Studies On The Enzyme Activity And Mechanism Of The Chelidonium Majus Againist Lymantria Dispar

Due to the benzyl-isoquinoline alkaloids of C.majus is broad spectrum of bioactivity,C.majus has been extensively studied in many fields,especially in the medical field.However,there are few studies on its insecticidal activity,and its specific insecticidal mechanisms are still unclear.In this study,the crude extract of C.majus was obtained by Soxhlet ultrasonic extraction,and the total akaloid was separated and obtained by macroporous resin,the total alkaloid composition was analyzed and identified by MS-HPLC.Then,we analyzed the effects of the crude extract of C.majus and its total alkaloid on the insecticidal effect and growth and development of 3rd instar larvae of L.dispar.Finally,we studied the effects of crude extracts and their total alkaloids on the activities of 5 kinds of enzymes and the level of transcription from the physiological and biochemical level and molecular level.The experimental results will provide the basic data for the further study of the molecular mechanism of the insecticidal effect.The results obtained in this paper are as follows:The results of the analysis of the active components of the insecticides of C.majus showed:The total alkaloid of C.majus can be successfully isolated and obtained by macroporous resin AB-8;the results of the test are further shown that Benzylisoquinoline alkaloids are the main insecticidal active components of C.majus.In the five measured alkaloids:the highest content of coptisine was 1624.23 mg/L,followed by celeryridine?16.01mg/L?,sanguinarine?3.45 mg/L?and berberine hydrochloride?5.21 mg/L?.While the content of tetrahydrocoptisine was the lowest,the tetrahydrocoptisine was only 0.47 mg/L;The results of determination of nutritional indicators showed:the total alkaloid of C.majus promotes the relative feed intake?RCR?,food conversion?ECD?and approximate digestibility?AD?of the third instar larvae of dispar,the food use efficiency?ECI?and food conversion rate?ECD?showed an inhibitory effect.In vitro enzyme activity results showed:?CECm?and total alkaloid?TACm?had significant inhibitory activity against 5 enzymes,which has the strongest inhibitory activity against glutathione S-transferase?GST?.The IC₅₀ was 7.058 and 5.333 mg/m L,followed by acetylcholinesterase?AChE?,the IC₅₀ was 7.122 and 7.827 mg/mL,while the carboxylesterase?CarE?inhibited the weakest.The sensitivity of CECm and TACm to the five enzymes of L.dispar larvae was GST>AChE>SOD>CAT>CarE;In vivo activity assay showed that:both CECm and TACm had different effects on the activities of five enzymes in the larvae of L.dispar;Among them,AChE and CarE were significantly inhibited at 12,24,48 and 72 h after CECm and TACm treatment;At 72 h of CECm treatment,GST is significantly inhibited by activity.While the activity of SOD and CAT after the treatment has a fluctuations.qRT-PCR results showed:TACm has different effects on the mRNA levels of the five enzymes.Among them,LaAChE and LaCarE transcription levels were lower in the treatment group than those in the control group at 3 time points.After 24 h of treatment,the transcription levels of LaAChE,LaCarE,and LaCAT were suppressed by TACm.Especially at 12 h,the mRNA levels of LaCarE and LaSOD were significantly decreased.