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Larvicidal and IGR activity of extract of Tanzanian plants against malaria vector mosquitoes.

Introduction

It has long been known that in Africa Anopheles gambiae s.s. Giles mosquitoes are the vectors that transmit malaria parasites. The disease's prevalence in Africa is estimated to constitute 300-500 million clinical cases every year, with a mortality rate of 1.4-2.6 million people as the disease is fatal if untreated (1). Although malaria fevers have been reasonably brought under control by the application of synthetic insecticides to kill the vector mosquitoes, there is currently recrudescence of the disease. This has been ascribed to the emergence of many of breeding places brought about by human activities, and the ever-increasing resistance of mosquitoes to currently available commercial insecticides. It is still believed that the only way to decrease the incidence of malaria is by combating the vector mosquitoes. Experience has shown that aerial toxicants for mosquito control are not effective, since mosquitoes are now highly domesticated and many adults rest indoors in hidden places such as closets. Therefore, it is envisioned that the only successful method of reducing mosquito densities to an appreciable level for which malaria epidemics can be controlled is by attacking the larval breeding places through the use of larvicides (2).

Chemical insecticides have continued to be commonly used for controlling mosquitoes in many parts of the world. Initially their use was focused on the control of mosquitoes, either by killing or repelling them. However, the appearance of mosquito resistance to conventional insecticides, together with public concern about the safety and availability of the insecticides have prompted the necessity to search for alternative insecticides that would be environmentally acceptable and less costly. Therefore, in recent years the use of environmentally friendly and easily biodegradable natural insecticides of plant origin has received renewed importance for malaria and other diseases control. Interest in this field is based on the fact that these substances are least phytotoxic and do not lead to the accumulation of chemical residues in flora, fauna, soil and the entire environment in general.

The above facts prompted us to undertake investigations of some plant species traditionally used as insecticidal agents, as well as other endangered Tanzanian plant species, with the aim of identifying lead compounds for the development of new plant based insecticidal agents. We now report results from these investigations.

Material & Methods

Plant materials: The investigated plant species are summarised in Table 1, which include parts investigated, locality and criteria used for their collection. All the plant species were identified on site and their identities confirmed at Herbarium of the Department of Botany, University of Dar es Salaam, where voucher specimens are deposited for future reference.

Extraction and isolation: Air-dried and pulverized root and stem barks, leaves, fruits or whole plant were extracted sequentially with pet ether, CH[Cl.sub.3] and MeOH, 2 x 48 h for each solvent. The extracts were stored at -18[degrees]C until further analysis or assay. Bioassay-guided isolation using the brine shrimp lethality test (BST) (3) yielded the active compounds from the relevant extracts. Fractionation of the concentrated extracts was carried out by VLC, followed by repeated column chromatography on silica gel and/or Sephadex[R] LH-20, eluting with pet ether and then pet ether containing increasing amounts of EtOAc, and mixtures of MeOH and CH[Cl.sub.3] (1:1, v/v) respectively. Structural determination was carried out based on interpretation of [sup.1]H and [sup.13]C NMR, and MS spectra, and upon comparison with the reported spectra data.

Larvicidal assay: The assay was performed by exposing the An. gambiae mosquito larvae in distilled water treated with a series of at least five concentrations of each test sample in DMSO, kept in beakers according to WHO protocols (4). Twenty late III or young IV instar larvae were used per beaker with three beakers per concentration (the water temperature being 25 [+ or -] 1[degrees]C) and for each test three 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 this was expressed as percent mortality (4), the lethal concentration at which 50% of the test larvae were killed ([LC.sub.50]) being worked out using a POLO PLUS computer package.

