| The storage work was the emphasis of foodstuff management in China, and how to effectively control the stored product pest was the key task for food storage. The botanical pesticide belongs to the bio-rational pesticide, and it is hard for pests to develop resistance to the botanical pesticide. As a result, the botanical pesticide answered for the impersonal demand of sustainable agriculture. The maize weevil Sitophilus zeamais Motsch., grain borer Rhizopertha dominica (Fab.) and red flur beetle Tribolium castaneum (Herbst) were the three key stored product pests in China, of which the maize weevil had been named as the first key stored product pest by national foodstuff department, and the grain borer was the primary pest of stored grain in south China. At present, the grain borer had developed serious resistance to some chemical pesticides, such as phosphine, etc. Thus, this research was effectively financed by the Chinese 15th key task project through the science and technology ministry of state (Application and Demonstration of Botanical Insecticide in Controlling Stored Product Pest, serial number: 2004BA523B03-2).1 Isolation and identification of effective insecticidal ingredients in Acorus gramineus Soland rhizome extract1.1 Isolation of effective insecticidal ingredients in A. gramineus rhizome extractThe dry powder of A. gramineus rhizome was firstly soaked using methanol in cool condition, and then the extract was pretreated by extraction method using organic solvent correspondingly. The results indicated that the methanol had better extraction effect to dry powder of A. gramineus rhizome as 19.2% extract yield after concentration, and the yield of petroleum ether fraction was up to 23.5% after concentration.Adopting the silica gel Column Chromatography (CC), the effective insecticidal ingredients of petroleum ether extract were separated as I2 vapor acted as display reagent step by step. Three active ingredients were obtained after concentration, and two ingredients with better activity were selected for purification in silica gel CC. At last, two active monomers were obtained, and the monomers were kind of yellowish oily liquid.1.2 Primary insecticidal effects of ingredients isolated from A. gramineus rhizome extractThe primary toxicities of insecticidal ingredients isolated from A. gramineus rhizome extract to S. zeamais, R. dominica and T. castaneum adults were tested using drug-film method. The results were listed as follows: As for four solvents extracts of A. gramineus rhizome extract, the petroleum ether extract had highest bioactivities to tested pests. At 314.40μg/cm2 dosage, the corrected mortalities of petroleum ether extract against three tested pests were 100.00% at 72h post-treatment. Furthermore, the water extract had some toxicities to R. dominica and T. castaneum adults with 88.51% and 56.98% corrected mortalities accordingly at 72h post-treatment.The results of bioassay for primary fractions at 157.20μg/cm2 dosage indicated the active ingredients belonged to the compound with low polarity.The C (number 61～72) fraction of 8 eluted fractions had highest toxicities against three tested pests as the corrected mortalities at 72 h post-treatment were more than 80.00%, these mortalities were significantly higher than that of other 7 eluted fractions. The bioassay results for sub-separated fractions at 157.20μg/cm2 dosage indicated ingredientsⅡ,ⅢandⅥhad better toxicities as the corrected mortalities to S. zeamais at 72 h post-treatment were 81.11%, 97.78% and 100.00% correspondingly, and the corrected mortalities to R. dominica at 72 h post-treatment were 57.78%, 97.78% and 97.78% correspondingly. At 157.20μg/cm2 dosage, the corrected mortalities of ingredientⅢandⅥto T. castaneum at 72 h post-treatment were 100.00%.Further primary bioassay for three selected ingredients demonstrated the ingredientⅢandⅥhad better insecticidal effects against pests. The insecticidal effect of ingredientⅢwas slow relatively, and the toxicity effect had close relationship with exposure time. IngredientⅢhad better bioactivities to R. dominica and T. castaneum adults with 64.44% and 90.00% corrected mortalities (72 h) accordingly at 39.30μg/cm2 dosage, and ingredientⅥhad better bioactivities to S. zeamais and R. dominica with 54.44% and 87.78% corrected mortalities (72 h) accordingly at 19.65μg/cm2 dosage.1.3 Primary analysis of effective insecticidal components for selected ingredients isolated from A. gramineus rhizomeActive components for three selected ingredients isolated from A. gramineus rhizome were primarily analyzed using Gas Chromatography-Mass Spectrometry (GC-MS).In ingredientⅡ, the molecular weight of compound 1～2 were 205, which were isomeric compounds. The molecular weight of compound 3～5 were 220, and were isomeric compounds as well. It was primarily concluded that ingredientⅡwas composed of 5 sesquiterpenoids. As for ingredientⅢ, compound 7 was the leading active component with 218 molecular weight. In ingredientⅥ, it was primarily concluded that compound 8 was elemine [1,2,3-trimethoxy-5-(2-propenyl) benzene], and compound 9 wasβ-asarone [cis-1,2,4-trimethoxy-5-(1-propenyl) benzene] acted as a leading active component in ingredientⅥ.1.4 Structure identification of active monomer isolated from A. gramineus rhizome1.4.1 Structure identification for compound 7Mass Spectrum (MS) data indicated the monomer 7 was a kind of guaiane-type sesquiterpene, and its molecular weight was 218. The monomer might contain methyl (CH3-), secondary carbon linked with tertiary carbon (＞CHCH2-) and ketonic bond (-C=O). Ultraviolet Spectrum (UV) showed monomer 7 contained conjugated double bond (-C=C-C=C-) or similar bond. Infrared Spectrum (IR) indicated the monomer 7 contained ketonic bond and conjugated unsaturated carbonyl unit (-C=C-C=O). 