Acaricidal and Cytotoxic Activities of Extracts from Selected Genera of Australian Lamiaceae

HORTICULTURAL ENTOMOLOGY

Acaricidal and Cytotoxic Activities of Extracts from Selected Genera of Australian Lamiaceae HEIDI L. RASIKARI,1 DAVID N. LEACH,1 PETER G. WATERMAN,1 ROBERT N. SPOONER-HART,2 ALBERT H. BASTA,2 LINDA K. BANBURY,1 AND PAUL I. FORSTER3

J. Econ. Entomol. 98(4): 1259Ð1266 (2005)

ABSTRACT Crude foliar extracts of 67 species from six subfamilies of Australian Lamiaceae were screened by whole organism contact toxicity on the polyphagous mite Tetranychus urticae Koch (Acari: Tetranychidae) by using a Potter precision spray tower. Cytotoxicity assessments against insect cell lines from Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) and Drosophila melanogaster (Meigen) (Diptera: Drosophilidae) also were made. The Spodoptera cell line was more susceptible to extracts than the Drosophila cell line. No direct correlation was observed between the two screening methods, but several interesting relationships were identiÞed. Extracts from subfamilies Ajugoideae, Scutellarioideae, Chloanthoideae, Viticoideae and Nepetoideae showed acaricidal activity, whereas only those from Ajugoideae and Nepetoideae displayed potent cytotoxic effects. A range of activities was observed for the 25 species of Plectranthus, 14 of which showed moderate-to-high contact toxicity against T. urticae. Overall, the lowest toxicity was observed for extracts from the plant subfamily Prostantheroideae, which showed little contact toxicity or cytotoxicity for the 18 extracts studied. KEY WORDS Tetranychus urticae, insect cell lines, Plectranthus, Glossocarya

PLANTS CONTAIN NUMEROUS secondary metabolites to deter attack from insect and generalist herbivores (Harborne 1988). The Lamiaceae or mint family is a large cosmopolitan family that comprises an estimated 250 genera with in excess of 7,000 species and has been cited as a potential source of insecticidal compounds (Sharma et al. 1992, Dev and Koul 1997). A review of the chemical characterization of species in the family Lamiaceae has revealed a range of chemical components, predominantly mono- and diterpenoids, of which a number possess a range of activities against numerous arthropods (Cole 1992, Simmonds and Blaney 1992). To date, there are few phytochemical reports that evaluate Australian ßora for insecticidal and acaricidal potential (de la Torre et al. 1997, Basta and Spooner-Hart 2002). Given that the Lamiaceae family is well distributed around the mainland states of Australia suggests that there is an untapped reserve of compounds with potential for antiarthropod activity. Several laboratory studies have highlighted the effectiveness of essential oils as alternatives to synthetic acaricides (Hay and Waterman 1993, Chiasson et al. 2001, Basta and Spooner-Hart 2002), in particular from Lamiaceae (Mansour et al. 1986). The modes of action 1 Centre for Phytochemistry and Pharmacology, Southern Cross University, Military Road Lismore, NSW Australia, 2780. 2 Centre for Horticulture and Plant Science, University of Western Sydney Hawkesbury campus, Richmond Road, Richmond, NSW Australia, 2753. 3 Queensland Herbarium, Environmental Protection Agency, Brisbane Botanic Gardens, Mt Coot-tha Rd., Toowong, Qld, Australia, 4066.

exhibited by C10 monoterpenes isolated from essential oils include fumigation (Dev and Koul 1997), genotoxicity (Franzios et al. 1997), and neurotoxicity, i.e., affecting the central nervous system of certain arthropods via interference with octopamine receptors (Enam 2001). In addition, highly reÞned horticultural mineral oils and plant oils also may act by blocking spiracles, thereby inhibiting gaseous exchange, or they may pass through the exoskeleton, dissolving fat bodies and destroying internal cellular structures (Taverner 2002). Diterpenes (C20 compounds) from Lamiaceae also exhibit a range of insecticidal effects across a spectrum of arthropods (Simmonds and Blaney 1992). Of particular importance are the antifeedant clerodane diterpenes, most notable in the genera Ajuga (Camps et al. 1987, Bremner et al. 1998, Ben Jannet et al. 2000), Scutellaria (Cole et al. 1990, Rodriguez et al. 1993), and Teucrium (Simmonds et al. 1989, Bruno et al. 1999). Also many Ajuga species contain ecdysone-like compounds or juvenile hormones that can disrupt or alter the normal growth processes of insects (Kubo et al. 1983, Camps and Coll 1993, Toledano 1998). Tetranychus urticae Koch, the twospotted spider mite, is an economically important pest worldwide (Helle and Sabelis 1985). In Australia, T. urticae is a key horticultural pest (Spooner-Hart 1989) and affects broad acre crops such as cotton (Redall et al. 2004), as well as small intensive commodities such as glasshouse roses (Nicetic et al. 2001). In the USA, T. urticae is a major economic pest of pome fruit orchards in the PaciÞc Northwest (Beers et al. 1998,