Results & Discussion

Results of investigations for susceptibility of the An. gambiae larvae to crude extracts of seventeen plant species are shown in Table 2. All the extracts showed activity against the late III and/or early IV instar An. gambiae s.s. Giles larvae. The larvae mortality was observed to increase as the concentration of the test samples was raised. This activity trend was also observed in the case of time elapsed mortality. Furthermore, some of the treated larvae showed sluggish movements and peculiar coiling. This suggested either neural or muscular effects being exerted by some of the active principles being the cause of the acute lethal effects exhibited by the extracts. Delayed lethal effects related to the duration of exposure to the test samples were also observed. These were attributed to the likely disturbance of the endocrine mechanisms that regulate moulting and metamorphosis, as it was previously postulated for the neem seed kernel extracts (5).

The larvicidal constituents were mostly less or moderately polar pet ether and chloroform soluble compounds. This activity trend was similar to the previously reported phenomenon for mosquitocidal constituents of Neorautanenia mitis (6). The root bark extracts were more active than those from the stem barks for most of the investigated plant species. This activity trend was rationalized by considering the fact that the more confined root-soil environment makes the roots to be more susceptible to pathogenic and/or predatory soil organism attacks. Therefore, this would compel the roots to produce more viable metabolites for self-defence as opposed to the aerial parts.

Table 2 shows that the Annona squamosa root and stem bark extracts were the most active among all the investigated plant extracts. In our investigations the root barks yielded the two ent-kaurane diterpenoids kaur-16-en-19-oic acid (1) (Structure depicted in Fig. 1) and 17-acetoxy-ent-kauran-19-al (2), all of which displayed larvicidal activity (Table 3). The fact that no further bioactive metabolites were isolated from the extracts led us to conclude that the ent-kaurane diterpenoids (1) and (2)were the major active principles of the root bark extract, probably other active metabolites not having been isolated because of their minute concentrations in the extract.

The stem bark extracts of A. squamosa are known to accumulate pesticidal annonaceous acetogenins (7). Therefore, the observed larvicidal activity of the stem bark extract was considered to have been exerted by such latter compounds, which however, were not isolated during these investigations, probably due to their low abundance in the investigated plant samples.

Extracts from the Uvaria (Annonaceae) species U. faulknerae, U. kirkii, U. leptocladon, U. lungonyana and U. scheffleri displayed larvicidal activity at varying levels (Table 2). Since both insecticidal and antitumour activities are known to exert similar modes of cell action by blocking the cellular oxygen transport system8, the larvicidal efficacy of the Uvaria species were considered to be due to the C-benzyl dihydrochalcones and flavanones metabolized by Uvaria species (9). Furthermore, U. lungonyana, which is endemic to Tanzania was first analysed chemically in these investigations and the root bark extract yielded polycarpol (3), chamanetin (4) and dichamanetin (5), melodorinol (6), acetylmelodorinol (7), benzyl benzoate (8), 2methoxybenzyl benzoate (9), pinocembrin (10) and 5-hydroxy-7-methoxyflavanone (11). Some of these compounds exhibited larvicidal activity (Table 3). The metabolites (6) and (7) are hereby being reported for the first time from the genus Uvaria.

Extracts from the two Asteranthe species occurring in Tanzania, A. lutea and A. asterias, upon analysis yielded the previously reported antifungal alkaloids 2',3'-epoxyasteranthine (12) and 2',3'-dihydroxyasteranthine (13) (10), all of which showed larvicidal activity (Table 3). These results demonstrate that compounds (12) and (13) that contain an indolepyranyl skeleton as a unique structural feature among the prenylated indoles from the family Annonaceae, have broad bioactivity spectra.

According to the Flora of Tropical East Africa, the four Uvariodendron species U. kirkii, U. gorgonis, U. pycnophyllum and U. usambarensis are endemic to Tanzania. So far there are no investigations describing the larvicidal or any biological activity of either of the plant species. Therefore, some of the Uvariodendron species were included in these investigations and crude extracts from U. pycnophyllum and U. usambaranse showed larvicidal activity (Table 2). The larvicidal activity was conceivably ascribed to the insecticidal phenylpropanoids such as eugenol and acetyl eugenol, which are the main constituents of some Uvariodendron species (11,12). This indicates the genus to be a useful source of mosquito larvicides.

