Studies On Acaricidal Action Mechanism And Bio-guided Isolation Of Bioactive Substance From Artemisia Annua L.

In the course of plant development and growth,a great number of metabolites were produced in the process of photosynthesis and respiration,and some of these products have physical activity or other use,have been exploited since ancient times.Plant components are important parts of natural drugs.Since ancient times,and during the fighting against the disease human body to test drugs,the natural medicinal plants mainly plant drugs application has accumulated a wealth of experience.In China,botanicals also known as the Chinese herbal medicines,and together with the traditional Chinese medicine constitute the cultural treasures of the Chinese nation,but also a valuable heritage of mankind.Artemisia annua L.is an annual herbaceous plant,belongs to Compositae Artemisia,is the main source of qinghao traditional Chinese medicine.The whole height was 40-150cm,specially over 200cm,and the growth period about 210d.Artemisia annua are distributed across in China, widely ecological adaptability,and often grew at the slopes,the woodlands and the wasteland.The medicine farmers often harvested the aerial parts when flowers in full bloom in the autumn,removed the old stems,dried,can be medicinal.Arternisia annua with high medicinal value and artemisinin derivatives can produce a lot of series medicines.Artemisinin indicated malaria,tuberculosis hot flashes,heat stroke,pruritus,urticaria,and seborrheic dermatitis,and other anti-mosquito.AS natural and geographical reasons,Chongqing is the world's largest basement of Artemisia annua,with "Artemisia annua hometown" reputation.The development of Artemisia annua industry has unique advantages in Chongqing.Mites,particularly phytophagous pest mites,are one of the important organisms which seriously harmful to our agricultural production.It has the characters of small individual size,fast breeding,the high population density,great harmness,and it can harm more than 150 crops. According to incomplete statistics,China's annual used for citrus,apples,pears,peaches,cotton, vegetables,wheat,tea etc 8 major crops,the acaricide control costs amounted to more than 9 billion yuan RMB.And as the elevation of mites resistance to chemicals,the control costs increase year after year.Faced with chemical pesticides generated by the various problems and insecticide or acaricide conventional means of research and development has become increasingly difficult, bioactive natural products coming into the mainstream of plant protection time has come.This study was funded by the National Natural Science Foundation(30671392)and the Chongqing Municipality scientific and technological project(2001-6599),the main research concerning grown in April,May,June,July and September of Artemisia annua root,stem,leaves, solvent extraction rate,and the bioactivity of various extracts against Tetranychus cinnabarinus, Panonychus citri and Petrobia harti,extract of Artemisia annua in July inhibition action of cowpea seeds radicle and hypocotyl growth,tracking acaricidal substances scopoletin isolated from Artemisia annua,and the effect on SOD,CAT,POD,GST,CarE,ACHE,MAO,Ca²⁺-ATPase of T. cinnabarinus treated by scopoletin.Through nearly three years of research,the main findings were followed:1 The extraction rate of Artemisia annuaThe materials of Artemisia annua plant roots,stems,leaves were extracted by conventional room temperature extraction,extraction at room temperature,stirring extraction at room temperature and microwave-assisted extraction etc using a series of solvents.Four kinds of extraction methods on the extraction rate do not differ greatly from the impact,and from an economic point of comparison,stirring extract at room temperature more appropriate.The roots,stems and leaves of Artemisia annua in April,May,June,July and September were extracted with different solvent,respectively,from the extraction rate,the parallel extract approach extraction rate generally higher than the extract of the sequenced.Water extraction rate is the highest (root stems extraction rate is higher than other solvent extraction rate),followed by acetone,and then ethanol.From different parts of the plant,the leaves the relatively high rate of extraction,followed by the roots,and stems the lowest.2 Suitable methods to botanical acaricide bioassayBioassay methods play a key role in the research and development of pesticide,new bioassay method is tantamount to the discovery of new pesticides.Based on this we compared a glass capillary method,slide impregnation