0022-0493/05/1259Ð1266$04.00/0 䉷 2005 Entomological Society of America

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Yang et al. 2001) and corn and sorghum crops in the Great Plains states (Yang et al. 2001, Bynum et al. 2004). Resistance to acaricides has been recorded in these areas of the United States as well as in Asia (Kim and Seo 2001), the United Kingdom (Skirvin et al. 2002), and Australia (Gough 1990), largely because of the reduced sensitivity of the target enzyme acetylcholinesterase (Stumpf et al. 2001). Other detoxiÞcation or target enzymes such as esterase, cytochrome P450-monooxygenase and glutathione S-transferase all play a role in metabolizing certain acaricides in T. urticae (Yang et al. 2001). Because resistance to synthetic means of control increases, the search for novel control agents and methods is paramount for future crop protection. Laboratory evaluation of arthropod pest species is dependent on many intrinsic and extrinsic factors for meaningful results. To obtain repeatable results, signiÞcant quantities of the test product as well as a large number of test organisms must be readily available. In addition, the test species also require stringent standardization in accordance to age, size, stage, and sex as well as experimental conditions such as temperature, illumination, and food supply because these factors inßuence product efÞcacy and detoxiÞcation (Busvine 1972). The use of insect cell lines, as a substitute for whole insect studies to minimize variability while determining efÞcacy with a relatively small amount of test product, is an area that has to date been largely overlooked (Mendki et al. 2001). In this article, we report the acaricidal effects of 67 endemic species of Lamiaceae against T. urticae. We further describe the use of an ATP luminescence cell-based proliferation assay (Cree and Androtti 1997) against two insect cell lines to assess for cytotoxicity and evaluate their use as a tool to screen for potential lead compounds. Materials and Methods Extraction Methods. Aerial plant material (leaves and stems) was collected from the Royal Botanic Gardens, Sydney, located at Mt. Annan (new South Wales; NSW) and the Brisbane Botanic Gardens, Mt Coot-tha (Queensland; QLD). Additional Þeld collections of many Plectranthus spp. were made in northern NSW and southern Queensland and vouchered specimens deposited at the Queensland Herbarium (BRI). Collection details and voucher numbers of all plants used in this study are provided in Table 1. Plant material was air-dried at 40⬚C, ground with a Waring blender, and cold extracted with AR grade methanol (Merck Pty. Ltd., Victoria, Australia) [1:4 (wt:vol) with sonication for 30 min, repeated three times], Þltered through a scintered glass funnel, and evaporated in vacuo (⬍40⬚C). Samples from Western Australian ßora were obtained from the commercial plant extract library of BioProspect Ltd. (Brisbane, QLD, Australia), which were routinely extracted with ethyl acetate [1:10 (wt:vol)] for 24 h, and rinsed with more solvent (20% of original volume) before being rotary evaporated (Buchi R220) to dryness.