[FIGURE 1 OMITTED]

The genus Tessmannia consists of 11 species that are either small or big trees and mainly found in the rain forests of some parts of Africa. In Tanzania, four Tessmannia species are reported, namely T. densiflora, T. martiniana var pauloi, T. martiniana var martiniana and T. burttii. Extracts from the three former species when assayed showed larvicidal activity. The methanol root bark extract of T. martiniana var pauloi also exhibited tail-like structural abnormalities for the larvae after 24 h exposure (Fig. 2). The latter structures emerged after 24 h of exposure and continued to grow until reaching the peak after 48 h, where the length of the larvae was equal to the length of the tail-like structures. The larvae also attained a dark brown colour and shaded during 48-72 h post exposure. Microscopic analysis suggested the tail-like structures to be part of the gut that had been elongated and extruded through the anal cavity.

The larvae that had shaded the tail and continued to survive were reared and monitored through their life cycle until adulthood. The morphological features of the emerged adults were normal as those from the control experiments. The males and females were allowed to mate and then fed with human blood. However, the females could not produce any batch of eggs. This suggested that factors that caused the larvae deformities had also interfered with the reproduction system in the adult mosquitoes. This process was repeated until the entire mosquito colony died. However, none of the isolated compounds could be assayed for this effect due to the paucity of the available samples. Furthermore, until now there is no literature describing the chemical constituents or any bioactivity information for the genus Tessmannia. In this regard, this study failed to associate the observed activity and the deformities with any class of compounds. As such there is great need to re-examine the extract and isolate the active compounds before the plants become extinct due to the on going deforestation process caused by harvesting the plant species for building poles and charcoal productions.

The crude pet ether, chloroform and methanol extracts from L. stellatus displayed larvicidal activity at varying levels, (Table 2). Previous investigations of L. stellatus indicated the crude extracts from the stem and root barks to have in vitro antimalarial activity, as well as weak toxicity against brine shrimp (BST) larvae (13). Among the compounds isolated from the extracts was insect juvenile hormone III (JH III), which was previously isolated from this and other plant species (14). The occurrence of JH III in plants has been quite intriguing since normally the compound is metabolised by insects in order to regulate their developmental processes (metamorphosis). Therefore, the compound when produces by plants may have similar roles, suggesting that the plants would be producing the compound in order to deter insect accumulation, as the insects would not prefer to acquire additional JH III doses beyond what is normally required for metabolism. Accumulation of this compound beyond biochemically allowable levels would disrupt the insects' development process. Hence, the compound would act as a bio-insecticide. Therefore, the presence of this compound in plant extracts would make the extracts act as readily biodegradable environmental larvicides.

[FIGURE 2 OMITTED]

However, when assaying the L. stellatus extract no sign of growth disruption was noticed, either in the larvae or in the adult stage of the insect. This could have been attributed to either small amount of the compound present in the extract or due to absence of the compound in the investigated extract as a result of seasonal and/or geographical location.

Crude extracts from H. opposita also showed some larvicidal activity (Table 2). In previous studies, such extracts showed no larvicidal activity (15), probably due to seasonal fluctuations in the biosynthesis of the active components. The results could also have been due to different methods of extraction, photosensitivity of some of the compounds in the extract or geographical origin of the plant. In previous studies, H. opposita materials were collected from a locality (Kwamngumi Forest Reserve in Muheza district) different from in the present studies (University of Dar es Salaam, Main campus), and probably explaining the difference in the larvicidal results. Previous chemical investigations of H. opposita revealed the presence of various types of compounds, including flavanones (16) and 3-O-benzoyl or 3-O-cinnamoylabiatane diterpenoids (17), some of which possess antitumour, insect antifeedant, antimicrobial and allelochemical activities. Since the physiological effects of the crude extracts on the tested larvae were not investigated, at this stage it is difficult to relate the observed larval toxicity to effects of some of the constituent compounds acting as allelochemicals.

