. Pharmazie, 59, 873-875. [5] Tamayo-castillo G, Jakupovic J, Bohlmann F, Castro V, King RM. (1989) Ent-clerodane derivatives and other constituents from representatives of the subgenus Ageratina. Phytochemistry, 28, 139-141. [6] Kihampa C, Joseph CC, Nkunya MHH, Magesa S, Hassanali A, Heydenreich M, Kleinpeter E. (2008) Larvicidal and IGR activity of extract of Tanzanian plants against malaria vector mosquitoes. Journal of Vector Borne Diseases, 46, 145-152. [7] Weenen H, Nkunya MHH, Bray DH, Mwasumbi LB, Kinabo LS, Kilimani VAEB, Wijnberg JBPA. (1990) Antimalarial compounds containing an α,β-unsaturated carbonyl moiety from Tanzanian medicinal plants. Planta Medica, 56, 371-373. [8] Rodriguez AM, Enriz RD, Santagata LN, Jauregui EA, Pestchanker MJ, Giordano OS. (1997) Structure-cytoprotective activity relationship of simple molecules containing an α,β-unsaturated carbonyl. Journal of Medicinal Chemistry, 40, 1827-1834. [9] Stahl E. (1969) Thin-Layer chromatography. A laboratory handbook. Springer Verlag, New York, p. 857. [10] Joseph CC, Moshi MJ, Sempombe J, and Nkunya MHH. (2006) 4-(Methoxy-benzo[1,3]dioxol-5-yl)-phenylmethanone: An antibacterial benzophenone from Securidaca longepedunculata. African Journal of Traditional, Complementary and Alternative Medicine, 3, 43-58. [11] WHO. (1996) Report of the WHO informal consultation on the evaluation and testing of insecticides. WHO, Geneva, pp 32-36 and 50-52. [12] Moshi MJ, Joseph CC, Innocent E, Nkunya MHH. (2004) In-vitro antibacterial and antifungal activity of extracts and compounds from Uvaria scheffleri. Pharmaceutical Biology, 42, 269-273.