Vol. 98, no. 4

Acute Toxicity Screening Bioassay. The extracts were initially dissolved in the minimum of UNIVAR (Asia PaciÞc Specialty Chemicals Ltd., NSW, Australia) methanol (99.8% purity) or ethyl acetate (99.5% purity). The concentration of extract was then adjusted to 1.0% (wt:vol) by using a preprepared solution (200 ppm) of surfactant Triton X-100 (octyphenol ethylene oxide condensate, Union Carbide, Sigma, St. Louis, MO) in distilled water to give a stock solution. Young adult (24 Ð 48 h) female twospotted spider mites were chosen for bioassay. They were collected from a mass culture of an organophosphate-susceptible colony maintained at the University of Western SydneyÕs Hawkesbury campus (BeneÞcial Bug Company Pty. Ltd., Richmond, NSW, Australia) and reared on insecticide free potted French bean, Phaseolus vulgaris L., seedlings in a glasshouse under conditions of 25 ⫾ 5⬚C, 65 ⫾ 5% RH, and a photoperiod of 14:10 (L:D) h. Mites (n ⫽ 20 mites, four replicates) of uniform size were randomly selected then transferred to French bean leaf discs (25 mm in diameter) mounted with the adaxial side uppermost on moistened cotton wool in petri dishes (90 by 15 mm). The test organisms on the food substrate were sprayed (5-ml aliquot) at two concentrations [1.0 and 0.5% (wt:vol)] as well as with a control blank comprising 10% (vol:vol) of solvent in Triton X-100 solution, by a Potter precision spray tower (Burkard, Rickmansworth, Herts, United Kingdom) at a pressure of 18.5 psi. The extract residue, when formulated to a 1.0% (wt:vol) solution, was calculated to be 57.5 ⫾ 2.8 ␮g/cm2 of product. After spraying, petri dishes containing mites were maintained at room temperature (23 ⫾ 5⬚C and a photoperiod of 14:10 [L:D] h). Mites were included in the mortality count (24 h posttreatment) if appendages did not move when prodded with a Þne artistÕs brush. Cell Culture Maintenance. Two cell lines were used in this study to test for differences in cell line response. The Spodoptera frugiperda (Sf9) cell line was obtained from Invitrogen (Mulgrave, Victoria, Australia) (catalog no. 11496) and was derived from pupal ovarian tissue. The semiadherent culture was maintained in serum-free Sf-900 II SFM (Invitrogen) media at 24⬚C on an orbital shaker at 50 rpm. A weekly subculture split ratio of ⬇1:10 was required to maintain line continuity. American Type Culture Collection (Manassas, VA) provided embryonic Drosophila melanogaster (SchneiderÕs D.mel-II) cell line (catalog no. CRL-1963). Using 90% SchneiderÕs Drosophila medium and 10% heat-inactivated fetal bovine serum (Invitrogen), this suspension culture was grown under the same conditions as described for Sf9 but with twice-weekly subcultures (1:10), as recommended by American Type Culture Collection. Cytotoxicity. The assessment of cytotoxic activity of Lamiaceae extracts against low passage number (⬍25) of Sf9 and D.mel-II cultures in vitro was carried out using the ATPLite-M luminescence kit assay (PerkinElmer, Rowville, Victoria, Australia). All extracts were dissolved in dimethyl sulfoxide (DMSO) and screened at 100 and 10 ␮g/ml Þnal concentration. Insect cells were seeded at

August 2005 Table 1.

RASIKARI ET AL.: BIOACTIVITY OF AUSTRALIAN LAMIACEAE

1261

Voucher specimen reference information Plant name

Area collected

Date collected

Voucher no.

Ajuga australis R. Br. Callicarpa pedunculata R. Br. Ceratanthus longicornis F. Muell. Clerodendrum floribundum R. Br. Clerodendrum inerme (L.) Gaertn. Clerodendrum tomentosum (Vent.) R. Br. Clerodendrum traceyi F. Muell. Faradaya albertissii F. Muell. Faradaya splendida F. Muell. Glossocarya calcicola Domin. Glossocarya hemiderma Benth. Gmelina leichardtii (F. Muell.) Benth. Hemiandra australis B.J. Conn Hemiandra leiantha Benth. Hemiandra pungens R. Br. Hemigenia humilis Benth. Hemigenia sericea Benth. Hemigenia westringioides Benth. Lachnostachys eriobotrya (F. Muell.) Druce Lycopus australis R. Br. Microcorys capitata (Bartl.) Benth. Microcorys sp. ÔStellateÕ Pityrodia bartlingii (Lehm.) Benth. Pityrodia verbascina (F. Muell.) Benth. Plectranthus actites P.I. Forst. Plectranthus alloplectus S.T. Blake Plectranthus amoenus P.I. Forst. Plectranthus argentatus S.T. Blake Plectranthus apreptus S.T. Blake Plectranthus cremnus B.J. Conn Plectranthus diversus S.T. Blake Plectranthus fasciculatus P.I. Forst. Plectranthus foetidus Benth. Plectranthus glabriflorus P.I. Forst. Plectranthus gratus S.T. Blake Plectranthus graveolens R. Br. Plectranthus habrophyllus P.I. Forst. Plectranthus leiperi P.I. Forst. Plectranthus mirus S.T. Blake Plectranthus nitidus P.I. Forst. Plectranthus omissus P.I. Forst. Plectranthus parviflorus Willd. Plectranthus sp. ÔBuchanans FortÕ Plectranthus sp. ÔPinnacleÕ Plectranthus sp. Hann Tableland Plectranthus sp. ÔKoonyum RangeÕ Plectranthus scutellarioides (L.) R. Br. Plectranthus spectabilis S.T. Blake Plectranthus suaveolens S.T. Blake Premna serratifolia L. Premna acuminata R. Br. Prostanthera incisa Benth. Prostanthera lasianthos Labill. Prostanthera nivea A. Cunn. ex Benth. Prostanthera rotundifolia R. Br. Prostanthera spinosa F. Muell. Prostanthera stricta R.T. Baker Scutellaria mollis R. Br. Teucrium racemosum R. Br. Teucrium sp. ÔMount AnnanÕ Teucrium sp. ÔOrmeauÕ Vitex lignum vitae Schauer Viticipremna queenslandica Munir Westringia eremicola A. Cunn. ex Benth. Westringia glabra R. Br. Westringia saxatilis B.J. Conn Westringia viminalis B.J. Conn & Tozer

