Studies On The Activities Of Five Types Of Natural Coumarins

Natural products especially the secondary metabolites in plants are not only a vital source of prevention and treatment of human’s diseases,but also an important resource for new pesticides discovery.Natural coumarins as a large class of plants’ secondary metabolites include five major subtypes as follows:simple coumarins,furanocoumarins(linear and angular),and pyranocoumarins(linear and angular).Coumarins and their derivatives exhibit wide-ranging biological properties against fungi,bacteria,insects,weeds and inflammatory activity.For finding potential active compounds this study pursued a detailed investigation of the insecticidal,antifungal and anti-inflammatory activities of coumarin derivatives(mainly nature coumarins)which are obtained from six plants or by synthetic methods,and further study the mechanisms of the active compounds.This work provided a theoretical basis for application of active compounds and a clue for the synthesis of coumarins with pesticide activity.1.Five types of natural coumarins from six plants.In my research,32 compounds were identified from six plants,including 22 coumarins covering five different types.From the roots of Angelica dahurica six compounds were identified as isoimperatorin,imperatorin,bergapten,phellopterin,oxypeucedanin,oxypeucedanin hydrate.From the roots of Angelica nitida nine linear furocoumarins were separated and identified as imperatorin,alloimperatorin,pabularinone,neobyakangelicol,byakangelicin,isoimperatorin,cnidilin,phellopterin,isopimpinellin.From the roots of Angelica pubescens six coumarins were obtained and identified as osthol,columbianedin,columbianetin acetate,columbianetin,xanthotexin,umbelliferone.Nodakenetin and 3’-acetoxy-4’-angeloyloxy-3,4-dihydroxanthyletin were obtained from the roots of Peucedanum decursivum.Praeruptorin E and 3’,4’-disenecioylkhellactone were obtained from the roots of Peucedanum praeruptorum.Nine flavonoids were obtained from the leaves of Murraya exotica L.2.The synthesis of the two types of coumarinsMany active natural chemicals in plants,generally as lead compounds,were commonnly changed its substituents by synthetic methods to improve its stability and activity to develop new pesticides.In this study pyran coumarin skeleton and osthol was considered as the lead structures,6 kinds of pyran coumarins and 8 simple coumarins were synthesized from 7-hydroxy coumarin and osthol by substitution,cyclization,epoxidation,ring opening reaction et al and identified as 8,8-dimethyl-2H,8H-pyrano[3,2-g]chromen-2-one,8,8-dimethyl-2H,8H-pyrano[2,3-f]chromen-2-one,6,7-dihydroxy-8,8-dimethyl-7,8-dihydro-2H,6H-pyrano[3,2-g]chromen-2-one,9,10-dihydroxy-8,8-dimethyl-9,10-dihydro-2H,8H-pyrano[2,3-f]chromen-2-one,8,8-dimethyl-2-oxo-9,10-dihydro-2H,8H-pyrano[2,3-f]chromene-9,10-diyl diacetate,10-hydroxy-8,8-dimethyl-2-oxo-9,10-dihydro-2H,8H-pyrano[2,3-f]chromen-9-yl acetate,7-Methoxy-8-(2’,3’-dibromo)-coumarin,Meranzin,Meranzin hydrate,7-Methoxy-8-(2’-hydroxy-3’-bromo)-coumarin,Yuehgesin C,Isomeranzin,Auraptenol,Murrayone.Among them a simple coumarin(7-Methoxy-8-(2’-hydroxy-3’-bromo)-coumarin)are new compound.This research made the preliminary attempt to synthesis coumarins especially pyran coumarins for new pesticides and provide theoretical basis for further development and application of coumarins.3.The study on the five different types of coumarins against Mythimna seperata.As people increasingly attention to the living environment,researches on pesticides are needed to develop new effective control ways which are both safe to humans and friendly to environment.Natural product-based botanical pesticides undoubtedly provide such an alternative because of their enormous structural diversity and their wide spectrum of biological activities.On this study 48 coumarins covering five different types and other compounds were tested against the oriental armyworm Mythimna separate by dipping leaf disc bioassays and topical application.Leaf disc bioassays results showed that 20 tested compounds had better antifeeding effect(more than 46.75%of antifeedancy)on M.seperata than Osthol(46.75%of antifeedancy),no significant differences existed in the five different types of coumarins,in each type of coumarins there were some coumarins