Biological Effects of Ecdysteroids and Their Non Steroidal Agonist Bisacylhydrazines A Review

RESHMA JOHN , RASHMI P.A , LINU MATHEW * School of Biosciences, Mahatma Gandhi University, Kottayam, Kerala Pin-686560 *Corresponding author email: linumathew@mgu.ac.in, mob: +919447505690

Abstract Ecdysteroids are polar steroid hormones controlling arthropod development and reproduction. Ecdysteroids in plants are called phytoecdysteroids, their concentration in plants are higher than that in insects. The isolation of phytoecdysteroids from plants has initiated fruitful research leading to the discovery of new phytoecdysteroids. Ecdysteroids possess a number of pharmacological, medicinal and agricultural applications. Ecdysteroids agonist and antagonists are potential insect control agents and valuable tool for studying ecdysteroid action. New agents to control insect pest species, interference with ecdysteroid action is an attractive, and little exploited target. The purpose of the review is to summarise the biological effects of ecdysteroids and its agonist and antagonists.

Key words: Ecdysteroids, agonist, antagonists, plant defences, bisacylhydrazines. Introduction Insect development is driven by the action of two hormone classes, the ecdysteroids and the juvenile hormones (Riddiford, 1994; Gilbert et al., 2000; Thummel, 2002) and they are primarily involved in the regulation of postembryonic development, which is characterized by moulting and metamorphosis. Ecdysteroids are produced in the prothoracic glands in larvae and in the reproductive organs in adults (ovaries and testis), and in the abdominal integument of some insects (Hoffmann & Gerstenlauer 1997). In the larval stages, 20-hydroxyecdysone initiates the moulting and metamorphosis process in larvae, and Juvenile hormone regulates these changes during the commitment period. Ecdysone, the first ecdysteroid was isolated by Butenandt and Karlson in 1954 (Dinan, 2001), and its structure

was identified by Huber and Hoppe (Hoppe & Huber 1965). Insects sporadically shed their rigid external exoskeleton and then replace it to grow. This process is called ecdysisis or molting (Gilbert, 2004). Insect molting is regulated via binding of the molting hormone (20hydroxyecdysone: 20E) to its receptor protein, the heterodimer of ecdysone receptor (EcR) and ultraspiracle (USP). The 20E-EcR/USP complex binds to the ecdysone response element in the promoter of target genes and regulates their transcription. The molting process is initiated by an increase in the titer of 20E, the larva stops feeding and apolysis of the epidermis from the old cuticle takes place leaving an ecdysial space that is filled with molting fluid containing inactive chitinolytic enzymes. Meanwhile massive protein synthesis takes place in the epidermal cells for deposition of a new cuticle (Riddiford, 1994; Dhadialla et al.,1998). Decline in the titer of 20E leads to the activation of enzymes for the digestion of procuticle underlying the old cuticle. After completion of this process moulting fluid is reabsorbed and pre ecdysal tanning of the new cuticle takes place (Reynolds, 1987). Finally, eclosion hormone and ecdysis-triggering hormone are released, when 20E titer is declined to the basel level and these events lead to the ecdysis of the larva leaving behind the remnants of the old cuticle (Truman et al., 1983; Dhadialla et al.,1998; Zitnanova et al., 2001). With the completion of ecdysis, feeding resumes and endocuticular deposition continues during the intermolt period. Every moult corresponds to the end of one growth stage and and the beginning of another (Fig 1).

Fig 1 The ecdysteroid titer in Drosophila development (Kozlova & Thummel , 2000) Phytoecdysteroids Phytoecdysteroids are analogues of invertebrate steroid hormones (zooecdysteroids) that occur in a wide variety of plant species (Bergamasco & Horn, 1983) (Table 1). Within a few years of the isolation of ecdysone from insects, an array of compounds having structural similarity to insect moulting hormones has been extracted from plants and because of plant origin these compounds are called phytoecdysteroids. Subsequent research has revealed that phytoecdysteroid occurrence is widespread (Dinan, 2004a) and their concentration in plants is higher than that in insects. It appears that 56% of terrestrial plant species contain significant levels of ecdysteroids ( Dinan, 2001). Most crop species do not contain detectable levels of ecdysteroids, spinach (Spinacia oleracea) and quinoa (Chenopodium quinoa) being notable exceptions (Bthory et al.,1982; Grebenok et al.,1991;Dinan, 1995,Dinan ,2001). A range of evidence exists in support that ecdysteroid concentrations are highest in tissues