MA Botanic Garden Nimbin, NSW QLD Nimbin, NSW MA Botanic Garden Kyogle, NSW MA Botanic Garden MA Botanic Garden MA Botanic Garden QLD QLD Nimbin, NSW MA Botanic Garden WA WA WA WA MA Botanic Garden WA QLD MA Botanic Garden WA WA WA QLD Nimbin, NSW QLD MA Botanic Garden QLD Byron Bay, NSW QLD QLD QLD QLD QLD MA Botanic Garden Upper Ormeau, QLD Diannas Bath, QLD QLD Nimbin, NSW West Gympie, QLD Nimbin, NSW Buchanans Fort, QLD The Pinnacle, NSW MC Botanic Garden Koonyum Range, NSW QLD QLD Boonoo Falls, QLD QLD QLD Nimbin, NSW MA Botanic Garden MA Botanic Garden MA Botanic Garden MA Botanic Garden MA Botanic Garden MA Botanic Garden MA Botanic Garden MA Botanic Garden Upper Ormeau, QLD Nimbin, NSW QLD MA Botanic Garden MA Botanic Garden MA Botanic Garden MA Botanic Garden

22/6/01 17/9/01 /2/02 17/9/01 25/3/02 21/10/03 /4/02 25/3/02 25/3/03 /2/02 /8/03 17/9/01 26/6/01 /1/02 /9/01 /9/01 /9/01 26/6/01 /1/02 /1/02 26/6/01 /1/02 /1/02 /1/02 /2/03 17/9/01 /2/02 26/6/01 /5/02 17/9/01 /2/03 /5/02 /2/02 /2/02 /5/03 26/6/01 15/2/03 15/2/03 /2/02 10/6/01 15/2/03 10/6/01 15/2/03 17/9/01 4/7/01 25/1/02 /2/02 /5/03 25/1/02 /4/02 /7/03 10/6/01 26/6/01 26/6/01 26/6/01 26/6/01 26/6/01 26/6/01 26/6/01 25/3/02 15/2/03 17/9/01 /8/03 26/6/01 26/6/01 26/6/01 26/6/01

# 942707 HLR AP01Ð1021 (BRI) PIF 28312 (BRI) HLR AP01Ð1020 (BRI) # 865572 HLR AP01Ð1088 (BRI) # L2994 # 862995 # 904314 PIF 28260 (BRI) PIF 29177 (BRI) HLR AP01Ð1015 (BRI) # 933150 BPL-612 BPL-107 CP-378 CP-329 # 893054 BP-555 PIF 28077 (BRI) # 893108 BPL-224 BPL-259 BPL-1071 PIF 7255 (BRI) HLR AP01Ð1014 (BRI) PIF 8366 (BRI) # 900151 RB 2958 (BRI) HLR AP01Ð1018 (BRI) PIF 12728 (BRI) RB 3077 (BRI) PIF 28252 (BRI) PIF 12184 (BRI) RB 2967 (BRI) # 820019 HLR CP03Ð107 (BRI) HLR CP03Ð105 (BRI) RB 2889 (BRI) AP01Ð907 (BRI) HLR CP03Ð111 (BRI) HLR AP01Ð908 (BRI) HLR CP03Ð106 (BRI) HLR AP01Ð1024 (BRI) RB 2663 (BRI) HLR AP02Ð110 (BRI) RB 2884 (BRI) RB 3089 (BRI) HLR AP02Ð108 (BRI) RB 3105 (BRI) PIF 29568 (BRI) HLR AP01Ð910 (BRI) # 840765 # 922939 # 865081 # 922950 # 873122 # 20000650 # 980216 # 990361 HLR CP03Ð110 (BRI) HLR AP01Ð1023 (BRI) PIF 28152 (BRI) # 922946 # 922933 # 823861 # 920521

WA, Western Australia; MA, Mount Annan.