The crude extracts from Croton sylvaticus also displayed larvicidal activity (Table 2). The genus Croton is known to constitute toxic plant species, making some Croton species to be used as sources of poison for hunting and fishing (18). As several antimicrobial cleorodane diterpenes and other compounds have previously been isolated from the genus (19). Similar compounds could have been responsible for the observed larvicidal activity in these studies, but such compounds not having been isolated possibly due to their low abundance.

Acknowledgement

We gratefully acknowledge the support by the Germany Academic Exchange Services (DAAD) through a study grant extended to C.K. through a collaborative research project with the International Centre for Insect Physiology and Ecology (ICIPE) in Nairobi, Kenya, and the Amani Medical Research Centre in Muheza, Tanga, Tanzania. We thank Mr Frank Mbago from the Herbarium, Department of Botany at the University of Dar es Salaam for locating and identifying the investigated plant species.

References

(1.) WHO expert committee report on malaria. Geneva: World Health Organization 2000.

(2.) WHO report of the WHO informal consultation on the evaluation and testing of insecticides. Geneva: World Health Organization 1996; p. 9-12.

(3.) Meyer BN, Ferrigini N, Jacobsen LB, Nicholas DE, McLaughlin JL. Brine shrimp: a convenient general bioassay for active plant constituents. Planta Med 1982; 45: 31.

(4.) WHO Malaria Fact Sheet No. 94. Available from: http:// www.who.ch/1998.

(5.) Zebitz CPW. Effects of three neem seed kernel extracts and azadirachtin on larvae of different mosquito species. J Appl Entomol 1986; 102: 455-63.

(6.) Joseph CC, Ndoile MM, Malima RC, Nkunya MHH. Larvicidal and mosquitocidal extracts, a coumarin, isoflavonoids and pterocarpans from Neorautanenia mitis. Trans R Soc Trop Med Hyg 2004; 98: 451-5.

(7.) Fontana JD, Lancas FM, Passos M, Cappelaro E, Vilegas J, Baron M, Noseda M, Pamilio AB, Vitale A, Webber AC, Maul AA, Peres WA, Foerster LA. Selective polarity and adsorption-guided extraction/purification of Annona sp polar acetogenins and biological assay against agricultural pests. Appl Biochem Biotechnol 1998; 70: 67-75.

(8.) Zafra-Polo M, Gonzalez MC, Estrornell E, Sahfaz S, Cortes D. Acetogenins from Annonaceae, inhibitors of mitochondrial complex I. Phytochemistry 1996; 42: 253-71.

(9.) Achenbach H, Hohn M, Waibel R, Nkunya, MHH, Jonker SA, Muhie S. Oxygenated pyrenes, their potential biosynthetic precursor and benzylated dihydroflavones from two African Uvaria species. Phytochemistry 1997; 44: 359-64.

(10.) Nkunya MHH, Jonker SA, Mdee LK, Waibel R, Achenbach H. New diprenylated indoles from Asteranthe asterias. Natl Prod Lett 1996; 9: 71-8.

(11.) Mohammed I, Waterman PG. Chemistry in the Annonaceae, XVII. Phenylpropenes from Uvariodendron connives seeds. J Nat Prod 1985; 48: 328.

(12.) Innocent E. Antimosquito terpenoids and other constituents of selected Tanzanian plants. Ph.D. Thesis. Dar es Salaam: University of Dar es Salaam 2007.

(13.) Nkunya MHH, Jonker SA, Makangara JJ, Waibel R, Achenbach H. Aporphinoid alkaloids and other constituents from Lettowianthus stellatus. Phytochemistry 2000; 53: 1067-73.

(14.) Toong YC, Schooley DA, Baker FC. Isolation of insect juvenile hormone III from a plant. Nature 1988; 333: 170-1.

(15.) Kihampa C. Novel quinonoids and other natural products from three mosquitocidal plant species. M.Sc. Thesis. Dar es Salaam, Tanzania: University of Dar es Salaam 2002.

(16.) Ngadjui BT, Ayafor JF, Sondengam BL, Connolly JD, Rycroft DS. Hoslundin, hoslundal, and hoslunddiol: three new flavonoids from the twigs of Hoslundia opposita (Lamiaceae). Tetrahedron 1991; 47: 3555-64.