which are most important for the survival of the plant or, in the case of annuals, of the species

into the next generation (Dinan,2001).The concentration of phytoecdysteroids in plants are increased by mechanical damage,insect herbivory or methyljasmonate treatment(Schmelz et al.,1998,1999,2002). Phytoecdysteroids are apparently non toxic to mammals and have a number of beneficial and pharmacological and medicinal applications (Dinan, 2001; Slama & Lafont, 1995) which explain the usage of ecdysteroids containing plants in medicine. These plant species are primarily represented by Leuzea carthamoides, Rhaponticum uniflorum, and Serratula coronate, Cyanotis vaga, Ajuga turkestanica, Pfaffia paniculata, P.

iresinoides(Lafont & Dinan ,2003).

Table 1 Medicinal plants containing phytoecdysteroids Plant species Family Parts containing phytoecdysteroids Achyranthes fauriei Amaranthaceae Roots 0.41.2mg/gdw Ogawa et al., 1974 Concentration Reference

Ajuga iva

Laminaceae

Aerial parts

4.5 mg/g dw

Wessner et al., 1992

Cyanotis vaga

Commelinaceae

Leaves

7 mg/g dw

Santos et al., 1970 Nishimoto et al., 1987

Pfaffia iresinoides

Amaranthaceae

Roots

6.2 mg/g dw 1020 mg/g dw

Asteraceae Serratula tinctoria

Aerial parts

Rudel et al., 1992

Sida rhombifolia

Malvaceae

Aerial parts

0.82 mg/g dw

Jadhav et al., 2007 Pathak et al., 1995

Tinospora cordifolia

Menispermaceae Aerial parts

0.25 mg/g dw

Chemical structure

Ecdysteroid carbon skeleton is termed as cyclopentano-perhydro-phenanthrene with a beta side chain at C 17,which is the product of terpene biosynthesis through mevalonic acid ,cholesterol and related sterols(Bathori & Pongracz, 2005). The whole side chain is kept in the case of C27-C29 ecdysteroids. C19,C21 and C24are formed by the cleavage of the side chain. Ecdysteroids contain cis A-B ring junction, a 7-en-6-one chromophore in the B ring,addition of -hydroxyl group at C14 and -hydroxyl group at C3.The B-C and C-D ring junctions are always trans. A-B ring junction is normally cis(5-H)and only rarely trans(5H). The methyl groups at C10 and C13 have a configuration(Adler & Grebenok 1999;Dinan, 2001;Bathori & Pongracz 2005; Reginaldo et al.,2008). The 14-hydroxy-7-en6-one chromophore result in a characteristic ultra violet absorption with max at 242 nm in methanol(Dinan, 2001).Usually two to eight hydroxyl group are present on ecdysteroids. Any carbon atom other than the C7, C15, and C18 carbons may be hydroxylated. Additional and alternative double bonds may exist between the4-5, 8-9,9-11,12-13,14-15,24-25,25-26,24-28 carbons. A second oxo group may be located on C2, C3,C12,C17,C20,C22. Numerous ecdysteroids exist with 5-OH,1-OH and 11-OH groups and all of these are phytoecdysteroids. Ecdysteroids hydroxylated at C9,C12,C17,C19,C21,C24,C28 and C29 occur in plants. Phytoecdysteroids may be found conjugated with both organic and inorganic acids,organic alcohol and glycosides(Bathori & Pongracz, 2005). Both the phytoecdysteroid level and structural diversity of plant ecdysteroids are surprising(Fig 2) . The number, location and the position of the hydroxyl substituents explain the structural diversity of phytoecdysteroids. Plants are capable of biosynthesising phytoecdysteroids from mevalonic acid. In many cases, the biosynthesis of phytoecdysteroids has been demonstrated to proceed via cholesterol and/or lathosterol (Adler & Grebenok, 1995). Current knowledge about the biosynthetic pathway(s) for ecdysteroids in plants is limited.