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JOURNAL OF ECONOMIC ENTOMOLOGY

⬇2 ⫻ 105 cells/ml (99 ␮l) into a 96-well plate (Nunc, Mt. Waverly, Victoria, Australia) before the addition (1 ␮l) of a test agent or control blank (DMSO). After incubation at 24⬚C for 24 h, the cells were lysed to release ATP, a nucleotide marker that directly relates to the number of living cells (Crouch et al. 1993), followed by the addition of a substrate solution containing luciferase and D-luciferin. The resulting emission of light is proportional to the ATP concentration and was measured by a Victor2 1420 multilabel counter (PerkinElmer Wallac, Turku, Finland). The inhibition of ATP and thus cell proliferation was expressed as a percentage of the DMSO blank and compared with the toxic effects of test agents chlorambucil (Sigma, Castle Hill, NSW, Australia) and rotenone (Sigma, Castle Hill, NSW, Australia) at 100 and 0.001 ␮g/ml, respectively. Data Analysis. Statistical analyses for efÞcacious samples were conducted using SPSS for Windows version 10. The Probit Analysis procedure in the SPSS package corrected for control mortality and obtained maximum-likelihood estimates of regression coefÞcients. If the Pearson goodness-of-Þt ␹2 test was signiÞcant (P ⬍ 0.05), a heterogeneity factor was used to calculate the lower and upper conÞdence limits (95% CL). The data from each treatment were plotted as probit-mortality against log dose (Busvine 1972) and the corresponding lethal concentration (LC50 and LC95) and inhibition concentration (IC50) values were calculated. Results and Discussion Acaricidal Activity. In the current investigations, we report acute contact toxicity (i.e., mortality within 24 h) associated with plant extracts delivered via a Potter tower. Aqueous formulations (5-ml aliquots) were delivered at concentrations between 0.25 and 1.0% (wt:vol). Phytotoxicity caused by some extracts was observed at concentrations above the higher rate and therefore not all were suitable for obtaining lethal concentration data. Approximately 58% of the extracts elicited a positive response at 1.0% (wt:vol) (Table 2) and 25% exhibited an appreciable level of acaricidal activity at 0.5% (wt:vol) screening concentrations (Table 3). Notably, 100% mortality was achieved after topical application of extracts [formulated to 1.0% (wt:vol)] of Clerodendrum traceyi, Ceratanthus longicornis, Plectranthus habrophyllus, Plectranthus sp. ÔHann TablelandÕ, and Premna serratifolia. Table 3 details results from Probit analysis of these and other efÞcacious extracts, provided sufÞcient material was available to conduct adequate doseÐresponse experiments. Promising acaricidal activity was obtained from subfamilies Ajugoideae, Viticoideae, Scutellarioideae, Chloanthoideae, and Nepetoideae, all of which showed a degree of acaricidal activity. No acaricidal activity was found for 14 of 18 representatives of the Prostantheroideae subfamily, and only minimal activity was observed from the remaining four species (Prostanthera lasianthos, Hemiandra australis, H. pungens, and Hemigenia humilis) was observed.

Vol. 98, no. 4

A range of acaricidal activity was observed for the 25 species of Plectranthus assessed in this study, with 64% showing a positive response compared with the control, 8% of which resulted in high mortality (⬎90%) at 1.0% spray rates. The genus Plectranthus is known to be rich in essential oils containing numerous volatile C10 monoterpenes (Abdel-Mogib et al. 2002), including terpinolene, fenchone, piperitenone oxide (Ngassoum et al. 2001), ␣-pinine, eremophilene, myrcene, and humulene (Kerntopf et al. 2002). These compounds have shown toxic effects via numerous modes of action against a range of arthropods (Dev and Koul 1997). In addition, oil-based sprays (botanical or petroleum derived) can have a behavioral effect (Basta and Spooner-Hart 2002) or a smothering effect (Taverner 2002) on mites, resulting in death by starvation, growth disruption, or asphyxiation. Diterpenes such as abietanes are abundant in Plectranthus, including royleanones, spirocoleons, and quinones (Abdel-Mogib et al. 2002), and have been associated with antibacterial activity (Rabe and van Staden 1998) and strong inhibitory effects on human carcinoma cell lines (Marquez et al. 2002), but they have shown little insecticidal activity. Research detailing the insecticidal effects of Plectranthus, in particular antifeeding activity against the aphid Schizaphis graminum (Rondani) has been observed from the abietane plectrin, isolated from East African species P. barbatus (Andrew) Benth (Kubo et al. 1984). Of interest, the exotic species Plectranthus ornatus Codd has yielded a novel clerodane derivative (Rijo et al. 2002), a class of diterpenes not previously found within this genus and often associated with insect antifeeding activity in other Lamiaceae, notably from the subfamily Ajugoideae (Camps et al. 1987). The possible synergistic/antagonistic effect and different mode(s) of action between mono- and diterpene compounds present in Plectranthus extracts is an area that needs to be considered in interpreting the results. Although the mode(s) of action against T. urticae cannot be ascertained at this stage, this study provides evidence that extracts of numerous Plectranthus species have an acaricidal effect on mites under laboratory conditions, and that further research into antiarthropod activities of Plectranthus would be worthwhile. Cell Line Differences. The two cell lines used in this study differ in insect order, tissue origin, and growth rates. It is also likely that the extracts screened would exhibit different modes of action at the cellular level. The positive control rotenone, a modiÞed isoßavonoid derived from the roots of Derris spp. (Fabaceae), is a respiration inhibitor ultimately leading to cell death and is extremely toxic to insects and Þsh (Gusmao et al. 2002). In this study, it is not surprising that the potent effects of rotenone were observed against both cell lines in the range of 0.1Ð 10.0 ng/␮l in the ATP-Lite bioassay. From initial screening of insect cell line responses to plant extracts, 100% of all extracts were toxic to Sf9 cells and 93% of extracts were toxic to D.mel-II cells when tested at the highest screening concentration of

