| Harmine is beta–carboline alkaloids isolated from Peganum harmala(harmal)that is distributed widely in the nature and exert various types of pharmaceutical properties in vitro and in vivo. Therefore it has been attracting by the innovation research of pesticide.Recently, many literatures report that harmine and its derivatives have insecticidal,bactericidal and nematicidal activity, and are focused on obtain new lead compounds of natural products–based agrochemical agents.In this study, we investigated the biological activity of 42 harmine derivatives which were synthesized from harmine against Aedes albopictus, Bursaphelenchus xylophilus and Plutella xylostella, and inhibitory activity of acetylcholinesterase in vitro and in vivo by the spectrophotometer, expression levels of acetylcholinesterase(ACh E), monoamine oxidase(MAO) and Glutathione(GSH) in vivo by the quantitative real––time PCR,inhibitory activity of Aa Nav1–1 sodium channel, from Aedes aegypti in Xenopus oocytes,by the two–electrode voltage clamp.The results showed that compound H–2–53, H–2–58, H–2–65, H–2–77, H–2–163,H–2–171, H–2–172, H–2–174, H–2–196 and H–9–3 displayed promising nematicidal activity against Bursaphelenchus xylophilus, with LC50 values of 1.63 ?g/m L, 1.63 ?g/m L,1.75 ?g/m L, 1.44 ?g/m L, 25.59 ?g/m L, 18.31 ?g/m L, 10.22 ?g/m L, 12.72 ?g/m L, 5.84?g/m L and 13.32 ?g/m L, respectively, higher than the parent compound, harmine(LC5027.50 ?g/m L); 22 compounds exhibited significant insecticidal activities against Aedes albopictus. Among all of the derivatives, especially compound H–2–196 displayed the best promising larvicidal activity with LC50 values of 2.58 ?g/m L, then compound H–2–174,H–2–53, H–2–44, H–3–7, H–2–65, H–2–58, H–2–77 and H–2–171, LC50 value were 6.68?g/m L–9.30 ?g/m L, compound H–2–25, H–2–27, H–2–172, H–2–197, H–3–9, H–3–55,H–3–81, H–3–83, S4, S6, J1, J2 and J3, LC50 value were 10 ?g/m L–29.55 ?g/m L, while the mortality of hamine was 6.25% at the concentration of 50 ?g/m L. It was disappointing to find that all derivatives display little or no biological activity against Plutella xylostella under our assay conditions.In order to determine the active compounds, 10 harmine derivatives, responsible for the potential inhibition of ACh Es, compounds were evaluated for the ACh E from Electrophorus electricus inhibiting assay in vitro. Compound H–2–77 showed the greatest potential for ACh Es inhibitory activity with IC50 0.07 ?g/m L, then compound H–2–53,H–2–58 and H–2–65, with IC50 0.92 ?g/m L, 090 ?g/m L and 0.82 ?g/m L, respectively,higher than harmine(4.52 ?g/m L); the IC50 values of compound H–2–163, H–2–171,H–2–172, H–2–174, H–2–196, H–9–3 were greater than 5.00 ?g/m L under our assay conditions. And further evaluated for the potential inhibition of ACh Es against B.xylophilus in vivo, compound H–2–53, H–2–58, H–2–65 and H–2–77 exhibited prominent inhibitory activity against ACh E, with IC50 values of 17.16 ?g/m L, 14.56 ?g/m L, 13.63?g/m L and 3.06 ?g/m L, respectively. The IC50 values of the positive control, harmine,against ACh E was 578.44 ?g/m L; Other compounds display little inhibitory activity in vivo under our assay conditions. After comparing the ACh E, MAO and GSH gene with the?–actin gene in B. xylophilus exposed to harmine and 10 harmine derivatives at 20 mg/m L concentration, respectively. After 24 h, compound H–2–53, H–2–58, H–2–65 and H–2–77significantly increased the level of ACh E m RNA by approximately 478%, 247%, 211%and 1120%, respectively; Treatment with compound H–2–53, H–2–65 and H–2–77significantly increased the level of MAO m RNA by approximately 564%, 652% and6622%, respectively; Treatment with compound H–2–77 significantly increased the level of GSH m RNA by approximately 337%; Treatment with harmine increased the level of ACh E,MAO and GSH m RNA by approximately 187%, 123% and 147% compared to the control group, respectively; The other treatments were no significant difference with control. The results showed that compound H–2–53, H–2–58, H–2–65 and H–2–77 probably interact with ACh E, MAO and GSH.After exposure to 160 ?M active compounds against Aedes aegypti, the Aa Nav1–1sodium channel currents, from Aedes aegypti in Xenopus oocytes inhibition rates less than50.52% by the two–electrode voltage clamp. The results showed that little decline of Aa Nav1–1 channel currents occurred in all treatment.The quantitative structure–activity relationship(QSAR) study on several series of harmine derivatives showed that the 2,9–position together as the electron–donating substituted and 7–position as electron–withdrawing substituted harmine displayed promising nematicidal activity against Bursaphelenchus xylophilus, ACh Es inhibitory activity in vitro and in vivo, and increased the level of ACh E, MAO and GSH m RNA in vivo; 2,9–position together as the electron–donating substituted or 7–position as electron–withdrawing substituted or 6–position as bromine substituted harmine displayed promising nematicidal activity against Bursaphelenchus xylophilus, ACh Es inhibitory activity in vitro, and down–regulated the level of ACh E, MAO and GSH m RNA in vivo.In conclusion, 2,9–positions together as the electron–donating substituted and7–position as electron–withdrawing substituted harmine appear to be useful as natural products–based nematicides for B. xylophilus through inhibition of acetylcholinesterase.For the practical use of the harmine derivatives as novel nematicides to proceed, further study is necessary on systemic action, phytotoxicity, and formulation for improving nematicidal potency and stability and reducing cost. Whereas 2,9–positions together as the electron–donating substituted or 6–positions as hydrogen or bromine substituted or7–positions as electron–withdrawing substituted harmines have potential as natural products–based nematicides for B. xylophilus, which action mechanism of nematicidal activity deserves further study. |
PDF Full Text Request