Fig 2 Structure of some Ecdysteroids

Role of phytoecdysteroids in plants

Phytoecdysteroids as Plant hormones 20-Hydroxyecdysone in nanomolar concentrations affected metabolic processes, including amylase activation, retardation of leaf senescence, having synergistic effect with auxin on coleptile elongation, antagonizing the effects of gibberelic acid (Golovatskaya, 2004). 20HE also altered germination, shoot growth, and root elongation in tomato (Bakrim et al., 2007).

Exogenous phytoecdysteroids produced weak gibberilin like activity in dwarf mung bean and rice bioassay(Drier &Tower,1988). There are examples of ecdysteroid induced growth and proliferation, but there is very little evidence supporting to consider ecdysteroids as plant hormone. Phytoecdysteroids in Defence Phytoecdysteroids may play a role in plant defense. Ecdysteroids (predominantly 20E) have been shown to have a range of detrimental effects on the development and survival of a number of insect species (Bombyx mori (Kubo et al., 1983), Pectinophora gossypiella (Kubo et al., 1981, 1983), Spodoptera frugiperda (Kubo et al., 1981), Acrolepiopsis assectella (Arnault & Slma, 1986; Harmatha, 2000), Agrius convolvulus (Tanaka & Naya, 1995), including inhibition of growth, supernumerary larval instars, death without moulting, death associated with promoted moulting and prothetely(Dinan et al., 2009). Detoxification of ecdysteroids occur in certain species by conjugating it with fatty acid and blocking the C-22 hydroxyl group, which is important for the biological activity of ecdysteroids (Dinan & Hormann, 2005; Dinan et al., 2009). Insect species like Heliothis virescens, H. armigera, Locusta migratoria, Manduca sexta, Spodoptera littoralis, Lacanobia olereaceae, Acherontia atropos are remarkably tolerant to ecdysteroids in their diet (Dinan, 1998; Dinan et al., 2009), while others are partially tolerent(e.g. Cynthia cardui, Tyria jacobaeae; Blackford & Dinan, 1997b), but higher levels are toxic to them. Ecdysteroid sensitive species are deterred from eating an ecdysteroid-containing diet that they die of hunger rather than consume ecdysteroid-containing food (e.g. Inachis io; Blackford & Dinan, 1997b).

Extraction and isolation of phytoecdysteroids Ecdysteroid extraction and purification is complicated due to their polar nature, poor crystallisation properties and the purification strategy comprises of multistep procedure

including extraction, prepurification and combination of one or more chromatographic steps. Dried plant parts were extracted exhaustive with large volumes of methanol. Ten fold excess of solvent usually methanol was used in this first step. The removal of both polar and apolar contaminants can be done using fractional precipitation and solvent-solvent partition. Fractional precipitation was done using acetone which removes the compound more polar than ecdysteroids. Solvent- solvent partition was done using aqueous methanol and hexane, the ecdysteroids remain in the aqueous methanolic phase, this process removes both lipohilic and hydrophilic contaminants. Next step is solid phase extraction(SPE) which removes certain compounds from ecdysteroids with similar polarity but difference in having their aromatic ring. Usually polyamides is used as stationary phase in SPE ( Bathori , 2002). Multistep chromatographic methods such as adequate combination of adsorption chromatography, partition chromatography, size exclusion and reverse phase chromatography are generally done for the final purification of ecdysteroid( Bathori ,1998). Ecdysteroid content of plant samples was generally determined by the use of bioassay, radio immunoassay (RIA), spectrophotometry, thin-layer chromatography or HPLC(Bathori et al.,2000) . HPLC is generally used in the last step of isolation of ecdysteroids for ,UV,MS and NMR analysis. Fig 3 explain method for extraction and isolation of ecdysteroid from Silence viridiflora( Toth & Bathori,2008). Phytoecdysteroid levels have been quantified by an ecdysteroid-specifc radioimmunoassay (RIA) which detects compounds structurally similar to ecdysteroids (Dinan, 1995a). Bioassay is dependent on biological activity detects compounds having ecdysteroid-like (agonist) activity (Clemment & Dinan, 1991; Clemment et al., 1993). Combination of these two assays provides a powerful tool for the analysis of phytoecdysteroids.