August 2005 Table 2.

1263

Comparison of the toxicity of Lamiaceae extracts on adult T. urticae against Spodoptera and Drosophila cell lines

Subfamily Ajugoideae

Scutellarioideae Viticoideae

Chloanthoideae Prostantheroideae

Nepetoideae

RASIKARI ET AL.: BIOACTIVITY OF AUSTRALIAN LAMIACEAE

Plant species

T. urticaea

Sf9b

D.mel-IIb

Ajuga australis Clerodendrum floribundum Clerodendrum inerme Clerodendrum tomentosum Clerodendrum traceyi Glossocarya calcicola Glossocarya hemiderma Teucrium racemosum Teucrium sp. Teucrium sp. ÔOrmeauÕ Scutellaria mollis Callicarpa pedunculata Faradaya albertissii Faradaya splendida Gmelina leichardtii Premna serratifolia Premna acuminata Vitex lignum-vitae Viticipremna queenslandica Lachnostachys eriobotrya Pityrodia bartlingii Pityrodia verbascina Hemiandra australis Hemiandra leiantha Hemiandra pungens Hemigenia humilis Hemigenia sericea Hemigenia westringioides Microcorys capitata Microcorys sp. ÔstellateÕ Prostanthera incisa Prostanthera lasianthos Prostanthera nivea Prostanthera rotundifolia Prostanthera spinosa Prostanthera stricta Westringia eremicola Westringia glabra Westringia saxatilis Westringia viminalis Ceratanthus longicornis Lycopus australis Plectranthus actites Plectranthus alloplectus Plectranthus amoenus Plectranthus argentatus Plectranthus apreptus Plectranthus cremnus Plectranthus diversus Plectranthus fasciculatus Plectranthus foetidus Plectranthus glabriflorus Plectranthus gratus Plectranthus graveoloens Plectranthus habrophyllus Plectranthus leiperi Plectranthus mirus Plectranthus nitidus Plectranthus omissus Plectranthus parviflorus Plectranthus sp. Buchanans Fort Plectranthus sp. Pinnacle Plectranthus sp. Hann Tableland Plectranthus sp. Koonyum Range Plectranthus scutellarioides Plectranthus spectabilis Plectranthus suaveolens

⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹⫹⫹ ⫺ ⫹ ⫹ NT NT ⫹⫹ ⫹ ⫺ ⫹⫹ ⫹⫹⫹ ⫹⫹⫹⫹ ⫹⫹⫹ ⫹⫹ ⫹⫹⫹ ⫺ ⫹⫹ ⫺ ⫹ ⫺ ⫹ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹⫹⫹⫹ ⫹ ⫹⫹ ⫹ ⫺ ⫺ ⫺ ⫹⫹ ⫹⫹⫹ ⫺ ⫺ ⫹⫹⫹ ⫺ ⫹⫹ ⫹⫹⫹⫹ ⫹⫹ ⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫺ ⫺ ⫹⫹⫹⫹ ⫹⫹ ⫹⫹ ⫺ ⫹⫹⫹

⫹ ⫹⫹ ⫹⫹⫹ ⫹ NT ⫹⫹⫹ ⫺ ⫹ NT NT NT ⫹⫹⫹ ⫹⫹ ⫹⫹ ⫹ ⫹⫹ NT ⫹⫹⫹ NT ⫹⫹ ⫹⫹⫹ NT ⫹⫹ ⫹⫹ ⫹⫹⫹ ⫹⫹ ⫹ ⫹⫹ NT ⫹⫹ ⫹ ⫹⫹⫹ ⫺ ⫹⫹⫹ ⫹ ⫹⫹⫹ ⫺ ⫹⫹ ⫹⫹ ⫹⫹ NT ⫹ NT ⫹⫹ ⫹⫹⫹ ⫹⫹ ⫹⫹ NT NT ⫹⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹ ⫹⫹⫹ NT NT ⫹⫹ ⫹⫹ NT ⫹⫹ NT ⫹⫹⫹ ⫹⫹ ⫹⫹⫹ ⫹⫹ ⫹⫹⫹ ⫹⫹