1H Nuclear Magnetic Resonance (1HNMR) spectrum indicated monomer 7 contained two methyl linked with tertiary carbon (CH3-CH＜), two methyl linked with double bond (CH3-C=C), a doubly allylic methylene group appearing as a tylical AB doublet (-C=C-CH2-C=C-), and a secondary carbon linked with ketonic bond (-CH2-CO-), without hydrogen linked with unsaturated carbon (-CH=CH-). 13C Nuclear Magnetic Resonance (13CNMR) spectrum reconfirmed monomer 7 had one ketonic bond, and two double bonds without hydrogen (＞C=C＜).Integrated with above spectrum characters, this monomer could be confirmed as a known sequiterpenoid, calamusenone named as 3,8-dimethyl-5-(1-methylethylidene)-1,2,3,4,5,6,7,8-octahydro-6-azulenone.1.4.2 Structure identification for compound 9MS data indicated the monomer 9 has much matching degree withβ-asarone, and its molecular weight was 208. UV spectrum showed monomer 9 and standard sampleβ-asarone contained a double bond conjugated with benzene. IR spectrum indicated the monomer 9 contained hydrogen linked with unsaturated carbon (-CH=CH-), methoxyl (CH3O-) and benzenoid form. 1HNMR indicated monomer 9 contained one methyl linked with unsaturated carbon (CH3CH=), three methoxyl groups, CH3CH=CH-group,-CH=CH-C= group and two hydrogen located in benzenoid form. 13CNMR reconfirmed monomer 9 had three methoxyl groups linked with benzenoid form as well as one methyl linked with unsaturated carbon. Integrated with above spectrum characters, monomer 9 could be confirmed as a known phenylpropanoid named as cis-1,2,4-trimethoxy-5-(1-propenyl) benzene.2 Bioactivities of petroleum ether extract,β-asarone and calamusenone isolated from A. gramineus rhizome extract to tested pests2.1 Bioactivities of petroleum ether extract isolated from A. gramineus rhizome extract to tested pestsThe bioassay results of drug-film method showed petroleum ether extract had better contact activities against R. dominica and S. zeamais adults. The corrected mortality of petroleum ether extract at 39.30μg/cm2 to R. dominica was 88.37% at 72 h post-treatment. LD50 (72 h) of petroleum ether extract to R. dominica, S. zeamais and T. castaneum were 8.85, 40.04, 64.88μg/cm2 correspondingly. Drug-paper fumigant experiment showed the petroleum ether extract had better fumigant activities against R. dominica, and the effect increased markedly with increasing of exposure time. After 120 h treatment, the corrected mortality to R. dominica at 25μL/L concentration hadn't significant (P＞0.05) difference to 100μL/L treatment. Petroleum ether extract had moderate fumigant activities against S. zeamais, and the corrected mortality at 100μL/L was 83.30% after 120 h treatment. The petroleum ether extract had excellent repellency action on T. castaneum by selection test on filter paper held with drug. As for three dosages, all the mean repellecy rates based on 24, 48, 72 h were V grade level. Petroleum ether extract had moderate repellency effect on R. dominica and 5. zeamais, and both of the mean repellency rates wereⅣlevel at 157.20μg/cm2 dosage.The mixture experiment of foodstuff and drug showed that petroleum ether extract had better population inhibition activity and control effect against R. dominica and S. zeamais, and better poisonous effect against R. dominica as well. At 250 mg/Kg dosage, the mortality of this extract to R. dominica was 97.73% at 3 d post-treatment, which was markedly higher than that of malathion treatment at 5 mg/Kg dosage. At 250 mg/Kg dosage, the control effect of petroleum ether extract against R. dominica was up to 100.00%, which was similar to the malathion treatment at 5 mg/Kg dosage. When mixed with wheat at 125 mg/Kg dosage, the population inhibition rate of petroleum ether extract to R. dominica was 96.47%, and there hadn't markedly difference with malathion treatment at 5 mg/Kg dosage. 2.2 Bioactivities ofβ-asarone isolated from A. gramineus rhizome extract to tested pestsβ-asarone had better contact activities against S. zeamais and R. dominica adults, and the LD50 (72 h) were 51.29μg/cm2 and 30.27μg/cm2 respectively.β-asarone had excellent fumigant activities against R. dominica. At 120 h post-treatment, the corrected mortality to R. dominica at 25μL/L concentration was 97.93%, which hadn't significant (P＞0.05) difference to 100μL/L treatment. The fumigant effect ofβ-asarone was relatively slow, which increased markedly with prolonging of exposure time.