(17.) Achenbach H, Waibel R, Nkunya MHH, Weenen H. Antimalarial compounds from Hoslundia opposita. Phytochemistry 1992; 31: 3781-4.

(18.) Krebs HC, Ramiarantsoa H. Phytochemistry 1997; 40: 931.

(19.) McChesney JD, Clark AM. Antimicrobial diterpenes of Croton sunderianus: 1-Hardwickic and 3,4-secotrachylobonic acids. J Nat Prod 1991; 54: 1625-33.

Corresponding author: Cosam C. Joseph, Department of Chemistry, University of Dar es Salaam, P.O. Box 35061, Dar es Salaam, Tanzania.

E-mail: Cosam@chem.udsm.ac.tz

Received: 18 October 2008

Accepted in revised form: 4 April 2009

Charles Kihampa (a,d), Cosam C. Joseph (b), Mayunga H.H. Nkunya (b), Stephen M. Magesa (c), Ahmed Hassanali (d), Matthias Heydenreich (e) & Erich Kleinpeter (e)

(a) Department of Environmental Science and Management, Ardhi University, Dar es Salaam; (b) Department of Chemistry, University of Dar es Salaam, Dar es Salaam; (c) National Institute for Medical Research, Amani Research Centre, Muheza, Tanzania; (d) Behavioural and Chemical Ecology Department (BCED), International Centre for Insect Physiology and Ecology, Nairobi, Kenya; (e) Institut fur Chemie, Universitat Potsdam, Potsdam, Germany
Table 1. Investigated plant species

Name of Criteria for
the plant Family Parts Locality selection

Uvaria AN SB, RB Selous Game Family
lungonyana Reserve activity *,
Vollesen Endemic plant
 species **

U. scheffleri AN SB, RB Maramba, Family
Diels. Muheza activity *
 district

U. faulknerae AN SB, RB Handeni, -do-
Verde Pangani
 district

U. leptocladon AN SB Korogwe -do-
Oliv. district

U. kirkii AN SB, RB UDSM Main -do-
Hook. F. campus

Uvariodendron AN SB, RB Amani Nature Family
usambaranse Reserve activity *
Diels.

U. pycnophyllum AN SB, RB Amani Nature Family
(Diels) R.E Fr. Reserve activity *,
 Endemic plant
 species **

Lettowianthus AN SB UDSM Main Family
stellatus Diels. campus activity *,
 (Planted) Literature
 reports

Annona AN SB, RB Kibanda -do-
squamosa L. village,
 Muheza
 district

Polyalthia AN RB Kichi hills, -do-
tanganyikensis Utete, Rufiji
Vollesen district

Asteranthe AN LS, SB Chalinze, Literature
asterias Coast reports
(S. Moore) region
Engl. & Diels.

A. lutea AN RB, SM Kwamngumi, Literature
Vollesen Muheza reports
 district

Tessmannia LG SB, RB Selous Game Family
densiflora Harms Reserve activity,
 Endemic plant
 species **

T. martiniana LG SB, RB Pugu forest, -do-
var pauloi Coast region
Harms

T. martiniana LG SB, RB Zaraninge -do-
var martiniana Forest
Harms Reserve

Croton EB SB, RB Selous Game Family and
sylvaticus L Reserve genus
 activity

Hoslundia LM LS, RB UDSM Main Ethnobotanical
opposita Vahl campus information

* The family Annonaceae is known to have compounds with
pharmacological, insecticidal, antimicrobial and
antiprotozoal activities; ** The plant is endemic to
Tanzania and neither chemical nor biological investigations
have been carried out; AN-Annonaceae; LG-Leguminosae;
EB-Euphorbiaceae; LM-Lamiaceae; SB-Stem Bark; RB-Root Bark;
LS-Leaves; UDSM-University of Dar es Salaam.