⫹ ⫺ ⫺ ⫺ ⫺ ⫹⫹⫹ ⫺ NT ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫺ NT ⫺ NT ⫹⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫺ ⫺ ⫺ ⫺ ⫹ ⫺ ⫹ NT ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹⫹⫹ ⫺ ⫺ ⫺ ⫺ ⫹⫹⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫺ ⫺ ⫹⫹ ⫺ ⫺ ⫹ ⫺ ⫺ ⫹ ⫹ ⫺

⫺, ⬍20%; ⫹, 20 Ð 49%; ⫹⫹, 50 Ð 89%; ⫹⫹⫹, 90 Ð99%; ⫹⫹⫹⫹, 100% mortality or inhibition of cell growth. NT, not tested. a Concentration sprayed 1.0% (wt:vol). b Concentration tested 10 ␮g/ml.

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JOURNAL OF ECONOMIC ENTOMOLOGY

Vol. 98, no. 4

Relative efficacy of selected Lamiaceae extracts against T. urticae Extract

n

Slope ⫾ SE

LC50 (wt:vol) (95% CL)

LC95 (wt:vol) (95% CL)

␹2

df

P

Ajuga australis Ceratanthus longicornis Clerodendrum inerme Clerodendrum traceyi Pityrodia bartlingii Plectranthus actites Plectranthus cremnus Plectranthus diversus Plectranthus glabriflorus Plectranthus graveolens Plectranthus habrophyllus Plectranthus nitidus Plectranthus scutellarioides Plectranthus suaveolens Plectranthus sp. Hann Tableland Plectranthus sp. Koonyum Range Premna serratifolia Premna acuminata Viticipremna queenslandica

529 469 383 313 460 315 460 398 385 449 397 386 478 465 389 389 403 385 392

2.84 ⫾ 0.229 4.42 ⫾ 0.378 2.43 ⫾ 0.298 13.10 ⫾ 2.92 2.46 ⫾ 0.268 4.05 ⫾ 0.455 3.39 ⫾ 0.358 3.03 ⫾ 0.290 6.66 ⫾ 0.661 1.87 ⫾ 0.245 3.84 ⫾ 0.334 2.36 ⫾ 0.409 4.17 ⫾ 0.393 3.69 ⫾ 0.310 5.32 ⫾ 0.686 3.75 ⫾ 0.407 5.13 ⫾ 0.462 5.07 ⫾ 0.469 4.48 ⫾ 5.05

0.49 (0.36Ð0.66) 0.49 (0.37Ð0.70) 0.56 (0.43Ð0.72) 0.58 (0.41Ð0.83) 0.63 (0.46Ð0.87) 0.60 (0.42Ð0.87) 0.38 (0.28Ð0.52) 0.25 (0.18Ð0.36) 0.57 (0.42Ð0.78) 0.76 (0.57Ð1.0) 0.33 (0.25Ð0.51) 0.81 (0.59Ð1.1) 0.46 (0.36Ð0.60) 0.41 (0.30Ð0.58) 0.29 (0.29Ð0.48) 0.58 (0.42Ð0.79) 0.32 (0.23Ð0.43) 0.55 (0.38Ð0.80) 0.73 (0.52Ð1.0)

1.40 (1.0Ð2.3) 1.2 (0.80Ð2.1) 2.65 (1.75Ð4.7) 1.50 (1.1Ð2.4) 2.10 (1.5Ð3.2) 2.0 (1.40Ð3.2) 1.30 (0.92Ð1.9) 0.85 (0.59Ð1.3) 1.50 (1.0Ð2.7) 2.5 (1.8Ð3.7) 1.10 (0.71Ð1.5) 2.7 (1.9Ð4.1) 1.2 (0.87Ð1.7) 1.4 (1.0Ð2.1) 0.96 (0.71Ð1.5) 1.9 (1.4Ð2.1) 0.83 (0.60Ð1.3) 1.2 (0.81Ð2.17) 1.6 (1.1Ð3.7)

24.20 54.17 14.56 0.314 15.38 15.71 26.8 58.2 25.59 27.19 27.3 5.1 32.1 20.5 11.1 21.8 30.9 41.0 32

19 15 15 7 15 7 19 11 10 15 11 11 15 15 11 11 11 11 11

0.188 0.000 0.484 1.000 0.425 0.028 0.108 0.000 0.004 0.027 0.004 0.926 0.006 0.154 0.434 0.026 0.001 0.000 0.001

