| Aim to study the bioactivity against the main stored pests including Sitophilus zeamais Motschulsk (S. zeamais), Rhyzopertha dominica Fabricius (R dominica), Tribolium castaneum (Herbst) (T.castaneum) and Oryzaephilus surinamensis (Linnaeus)(O. surinamensis), several solvents and methods were selected to extract the bioactive substance efficiently from Legumiosae plant TFG seeds. From the year of 2003 to 2006, a series of studies concerned plant TFG have been conducted including its contact toxicity, repellency and its phytochemistry, the research results are outlined as follows:1. The powderded seeds of TFG was extracted with alcohol, chloroform, ethyl acetate, acetone and petroleum ether at relative high temperature by dipping the extract flask in warm water, the extracts rate of TFG seeds in alcohol was 11.48%, which is the highest rate among these solvents, while petroleum ether had the lowest extract rate which was only 4.91%. The extracts rate of TFG in chloroform, ethyl acetate and acetone was 7.24%, 7.54% and 5.29%.The five extracts obtained from different solvents all had a certain repellency against S. zeamais, the chloroform extract had the most effective repellency among those extracts, and its repellency level against S. zeamais could reach toâ…¡when dosage was at 1000μg/cm2, the second was acetone extracts.All the extracts had a significant repellency to T.castaneum, average repellency of different extracts all exceeded 80%, and repellency level wereâ…¤, acetone extracts had the most effective repellency to T.castaneum. the second one was chloroform extracts, the one had the most poor repellency was ethyl acetate. The difference of repellency rates between S. zeamais and O. surinamensis did not caused obviously by TFG extracts, while the repellency rate between S. zeamais and T.castaneum was notable, and the repellency rate between R. dominica and O. surinamensis was also notable. The order of repellency against stored pests by TFG solvent extracts was T.castaneum＞S. zeamais＞O. surinamensis＞R. dominica.All the five extracts possessed strong contact activity to S. zeamais, the adults, treated by extracts for three days and then cultured by clean wheat for one week, nearly all died. The alcohol extracts had the most effective contact toxicity to S. zeamais, its LD50 (effective dosage causing 50% mortality rate)to S. zeamais was 0.362mg/cm2 and 0.328mg/cm2, respectively. The second was ethyl acetate extracts and its LD50 to S. zeamais within 48 hours was 0.334mg/cm2, acetone extracts also had excellent contact toxicity to S. zeamais and its LD50 within 24 hours to S. zeamais was 0.380mg/cm2.The petroleum ether extract had the lowest contact toxicity to S. zeamais among the five TFG extracts. The LD50 of petroleum ether extract to S. zeamais was 0.772 mg/cm2 and 0.680 mg/cm2 by treatment within 24 hours and 48 hours, respectively.All the TFG five extracts had poor contact toxicity to T. castaneum adults. The mortality of T. castaneum changed very little after treated for three days and then cultured for one week. TFG alcohol extracts showed the most effective activity to R. dominica among these extracts, its LD50 to R. dominica adults was 0.482 mg/cm2, 0.450mg/cm2,0.400mg/cm2 and 0.386mg/cm2 within 24 hours, 48 hours, 72 hours and 10 days; the TFG petroleum ether extracts had the hightest LD50 to T. castaneum.TFG actone extract had strong toxicity to R. dominica with the LD50 of 0.240mg/cm2 by treatment within 24 hours, it showed more efficient than alcohol extract and chloroform extract, the mortality of R. dominica adults increased rapidly from 24 hours to 48 hours after treatment. The mortality of O. surinamensis changed differently according to the different TFG extracts and treatment dosage, alcohol extracts and chloroform extracts had more active contact toxicity than