Fig 3 The scheme for the ecdysteroid isolation from Silence viridiflora( Toth & Bathori,2008) Bioassay systems for ecdysteroids Sensitivity of various dipterans, including Calliphora, Musca and Sarcophaga, are used for the bioassay of ecdysteroids. The sclerotization bioassay using mature larvae of the blowfly Calliphora erythrocephala was instrumental in the first isolation of ecdysteroids (Thomson et al.,1970). Musa bioassay using Musa domestica is more sensitive compare to the Calliphora test (Kaplsnis et al.,1996). The lepidopteran Chilo suppressalis dipping test was used for the screening of plant extracts for phytoecdysteroids(Imai et al.,1969). Insect cell lines from different insect species and tissue origins have been used to understand mode of action ecdysteroids. The Kc cell line, derived from 12-h Drosophila embryos, exhibits a number of well characterized responses to the application of ecdysteroids (Courgeon, 1972; Cherbas et

al., 1977). The Drosophila Kc cell line is one of the most well documented 20E-responsive cell lines currently available and has been used in numerous studies examining the effects of 20E. The epithelial cell line from Chironomus tentans established by Wyss (1982), is considered as a simplified model to study early events in ecdysteroid induced tissue differentiation (Spindler-Barth et al., 1989, 1995; Spindler-Barth, 1991, 1993; LammerdingKtippel et al., 1994) and cuticle formation. Chironomus cells do not synthesise moulting hormone but metabolise added ecdysteroids very slowly. The Drosophila imaginal disc cell lines (Cottam &Milner1997) and Spodoptera frugiperda Sf9 (Lynn & Oberlander 1983; Vaughn et al.,1997) cell line was also used to study and compare the effect of ecdysteroids and bisacylhydrazenes. Dinan and co-workers (2001a) used D. melanogaster BII tumorous blood cells to test different ecdysteroids and extracts with ecdysteroid agonistic or antagonistic activities. This BII cell line did not metabolize ecdysone, (Dinan et al., 1985). Changes in cell morphology and a reduction in cell density occur in the presence of ecdysteroid agonist that can be assessed turbidometrically. In vitro production of phytoecdysteroids Cell culture technique seems to be a good alternative for production of biologically active ecdysteroids. The ability of several plant species to produce phytoecdysteroids in vitro was first demonstrated in callus cultures of Achyranthes fauriei and Trianthema portulacastrum (Hikino et al., 1971, Ravishankar & Mehta, 1979). Phytoecdysteroids have also been isolated from the culture filtrates of Ajuga turkestanica (Lev et al., 1990), A. reptans var. atropurpurea (Matsumoto & Tanaka, 1991), Pteridium aquilinum (McMorris & Voeller, 1971) and Serratula tinctoria (Corio-Costet et al., 1996). Callus and suspension cultures of A. turkestanica, synthesised 20E and turkesterone, the 20E concentration in the cells being several times higher than that in the roots and leaves of the plants, whereas the turkesterone concentration was somewhat lower than in the intact plant (Lev et al., 1990). Hairy root

cultures of Ajuga reptans var. artropurpurea, Achyranthes fauriei, Pfaffia iresinoides and Vitex strickeri (Matsumoto & Tanaka, 1991) produced phytoecdysteroids. A. reptans hairy root clones were shown to synthesize 20E, norcyasterone, cyasterone and isocyasterone, which are characteristic ecdysteroids of intact plant roots. The cell culture that produced secondary metabolites are considered interesting model system for studies of biosynthesis and physiological role of phytoecdysteroids, the available information on which is scanty. Commercial availability of phytoecdysteroids Today more than 140 different ecdysteroid-containing preparations are available on the market(Fig 4). They ususally contain 20E, sometimes in combination with other ecdysteroids, and they are proposed in particular for use by bodybuilders (Lafont & Dinan, 2003). These preparations contain ecdysteroids isolated from just a few plant species, mainly Leuzea carthamoides, Cyanotis arachnoidea , Pfaffia sp.