method,leaf impregnation method.The results showed that glass capillary method was no significant difference with the FAO recommended slide impregnation method.And its pesticide usage was a significant decline and suitable for the botanical acaricide bioassay.3 Acaricidal activity of Artemisia annua extracts3.1 Bioactivities against Tetranychus cinnabarinusThe aim of this study was to determine the best extraction technique,the suitable solvent and the optimal plant parts and the acaricidal activities of Artemisia annua L.The acaricidal bioactivities against Tetranychus cinnabarinus of total 135 extracts of petroleum ether(30-60℃),petroleum ether (60-90℃),ethanol,acetone and water parallel and sequenced extracts from the leaves,stems and roots of Artemisia annua L.in different periods of April,May,June,July and September were determined by slide-capillary method in laboratory.The results showed that the acaricidal bioactivities elevated with the development of A.annua plant at the concentration of 5 mg·mL^-1.The general tendency exhibited the sequence of July＞June＞May＞April,but September decreased comparing with July.However,the most effective extracts in five months were all acetone parallel extract from A.annua leaf,and the corrected mortalities after treatment 48h were ranged from 74% to 100%.The median lethal concentrations(LC₅₀)against T.cinnabarinus of acetone parallel extracts from A.annua leaves in April,May,June,July and September were 1.3817,0.9443,0.8376, 0.4341,and 0.5986 mg·mL^-1,respectively after treatment48h.The Artemisia annua in July acetone parallel extract showed the highest activity,the growth cycle of Artemisia annua,the vegetative growth period is a crucial period of the defensive ability of the outside world is the strongest,with the experimental findings anastomosis.3.2 Bioaetivities against Panonychus citriLeaves of Artemisia annua in June except petroleum etherâ…¡sequenced extract and water sequenced extract,other than the activity of the extracts of the root extract significantly improved. One acetone parallel extract against Panonychus citri at the concentration of 5 mg·mL^-1treatment for 48 h,the corrected mortality was 88.84%,and petroleum etherâ…¡under the same conditions against Panonychus citri also reached 84.27%.The different solvent extracts of leaves of Artemisia annua in July against Panonychus citri and their biological activities were significantly higher than the roots and stems.Leaf acetone parallel extract with the highest bio-activity,at the concentration of 5 mg·mL^-1for 48 h,the Panonychus citri corrected mortality was 100%.Leaves of Artemisia annua in July of the acetone parallel extract of Panonychus citri,biological activity was significantly higher than that in June,the Panonychus citri 48h after treatment,the LC₅₀(0.4222mg·mL^-1)in July,only 44%of June(0.9489mg·mL^-1).3.3 Bioactivities against Petrobia hartiThe acaricidal bioactivity against Petrobia harti of all solvents extracts of Artemisia annua in June was generally not too strong.Different parts of Artemisia annua in July by using several different polar solvent extracts,the biological activity against Petrobia harti shown superior to in June.Of which leaves in July parallel acetone extract was the highest biological activity,treatment for 48h,the corrected mortality was 95.00%.The acetone parallel extract of Artemisia annua in July against Petrobia harti treatment after 48h,the LC50 was 0.4715 mg·mL^-1,and in June was 0.9083 mg·mL^-1.4 Phytotoxicity of Artemisia annua extracts in July to cowpea seedsIt has been confirmed that the plant Artemisia annua in July showed a relative strong acaricidal activity against Tetranychus cinnabarinus,Panonychus citri and Petrobia harti.However,in order to clarify the toxic effects of plant extracts on the seed germination rate of cowpea,which was principal host plant of the spider mite,and also the radicle and hypocotyl growth inhibition.These extracts were the parallel and sequenced extracts of different solvents from the roots,stems and leaves of Artemisia annua in July.The results showed that,the impact of cowpea seed germination of Artemisia annua in July roots,stems and leaves different solvents extract varied with the solvent species and different extraction methods.Of which the impact of stem ethanol sequenced extract against cowpea seed germination rate was greatest,and that showed significant inhibition effect,and the germination rate was only 23.81%.Otherwise,the leaf acetone sequenced extract also showed strong inhibition effect on cowpea seed germination rate,of which the