other extracts.2. Since alcohol was the optimum solvent to extract bioactive substance according to the results of the first experiment, it was selected as extract solvent for different extracting method including method of dipping powered TFG in alcohol at room temperature (TFGR), method of dipping powdered TFG in alcohol at relative high temperature (TFGH), method of Soxhlet extraction (TFGS), method of TFGR and TFGS (first, dipping powered TFG in alcohol at room temperature to get some extracts, then extracting remainder with Soxhlet extraction apparatus to obtain more extracts, TFGR-TFGS), method of Supercritical CO2 Fluid Extraction (TFG-SCFE), no modifier was used in TFG-SCFE. The results showed that the extracts rate of TFGR, TFGH, TFGS, TFGR-TFGS, TFG-SCFE was 4.65%, 11.48%, 12.99%, 8.14%, 11.35%, respectively.All the extracts obtained from those five methods were good at repelling T.castaneum, the repellency rates varied obviously when the extract method changed. TFG-SCFE extracts could repel T.castaneum at very little dosage. Treatment with TFG-SCFE extracts at dosage 0.16 mg/cm2, the average repellency rate within 4 days was 87.9%; treatment with TFGH extracts at dosage about 0.96 mg/cm2 to 1.60 mg/cm2, the average repellency rates of everyday all exceeded 80%, and the average repellency rates within 4 days was high to more than 90%, TFGR extracts, TFGS extracts and TFGR- TFGS extracts all had relative poor repellency to T.castaneum, the highest dosage for testing was 16 mg/cm2, which was 100 times to the testing dosage of TFG-SCFE extracts.The LD50 values of extracts obtained from those five methods to T.castaneum increased gradually from TFGH, TFG-SCFE, TFGR, TFGR-TFGS and TFGS. TFG-SCFE extracts had similar contact toxicity as TFGH extracts, treatment with it at dosage 0.91 mg/cm2 on T.castaneum, the mortality achieved to 90% within 72 hours, and the mortality all exceeded 80% when dosage above 0.52 mg/cm2.The toxicity order to R. dominica was TFGH＞TFG-SCFE＞TFGR＞TFGS＞TFGR-TFGS. TFGH extracts and TFG-SCFE extracts showed effective contact toxicity to R. dominica, the LD50 of TFGH to R. dominica within 24 hours, 48 hours,72 hours and 10 days was 0.255 mg/cm2, 0.144 mg/cm2, 0.112mg/cm2 and 0.100mg/cm2, respectively. The LD50 of TFG-SCFE to R. dominica within 24 hours, 48 hours,72 hours and 10 days was 0.256 mg/cm2, 0.156 mg/cm2, 0.142mg/cm2 and 0.130mg/cm2, respectively.3. Alcohol, ethyl acetate, chloroform was used as modifier to extract bioactive substance from TFG seeds powder, and the extract rates of TFG-SCFE with alcohol (TFG-SCFE-A), TFG-SCFE with ethyl acetate (TFG-SCFE-E), TFG-SCFE with chloroform(TFG-SCFE-C), and TFG-SCFE without modifier was 14.17%,13.31%,10.74% and 10.18%. Among those four extracts, TFG-SCFE-A extracts was the best pesticide to kill 5. zeamais. The LD50 of TFG-SCFE-A to 5. zeamais within 24 hours, 48 hours, 72 hours was 231.76μg/cm2, 194.43 mg/cm2, 141.06μg/cm2 respectively. The second was TFG-SCFE-E extract, its LD50 to S. zeamais within 24 hours, 48 hours and 72 hours was 228.67μg/cm2,180.09μg/cm2 and 152.61μg/cm2, the third was TFG-SCFE-C, and the last one was TFG-SCFE, the LD50 of TFG-SCFE to S. zeamais within 72 hours was the largest one, and the value was 218.74μg/cm2.The contact toxicity order of the SCFE extracts with different modifier against T. castaneum was TFG-SCFE-C＞TFG-SCFE-A＞TFG-SCFE＞TFG-SCFE-E, the LD50 of them to T.castaneum was 278.57μg/cm2,303.89μg/cm2,314.74μg/cm2 and 429.45μg/cm2.Among these four extracts, TFG-SCFE-A extracts possessed strong pesticidal effect to R. dominica, the LD50 of it to R. dominica within 24 hours and 10 days was 115.14μg/cm2 and 78.25μg/cm2, respectively; TFG-SCFE extracts was the one that showed poor contact toxicity, the LD50 of it to R. dominica within 24 hours and 10 days was 174.71μg/cm2 and 