Fig

Examples

of

commercially

available

preparations

containing

phytoecdysteroids

and

their

Web

address.(http://www.myprotein.com/sports-nutrition/beta-ecdysterone/10529949.html ,http://www.rocksolidbodybuilding.com/Beta-Ecdysterone-supplement.html, http://astronutrition.com/sni-20-beta-hydroxy-120-

caps.html,http://www.rebodybuilding.com/forum/scifit/13423-scifit-ecdysterone-300-reviews.html,Indiaprotein.com/BetaEcdysterone,www.nutrend-supplements.com)

Applications of phytoecdysteroids Phytoecdysteroids are apparently non toxic to mammals and have a number of beneficial and pharmacological and medicinal applications (Dinan, 2001; Slama & Lafont, 1995). Phytoecdysteroids display a wide range of biological activities which explain the usage of ecdysteroids containing plants in medicine (Table 2). Table 2 Medicinal applications of phytoecdysteroids
Pharmacological effects
Protein synthesis Stimulatory effect on protein synthesis, ingested ecdysteroids(5mg/Kg) increased mRNA translation efficiency Antidiabetic effects 20E reduced induced hyperglycemia,stimulates glucose incorporation into glycogen and enhance glucose utilisation by tissues. Antidiabetic effects are known for ecdysteroid containing plants used in traditional medicine. In vitro experiments demonstrate that ecdysteroids increase glucose consumption in an insulin independent fashion. Hypocholesterolaemic effects Ecdysteroids reduce cholesterol level by reducing biosynthesis,enhancing catabolism, and promoting its conversion into bile acid. Rats Uchiyama & Yoshida, 1974, Dinan ,2009, Lupien et al., 1969, Dinan & Lafont ,2006 Humans Detmar et al .,1994, Lafont &Dinan,2003 ,Dinan & Lafont ,2006 Human Hepatocytes Dinan ,2009 Rat Mouse Uchiyama & Yoshida, 1974,Yoshida et al., 1971 Kutepova et al .,2001, Wessner et al .,1992,Chen et al., 2006. Dinan & Lafont ,2006

Effect of ecdysteroids

Species/ cells
Mice

Reference

Otaka et al.,1968, Dinan & Lafont ,2006

Wound healing

Ecdysteroid containing liposome promote wound healing, 20E stimulate keratinocyte differentiation. In vitro,it has psoriasis inhibiting activity

Growth proliferation

and

cell

Ecdysteroids have been shown to increase growth in a wide variety of animals 20E (5mg/Kg) administered accelerates

Mice,Rats Sheep,Pigs Quails Rats Lafont &Dinan , 2003 Feldman et al.,2008

healing process after experimental bone fracture. Stimulates proliferation of human umbilical vein endothelial cells Stimulates erythropoiesis

Rats

Rats

Immuno protective

Ecdysteroids

have

immunomodulatory

Rats

effects,20E, Pol B,Turkesterone increase the concentration of antibody forming cells in spleen. 20E induce activation of human lymphocytes Hepatoprotective agent 20E improves liver regeneration after Rats Humans

Lafont & Dinan , 2003 Dinan ,2009 Trenin & Volodin, 1999

chemically induced damage and stimulates bile secretion Nephroprotective 20E(5mg/Kg) restore normal glomerular Rats

Lafont & Dinan , 2003 Dinan & Lafont ,.2006

Dinan &Lafont ,2006

filtration rate and suppress albumineria Antioxidant 20E has anti oxidant , anti free radical properties and reduce lipid peroxidation Antibiotic activity Administration of 20E to rabbits reduces infection by protozoa (Lamblia duodenalis, Flagellates) Rats Mice Rabbits Lafont &Dinan , 2003 Lafont &Dinan ,2003

Adaptogenic effects

Ecdysteroids enhance the ability to cope with stress and enhance resistance to tiredness.

