Studies On The Biological Activity Of Deoxypodophyllotoxin And Its Mechanism

Part one: Study on the insecticidal activity of deoxypodophyllotoxinand its effects on enzymesThe toxicity of deoxypodophyllotoxin to newly-hatchedPeriplaneta americana larvae and 3rd instar larvae of Pieris rapae L. wastested by the method of drug film and leaf-dipping. Its effects on theactivities of AChE and ATPase in adult P. americana in vitro and in 3rdinstar larvae of Pieris rapae L. in vivo were investigated as well. Theinsecticidal activity of deoxypodophyllotoxin to P. americana wasdose-time-dependent with LC₅₀ values of 26.26＞4.68＞1.51＞0.62μg/cm² at 24, 48, 72, 96 h. LC₅₀ values of deoxypodophyllotoxin to 3rd instar larvae of P. rapae L. are 151.87＞39.99＞10.60 mg/L at 48, 72,96 h respectively. Deoxypodophyllotoxin did not affect the activity ofAChE either in vitro or in vivo. Deoxypodophyllotoxin inhibitedNa⁺-K⁺-ATPase in dose-dependent manner at the concentration range of5～625μmol/Lwith an IC₅₀ of 44.9μmol/L in vitro. It activatedNa⁺-K⁺-ATPase at lower concentration while inhibited it at higherconcentration in vivo. Deoxypodophyllotoxin activatedCa²⁺-Mg²⁺-ATPase at lower concentration while inhibited it at higherconcentration either in vitro or in vivo. The results indicated thatdeoxypodophyllotoxin exhibited high toxicity against P. americanalarvae and 3rd instar larvae of P. rapae L. AChE was not the target ofdeoxypodophyllotoxin while ATPases may be one important target ofdeoxypodophyllotoxin.Part two: Study on the mechanism of toxicity ofdeoxypodophyllotoxin against Brochydaino rerioWith the fenvalerate as the positive control, the toxicity ofdeoxypodophyllotoxin and podophyllotoxin against Brochydaino reriowas assayed by the acute toxicity test. Four gradient treatments ofdeoxypodophyllotoxin were set up under the 96 h LC₅₀. The activities ofATPase, CAT and SOD in the muscle of Brochydaino rerio weremeasured with assay kits after 48h. The LC₅₀ (96h) ofdeoxypodophyllotoxin, podophyllotoxin and fenvalerate against Brochydaino rerio were 3.49, 3.70 and 0.00775mg/L and the safeconcentrations were 0.36, 1.61 and 0.000803 mg/L respectively.Deoxypodophyllotoxin activated total ATPase, Na⁺-K⁺-ATPase andCa²⁺-ATPase at concentration of 1～2 mg/L while inhibited them at theconcentration of 4mg/L. Deoxypodophyllotoxin inhibited activity of SODat 0.5 mg/L, while had no effect on CAT, activated SOD and CAT at 1and 2 mg/L and inhibited them at 4 mg/L with inhibitory rate of 34.1%and 31.9%. The results showed that deoxypodophyllotoxin weremoderately hazardous to Brochydaino rerio and ATPase, meanwhile CATand SOD may be one important target of deoxypodophyllotoxin.Part three: Effects of deoxypodophyllotoxin on calcium homeostasisin rat dorsal root ganglion neuronsThe effects of deoxypodophyllotoxin on [Ca²⁺]_i in dorsal rootganglion neurons of rats were investigated with laser scanning confocalmicroscopy (LSCM) and its effects on the activities of AChE and ATPasewere determined with AChE and ATPase assay kits.Deoxypodophyllotoxin elevated [Ca²⁺]_i in Ca²⁺-containing medium in aconcentration-dependent manner with an EC₅₀ of 22.2μmol/L. And it alsoraised [Ca²⁺]_i in Ca²⁺-free medium. Deoxypodophyllotoxin did not affectthe activity of AChE. Deoxypodophyllotoxin inhibited eitherNa⁺-K⁺-ATPase or Ca²⁺-ATPase at the range of 1～625μmol/Lconcentration. These results indicated that deoxypodophyllotoxin induced [Ca²⁺]_i transient rise in rat DRG neurons via stimulating both extracellularCa²⁺ influx and intracellular Ca²⁺ release and reducing the ejection ofCa²⁺ by inhibited activities of ATPase.