germination rate was 40.48%. The different solvent extracts of root stems and leaves Artemisia annua in July were tested their phytotoxicity against cowpea seeds,and the results showed that weak inhibition effect on radicle and hypocotyl growth,and maybe also exist some material which promoting radicle,hypocotyl growth.5 Bio-guided isolation of acaricidal active material from Artemisia annuaWith the bio-guided isolation method,the acaricidal activity of the different components isolated from Artemisia annua July parallel leaf acetone extracts by column chromatography were determined.Separated at the end of the 20 kinds of components,the acaricidal activity of component 17 was strongest,followed by component 18 and 8.LC₅₀(48h)of components 17,18 and 8 were 0.1675,0.4368 and 0.3753 mg·mL^-1,the active component 17 arising about 2.6 times compared with July of the acetone extract.The acaricidal activity against Panonychus citri of different components isolated by column chromatography from the acetone parallel extract of Artemisia annua leaf in July were determined in laboratory,in which the acaricidal activity of 17^thcomponent was highest treatment for 48 h, corrected mortality 96.65%,and there was significant differences with other components.Use bio-active guided isolation from the acetone extract of Artemisia annua by column chromatography,the acaricidal activity of the total 20 kinds components were also determined in laboratory,acaricidal activity of the 19^thcomponent was highest at 48 h after the treatment and the corrected mortality was 69.28%. Considered from the two factors of effective content and biological activity,component 17 is the most valuable component,17^thgroup contains three main components,of which the content of 17-3 was maximum 77.64%,17-1 second 16.21%,17-2 only 5.31%.TLC on the three components of the color of iodine, visible pot only one,and no smearing.The acaricidal activities against Tetranychus cinnbarinus of three components were determined in laboratory,the results showed that 17-3 has the strongest acaricidal activity treatment for 48 h,the LC₅₀for T.cinnabarinus was 0.1014 mg·mL^-1,elevated compared to the LC₅₀of 17^th0.1657 mg·mL^-1.According to the selected 17-3 re-crystallization experiments,select a suitable temperature with methanol,acetone,chloroform,ethyl acetate,n-butane dissolved,recrystallization,then put in iceberg cooling and crystal,and to choose the best temperature and solvent.According to crystal shape and the amount of solvents used,selected the best condition was ethyl acetate in 55℃. Followed by HPLC-MS,¹H-NMR,IR,and other means to determine 17-3 for the scopoletin, molecular weight:192.17,molecular formula:C₁₀H₈O₄.Bioassay results showed that the acaricidal activity against T.cinnabarinus of scopoletin was better than 17-3.6 Acaricidal mechanism of scopoletin against Tetranychus cinnabarinusTreatment after by scopoletin,the SOD activity of Tetranychus cinnabarinus was decreased compared with the control,and varied with the scopoletin concentration changed.Along with the activity of the extension of treatment time increased at first and then a downward trend,but the overall performance of the activity by the pharmaceutical treatment lower than that of control.All treatment measured by the activity of the catalase increase,which could be activated after treatment by scopoletin in T.cinnabarinus.POD activity decreased significantly compared with the control, and varied with the changes of scopoletin concentration.Overall,T.cinnabarinus treatment by scopoletin,the POD activity in vivo was inhibited obviously.With the extension of the treatment time,the overall vitality of GST on a declining trend, especially treatment after 48h,the activity to a lower level.But virtually all of the treatment groups were higher activity than control,in other words,GST activity of T.cinnabarinus treatment by scopoletin has improved.Scopoletin activated T.cinnabarinus carboxylesterase activity,and with the increasing concentration of scopoletin activity of the extent of activation also increased. Acetylcholinesterase activity increased first and then showed downward trend.Treatment by scopoletin the Ca²⁺-ATPase activity of T.cinnabarinus,compared with the control,had declined in certain degree,that is to say scopoletin could inhibit Ca²⁺-ATP enzyme activity in a certain extent. On monoamine oxidase activity,the treatment group and control group of MAO close to the vitality of the trend changes,but were all lower than the control group,therefore,the MAO activity of T. cinnabarinus was inhibited in a certain extent.