Dinan ,2009

Agricultural application Phytoecdysteroids can be commercially exploited to improve production in sericulture. Administration of phytoecdysteroids affect growth and development of silkworms.The exogenous phytoecdysteroids administration to the last instar larvae of Bombyx mori,

hastens maturation in silkworm and synchronise cocoon spinning(Chou & Lu 1980). The doses used to bring about the beneficial effects on silkworms are low relative to those having detrimental effects on insects .Ecdysteroids have also been proposed as a treatment for the enhancement of fecundity of honey bees in apiculture (Lafont & Dinan2009) and improving prawn/shrimp aquaculture. When Marsupenaeus japonicas was exposed to diet incorporated with ecdysteroid-containing extract of Achyranthes spp. the treated shrimps showed improved weight gain over the control animals (Cho & Itami, 2004).

Gene switches The heterodimer of nuclear receptors,ecdysteroid receptor(EcR) and ultraspiracle(USP) an homologue of the vertebrate retinoid X receptor considered as the functional ecdysteroid receptor. The homologue of USP in animals and humans is the retinoid X receptor (RXR) . EcR/USP complex interacts with specific ecdysteroid responsive elements in the promoter region of ecdysteroid responsive gene to alter gene transcription activity( Dinan & Lafont ,2006). Ecdysteroid receptor is not a natural component of vertebrate cell,ecdysteroids are nontoxic to vertebrates and ecdysteroids do not activate vertebrate steroid hormone receptors even at very high concentrations. Ecdysteroids are successfully developed as an effective inducer for EcR based gene expression system and are suitable technologies for tight control transgene expression in mammalian cells(Lafont & Dinan ,2003). EcR based gene switches have been successfully applied in animal models (Karzenowski et al.,2005,Galimi et al.,2005,Hoppe et al.,2000), in functional genomics(Takeda et al.,2000), disease models(Albanese et al.,2000) and are candidate systems for switch controlled human gene therapy(Palli et al.,2005,Toniatti et al,2004,Goverdhana et al.,2005,) and feasibility has been demonstrated for two ecdysteroid ligands: ponasterone A and muristerone A(Lapenna& Dinan,2009).The non steroidal ecdysteroids agonist bisacylhydrazene are considered as an alternative ligand to generate an ecdysteroid inducible gene switch systems. Bisacylhydrazines Disturbances in the normal course of molting and metamorphosis under the action of exogenous ecdysteroids or substances mimicking their biological activity usually lead to the insect death. Ecdysteroids has limited application in controlling arthropod pest because of its chemical and metabolic instability, they are too polar to penetrate insect cuticle and too expensive to synthesise on large scale (Dinan, 198;Lafont & Dinan 2009).

Bisacylhydrazines are non steroidal agonist of 20E and exhibit their insectisidal activity via interaction with the ecdysteroid receptor proteins. Bisacylhydrazines are more metabolically stable in vivo than ecdysteroids and because they are true ecdysone agonists at the receptor level, ingestion of bisacylhydrazines creates hyperecdysonism in susceptible insects, thus inducing effects and symptomology of a molt event (Dhadialla et al.,1998, 2005). The larvae ultimately die as a result of their inability to complete a molt, starvation, and desiccation due to hemorrhage. Four potent analogs (tebufenozide, methoxyfenozide, halofenozide and chromofenozide) are currently on the market (Fig 5, Table 3) as safer insecticides with reduced mammalian toxicity. These insecticides have very good ecotoxicological profile, having virtually no impact on most non target organisms including beneficial insects and pollinators, (bees, predators and parasitoids), birds, fish and terrestrial invertebrates (Dhadialla et al., 2005). The BAH insecticides also have low-toxicity to mammals making them reduced risk materials for humans handling the products.

tebufenozide methoxyfenozide

Chromofenozide halofenozide Fig 5 Chemical structure of bisacylhydrazine insecticides