Table 2. Activity of crude extracts against III/IV instar
larvae of Anopheles gambiae s.s. Giles after
24 h exposure (ppm)
 PE
Botanical Plant
name part [LC.sub.50] 95% CL

Uvaria SB ne ne
 lungonyana RB ne ne
U. scheffleri SB 130 84-189
 RB 209 135-339
U. faulknerae SB 162 109-239
 RB 27 17-46
U. leptocladon SB 153 98-228
U. kirkii SB 48 34-66
 RB 76 51-113
Uvariadendron SB 188 137-262
 usambaranse RB 439 334-707
U. pycnophyllum SB ne ne
 RB ne ne
Lettowianthus SB 93 65-127
 stellatus SB 50 38-67
 Annona RB 44 29-66
 squamosa
Polyalthia RB 96 69-132
 tanganyikensis
Asteranthe lutea RB 59 32-95
 SB 335 246-553
A. asterias LS 444 319-898
 SB 238 150--349
Tessmannia SB ne ne
 densiflora RB ne ne
T. martiniana SB ne ne
 var pauloi RB ne ne
T. martiniana SB ne ne
 var martiniana RB ne ne
Croton sylvaticus SB 246 195-372
 RB 110 76-157
Hoslundia LS 171 6-288
 opposita RB 375 276-583

 CH ME
Botanical
name LC50 95%CL [LC.sub.50] 95%CL

Uvaria 245 185-337 373 260-781
 lungonyana 93 22-155 161 116-227
U. scheffleri 224 150-355 250 153-545
 363 243-880 164 104-252
U. faulknerae 33 23-48 82 61-111
 24 17-34 165 122-222
U. leptocladon 88 26-142 393 263-1085
U. kirkii 52 38-75 70 52-97
 95 64-139 129 83-209
Uvariadendron 188 29-318 357 258-554
 usambaranse 150 106-207 494 394-775
U. pycnophyllum 56 34-87 109 66-192
 56 34-86 56 35-86
Lettowianthus 256 149-708 355 265-500
 stellatus 17 9-25 24 10-40
 Annona 13 8-18 21 38-48
 squamosa
Polyalthia 133 90-199 70 50-100
 tanganyikensis
Asteranthe lutea 326 214-616 488 334-707
 212 138-342 582 482-802
A. asterias 267 186-384 494 394-775
 439 334--707 294 220-405
Tessmannia 104 60-150 192 130-285
 densiflora 162 113-232 383 243-402
T. martiniana 83 40-120 122 82-173
 var pauloi ne ne 114 44-186
T. martiniana 256 149-708 353 213-402
 var martiniana 204 133-340 148 74-254
Croton sylvaticus 232 170-342 238 184-354
 163 117-232 164 115-239
Hoslundia 369 257-659 191 69-301
 opposita 439 334-707 368 276-537

SB-Stem bark; RB-Root bark; LS-Leaves; PE-Pet ether;
CH-Chloroform; ME-Methanol; ne-Not extracted.

Table 3. Larvicidal activity of the compounds against III
and/or IV instars larvae of An. gambiae s.s. Giles
after 24 and 48 h exposure

Plant Duration Part/ [LC.sub.50]
source Compound (h) Extract (ppm) 95% CL

Annona 1 24 RC 61 29-95
squamosa 48 20 5-33
 2 48 RC 173 123-247
 72 120 84-189
Uvaria 3 24 RC 393 263-1085
lungonyana 48 150 83-242
 4+5 24 RC 122 44-209
 48 50 38-67
 6 24 RC 80 52-191
 48 21 14-33
Asteranthe 12 24 RC 2.3
lutea 48 0.5
 13 24 77.5
 48 33.8

RC--Root bark chloroform extract; CL-Class limits.
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Article Details
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Author:Kihampa, Charles; Joseph, Cosam C.; Nkunya, Mayunga H.H.; Magesa, Stephen M.; Hassanali, Ahmed; Heyd
Publication:Journal of Vector Borne Diseases
Article Type:Report
Geographic Code:6TANZ
Date:Jun 1, 2009
Words:3653
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