β-asarone had better fumigant activities against S. zeamais, and the corrected mortality at 25μL/L concentration was 89.81% after 120 h treatment.β-asarone had excellent repellency action on T. castaneum, all the mean repellecy rates based on 24 h, 48 h and 72 h were no less thanⅣgrade level.Theβ-asarone had better poisonous activity, population inhibition action and control effect against R. dominica and S. zeamais. At 200 mg/Kg dosage, the mortality ofβ-asarone to R. dominica was 99.00% at 3 d post-treatment, which was markedly higher than that of malathion treatment at 5 mg/Kg dosage. At 100 mg/Kg dosage, the population inhibition rate and control effect ofβ-asarone against R. dominica was 85.25% and 90.35% accordingly, which was similar to the malathion treatment at 5 mg/Kg dosage. When mixed with wheat at 250 mg/Kg dosage, the population inhibition rate and control effect ofβ-asarone against 5. zeamais was 98.82% and 88.08% accordingly, which hadn't significant (P＞0.05) difference to malathion treatment at 5 mg/Kg dosage.2.3 Bioactivities of calamusenone isolated from A. gramineus rhizome extract to tested pestsCalamusenone had moderate contact activities against S. zeamais and R. dominica adults, and the LD50 (72 h) were 61.84μg/cm2 and 71.34μg/cm2 respectively. Calamusenone had better fumigant activities against S. zeamais and R. dominica adults. At 120 h post-treatment, the corrected mortalities to S. zeamais and R. dominica at 166.67μL/L concentration were 72.52% and 86.43% correspondingly. The fumigant effect of calamusenone against R. dominica increased markedly with prolonging of exposure time. Furthermore, calamusenone had excellent repellency action on T. castaneum, the mean repellecy rate during 72 h at 19.65μg/cm2 dosage wasⅤgrade level.The calamusenone had better poisonous activity, population inhibition action and control effect against R. dominica. At 500 mg/Kg dosage, the mortality of calamusenone to R. dominica was 94.47% at 3 d post-treatment. The population inhibition rate (250 mg/Kg) and control effect (125 mg/Kg) of calamusenone against R. dominica were 98.70% and 100.00% accordingly, which hadn't significant (P＞0.05) difference to the malathion treatment at 10 mg/Kg dosage.2.4 Combined toxicities ofβ-asarone and calamusenone to tested pestsTest of drug-film method showed mixted application forβ-asarone and calamusenone had moderate synergism to tested pests, and the synergism was gradually enhanced with increasing ofβ-asarone content. When the ratio (W/W) ofβ-asarone and calamusenone was 4:1, the co-toxicity coefficient (CTC) was 127.53 (＞120), it denoted there had marked synergism to R. dominica. As for S. zeamais, there had marked antagonism (CTC=69.42＜80) when the ratio (W/W) ofβ-asarone and calamusenone was 1:1, whereas marked synergism (CTC=142.33) was given when the ratio (W/W) was 2:1.3 Insecticidal mechanism ofβ-asarone to S. zeamais and R. dominica adults3.1 Insecticidal mechanism ofβ-asarone to S. zeamais adultWhen S. zeamais was treated withβ-asarone at LD50 (485.00 mg/Kg) by mixture test for foodstuff and drug, the activities of acetylcholinesterase (AChE) and glutathione S-transferase (GST) were restrained at most times, and their activities were at lowest level at 24 h post-treatment. With the prolonging of exposure time, the induced action ofβ-asarone on Carboxylesterases (CarE) intro-pest was strengthened accordingly. The exposure time ofβ-asarone had some effects on the activity of est isozyme. At 12 h post-treatment, expression of isozyme E4 and E5 were restrained in some extent, whereas expression of isozyme E1, E2 and E3 were enhanced somewhat at 36 h post-treatment.After treated in five dosages, theβ-asarone at low dosage (=250.00 mg/Kg) showed induced action to AChE of intro-pest, whereas theβ-asarone at high dosage (=500.00 mg/Kg) gave inhibiting action to AChE.β-asarone had some inhibiting action to GST, but the inhibiting effect hadn't close relationship with dosage. With the enhancing of dosage forβ-asarone, the activity of CarE was significantly promoted. On the contrary, activity of Est E5 was somewhat restrained as the dosage was enhanced.3.2 Insecticidal mechanism ofβ-asarone to R. dominica adult When treated withβ-asarone at LD50 (94.49 mg/Kg), the activity of AChE to R. dominica was induced at 12 h, yet it was mostly restrained at (after) 24 h post-treatment. The exposure time ofβ-asarone to R. dominica hadn't distinct effect on GST. Whereas, the inducement ofβ-asarone on est isozyme was somewhat related with exposure time, and the isozyme E4 was induced distinctly at 12 h post-treatment.Theβ-asarone at low dosage (67.5 mg/Kg) showed marked induced action to AChE of intro-pest. With the increasing of dosage, the activity ofβ-asarone on AChE intro-pest was mostly restrained.β-asarone showed induced action to GST at low dosage (=100.0 mg/Kg), but inhibiting action to GST at high dosage (=133.3 mg/Kg). With the enhancing of dosage forβ-asarone, the activity of CarE was somewhat promoted.β-asarone had induced action to Est isozyme intro-pest at different dosages, whereas the induced effect hadn't close relationship with treated dosage.
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