Table 3 Bisacylhydrazine ant their pest spectrum. Common name

Tebufenozide ( RH-5992)

Registered names
MIMIC, CONFIRM, ROMDAN

Industry
Rohm and Haas Co.,a Dow AgroSciences LLCb

Pest spectrum
Lepidoptera specific Chilo suppressalis,Cnapholocrocis medinalis Lymantria dispar, Plusia spp., Pseudoplusia includes, Spodoptera spp. Trichoplusia ni

Methoxyfenozide (RH-2485)

INTREPID RUNNER PRODIGY FALCON MATRIC KILLAT

Rohm and Haas Co.,a Dow AgroSciences LLCb Nippon Kayaku, Saitane, Japan and Sankyo, Ibaraki, Japan

Lepidoptera specific Heliothis spp.,Helicoverpa zea

Chromofenozide (ANS-118, CM-001)

Lepidoptera specific Spodoptera littura, Spodoptera exigua Spodoptera littoralis , Cnaphalocrosis medinalis,Heliothis virescens Chilo suppressalis

Halofenozide (RH-0345)

MACH 2

Rohm and Haas Co.,a Dow AgroSciences LLCb

Lepidoptera, Coleoptera specific Agrotis ipsilon,Spodoptera frugiperda Popillia japonica

Ecdysterone antagonists Ecdysterone antagonists inhibit the effect of ecdysterone. The compound azadirachtin from the seeds of the tree Azardirachta indica,is a feeding inhibitor and growth disrupting compound for most insect orders. Treatment of insects with azadirachtin frequently elicits a delay or permanent block of molting. Molting failures are due to reduced ecdysteroid titre,( Redfern et al., 1982) , and azadirachtin inhibits ecdysteroid secretion from the prothoracic glands ( Sieber & Rembold (1983). Azadirachtin may also affect the metabolism of ecdysteroids, for example by hindering the conversion of ecdysone into the hormonally more active 20-hydroxyecdysone (Smith & Mitchell, 1988). Imidazole compounds, KK-42 (1benzyl-5-[(E)-2,6-dimethyl-1, 5-heptadienyl] imidazole) and KS-175 (4-

phenoxyphenoxypropyl imidazole), have been reported to inhibit ecdysteroid synthesis,

leading to disrupted development( Smagghe ,2007). Cucurbitacins B and D, isolated from seeds of Iberis umbellata (Cruciferae) and shown to be responsible for the antagonistic activity of a methanolic extract of this species in preventing the 20-hydroxyecdysone (20E)induced morphological changes in the Drosophila melanogaster BII permanent cell line (Dinan et al.,1997). Other compounds being considered for anti-ecdysteroid effects include the brassinosteroids, , limonoids and triterpenes (Richter & Koolman 1991; Decombel et al.,2005; Dinan & Hormann, 2005). Antagonists of ecdysteroid action in invertebrate systems will be useful as biochemical probes for the investigation of the control of gene expression by ecdysteroids as well as lead compounds for the development of new insect pest control agents (Dinan, 1995; Keckeis et al., 2000). Conclusion Phytoecdysteroids are structural analogues of insect molting hormone ecdysone. They exert a number of beneficial pharmacological effects on mammals and hundreds of ecdysteroid containing anabolic preparations are available in the markets. Some ecdysteroids may provide promising alternative to anabolic androgenic steroids in therapy, but their mode of action is completely unknown. So it would be worthwhile to perform detailed clinical trials to evaluate its importance as a medicine. In vitro culture with high potential activity seems to be a good source for the production of biologically active phytoecdysteroids. Bisacylhydrazenes are non steroidal agonist of 20-hydroxyecdysone and exhibit their activity via interaction with ecdysteroid receptor proteins. These insecticides are safe to beneficial insects and have benign ecotoxicological properties,they are selective in their mode of action and potentially act only on target species.The effect of insect hormone ecdysterone analogues or agonists has been widely studied to understand the regulation of insect development and recent developments in these technologies should help in developing novel pest management methods.

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