100 ␮g/ml (data not shown). At a reduced concentration of 10 ␮g/ml (Table 2), almost all plant extracts remained toxic to Sf9 cells (94%), but they had minimal effect on the D.mel-II line (22%). A similar susceptibility pattern was noted for these two cell lines when screened against Bacillus thuringiensis endotoxins (Kwa et al. 1998), but it is in contrast to results obtained from neem seed limonoids (Cohen et al. 1996). The results from our study provide evidence for concentration-response differences between the two cell lines, and indicate that overall, the Spodoptera cell line is more susceptible to these extracts than the Drosophila cell line. Cytotoxicity versus Acaricidal Effects. One of our aims in this study was to assess an insect cell-based bioassay to screen crude extracts for targeting lead compounds with antiarthropod activity. Previously, the use of a cell-based bioassay against the D. melanogaster (BII) line has been successfully demonstrated for the detection of antilepidopteran ecdysteroid compounds in plant extracts (Clement et al. 1993). Our analysis of 67 extracts against whole mite and insect cell lines highlighted a range of toxicity combination results for the two bioassay methods. However, no direct association (high cytotoxicity/high acaricidal effect) was observed for the 67 extracts presented here. A high acaricidal response coupled with a low cytotoxic response was observed for extracts of Ceratanthus longicornis [LC95 ⫽ 1.2% (0.80Ð2.1) (wt:vol)], Plectranthus sp. Hann Tableland [LC95 ⫽ 0.96% (0.71Ð1.5) (wt:vol)], and Premna serratifolia [LC95 ⫽ 0.83% (0.60Ð1.3) (wt:vol)]. Extracts from subfamily Ajugoideae, Glossocarya calcicola, and Nepetoideae, Plectranthus fasciculatus, were both highly toxic to both Sf9 and D.mel-II but exhibited low acaricidal activity. G. calcicola was the most cytotoxic extract studied against the resistant cell line D.mel-II (IC50 ⫽ 1.22 ␮g/ml), followed by P. fasciculatus (D.mel-II IC50 ⫽ 5.50 ␮g/ml). These extracts showed no adverse effect against T. urticae, which resumed feeding and oviposition several hours after the spray treatment. Details of the chemistry and

effects of these extracts against the diamondback moth Plutella xylostella (L.) are being published elsewhere. It is interesting to note the low acaricidal/low cytotoxic activity from 14/18 members of the subfamily Prostantheroideae (Table 2). This result is surprising because Prostanthera is reputed to contain essential oils that possess a range of mono- and sesquiterpenes (Southwell and Brophy 2000). In conclusion, to date very little is known regarding the distribution of insecticidal compounds in Australian Lamiaceae. From this study, we have found that crude foliar extracts of some previously unstudied Australian species have either acaricidal or cytotoxic effects and that the probability of uncovering some novel bioactive compounds from subfamilies Ajugoideae, Viticoideae, and Nepetoideae is high. Some Plectranthus species are often available in large quantities so that signiÞcant amounts of active compounds could readily be obtained for comprehensive studies, and they would seem to be ideal targets for future studies. It seems that botanical sources of pesticides will play an important role in future crop protection strategies in response to insect resistance, public health, and environmental concern (Rice et al. 1998). From an industrial perspective, botanical compounds need to be novel and subject to stringent and robust tests to provide reliable results on a given spectrum of pests, in a range of agronomic and climatic conditions (Mendki et al. 2001). As such, the evaluation of a potential lead product requires subjection to a range of bioassay procedures, Þeld trials, and toxicity studies, all of which have their own merits and demarcations. The number of active extracts (94%) observed for Sf9 cells was high compared with the active extracts (22%) observed for D.mel-II cells. In terms of the whole mite bioassay in which 58% of extracts were active, these two cell lines over- or underestimated the toxicity relative to the whole organism and were therefore not suitable for direct comparison. Unfortunately, we were unable to develop or obtain a commercial cell line culture for T. urticae for such pur-

August 2005

RASIKARI ET AL.: BIOACTIVITY OF AUSTRALIAN LAMIACEAE

poses. However, cell-based bioassays do have an important role in bioassay-guided fractionation alleviating the high cost, time, and material required to perform such work on whole organisms. In summary, this study demonstrated that cell-based bioassays have potential for assessing insecticidal leads; however, at this stage whole arthropod bioassay remains a preferred option. Acknowledgments We thank Glen Leiper (Beenleigh, QLD) and the late Barry Walker, Nimbin (NSW) for providing access to many local Plectranthus species. We appreciate the assistance with the Þeld collection of samples in Queensland that was provided by Ron Booth and Rigel Jensen We acknowledge Þnancial support for this work from BioProspect Ltd. and the Australian Research Council.

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