135.22μg/cm2.4. Used extract rate as standard values, extract pressure (EP), extract temperature (ET), extract duration (ED), modifier volume (MV), modifier consistence (MC) were investigated to settle down optima range, the results showed range for EP was 20MPa～30MPa, range for ET was 45℃～55℃, range for ED was 1h～2h, range for MV was 20mL～40mL and range for MC was 60%～95%.Contact toxicity and extract rate were used as standard values to optimize extraction conditions, which were achieved at 55 ΩC under 25 Mpa of pressure. The extraction efficiency for 200 grams of dry sample reached 16.96% with 30 mL of 95% alcohol. Extracts loaded on filter paper showed dose and time dependent toxicities to adult R. dominica with a LD50 value of 65.10μg/cm2 after 3d treatment and 7d culturation.5. TFG-1 extract was first separated into five parts by partition in liquid extraction method, petroleum ether, chloroform, ethyl acetate, n-butanol and water were used, extract rate of them was 61.32%,9.22%,9.16%,8.39% and 17.08%. the petroleum ether fraction was identified to be the most active part as result of contact toxicity; the second, chloroform fraction, and the third, ethyl acetate, treatment with TFG-1 n-butanol extracts and TFG-1 water extracts to R. dominica showed no contact toxicity. and the LD50 of TFG-1 petroleum ether extract to R. dominica within 24 hours, 48 hours, 72 hours and 10 days was 175.05μg/cm2,133.65μg/cm2,113.37μg/cm2 and 79.34μg/cm2, respectively.Separation and purification of the petroleum ether part was performed through silica gel column for several times. Seven parts were got by TLC testing, the separation rate of partâ… was the highest one, and the value was 75.13%, which mean partâ… was the main ingredients of TFG-1 petroleum ether extract. Treatment with extracts of partâ… , partâ…¥and partâ…¤at dosage 519.95μg/cm2 on R. dominica, the mortality rate of adults was 100%, 80.76% and 46.67% within 24 hours, partâ…¡, partâ…£and partâ…¦had little contact toxicity to R. dominica, partâ…¢showed no toxicity to R. dominica by those testing treatment dosages. Five samples were obtained from partâ… by column chromatography, that was sample a, sample b, sample c, sample d and sample e, and results of separation rates showed sample e was the main components of partâ… . Four samples were got from partâ…¥,and sample i was the main component of partâ…¥. Treatment with sample e and sample i at dosage 259.98μg/cm2 to R. dominica, the mortality of adults was 77.78% and 41.11%, respectively.One monomer compound (compoundâ… )was isolated from the active sample e, the molecular structure of the compound was definitely identified as 1,2-dilinoleic acid-3-stearic acid-triglyceride by the spectrum analysis including MS, 1H-NMR, 13C-NMR and IR. One crystal (compoundâ…¡) was isolated from sampleâ… , and was named as 4a, 14a-dimethyl-cholesterol.6. It was found that compoundâ… possessed strong contact toxicity to R. dominica and S. zeamais, treatment with dosage 129.99μg/cm2, the average mortality of R. dominica and S. zeamais within 10 days was 70.00% and 100.00%, respectively, compoundâ…¡also showed contact toxicity to R. dominica and S. zeamais, treatment with dosage 259.98μg/cm2, the average mortality of R. dominica and S. zeamais within 10 days was 4.44% and 97.78%. After having mixed compoundâ… and compoundâ…¡at the weight rate 1:1, the mixture was tested its contact toxicity to R. dominica and S. zeamais. The results showed treatments of mixed compound had pesticidal effects on R. dominica and S. zeamais, whose LD50 were 217.60μg/cm2 and 45.44μg/cm2 within 10 days; the co-toxicity coefficient of mixed medicament to R. dominica and S. zeamais were 60.57 and 30.23, the values were much smaller than 80, so their medicamental avail relaxational effect.
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