with good antifeeding effect on M.seperata,such as simple coumarin Murrayone(77.91%of antifeedancy),linear furancoumarin bergapten(94.30%of antifeedancy),angular furancoumarin Columbianadin and Columbianetin(82.70%and 81.07%of antifeedancy),linear pyrancoumarin Pd-D-V(86.65%of antifeedancy),angular pyrancoumarin Disenecioyl Khellactone(70.86%of antifeedancy).Leaf disc bioassays results also showed that in five different types of coumarins,angular pynancoumarins(more than 20%of mortality)had better insecticidal activity against 2rd instar larvae of M.seperata than linear furancoumarins and angular pynancoumarins(less than 20%of mortality).Simple coumarin Murrayone(70.00%of mortality),angular furancoumarin Columbianadin(70.00%of mortality)and Columbianetin(70.00%of mortality)had better insecticidal activity than Osthol(0 of mortality),the three coumarins had better insecticidal activity with the EC50 values 0.704、0.766、0.801 mg·mL-1 against 2rd instar larvae of M.seperata than Osthol but weaker than Rotenone by Leaf disc bioassays.Topical application results showed five different types of coumarins of angular furan coumarins had better insecticidal activity than other types of coumarins,but all compounds had weak or no contact poisoning effect with mortality rate less than 50%on M.seperata larvae relative to Rotenone with 100%of mortality rate at 1 mg·mL-1.In conclusion,this study found Murrayone,Pd-D-V,Columbianadin and Columbianetin had better antifeeding effect and insecticidal activity than Osthol,but not as good as Rotenone.4.The study on the five different types of coumarins against Plant pathogenic fungi.Plant pathogenic fungi are the primary infectious agents of plants during thelr developmental stages and have caused devastating million-dollar crop losses worldwide.Researches on fungicides are challenged by the urgent needs to develop new effective control ways which are both safe to humans and friendly to environment.Natural product-based botanical fungicides undoubtedly provide such an alternative,not only because of their enormous structural diversity but also for their wide spectrum of biological activities.This study aimed to pursue a detailed investigation of the antifungal activities of coumarin derivatives(mainly nature coumarins).In this report,we describe an in vitro antifungal evaluation of 41 coumarin derivatives against Sclerotinia sclerotiorum,Thanatephorus cucumeris,Botrytis cinerea,Fusarium graminearum,and Colletotrichum capsici.The results showed that coumarins from each of the five major subtypes had either good or weak fungicidal activities,no significant differences existed in the five different types of coumarins,in each type of coumarins there were some coumarins w good antifungal activity,such as Murrayone(a simple coumarin)exhibited better activity against C.capsici with EC50 values of 24.9 μg·mL-1 than commercial fungicide Osthol with EC50 values of more than 500μg·mL-1.Imperatorin(a linear furancoumarin)exhibited better activity against S.sclerotiorum with EC50 values of 21.9 μg·mL-1 than commercial fungicide Osthol with EC50 values of more than 50μg·mL-1.Libanorin and Cniforin B(both angular furanocoumarins)had activities against S.sclerotiorum,B.cinerea,F.graminearum,and C.capsici with EC50 values of 18.8,18.8,25.7,29.5μg·mL-1 and 26.5,34.5,45.5,32.2μg·mL-1,and both demonstrated their higher potencies against S.sclerotiorum and C.capsici(EC50<50 μg·mL-1)compared to the commercial fungicide Osthol(EC50>50 μg·mL-1).Pd-D-V(a linear pyranocoumarin)showed more excellent activity against S.sclerotiorum(ECso=13.2 μg·mL-1)than commercial fungicide Osthol(EC50>50 μg mL-1)and moderate activity against B.cinerea,F.graminearum and C.capsici(EC50=35.5,33.5,37.3 μg·mL-1).Disenecioyl Khellactone(an angular pyranocoumarin)showed good activity against S.sclerotiorum,T.cucumeris,B.cinerea and F.graminearum(EC50=29.1,36.2,11.0,40.1 μg·mL-1),and demonstrated higher potencies against S.sclerotiorum and B.cinerea(ECso<50 μg·ML-1)compared to the commercial fungicide Osthol(EC50>50,23.5μg·mL-1).6 synthetic pyranocoumarins exhibited more significant antifungal activities against S.sclerotiorum,T.cucumeris,and B.cinerea with EC50 between 4.7 and 9.9 μg·mL-1 than commercial fungicide Osthol(EC50>10.0 μg ·mL-1).Several linear and angular pyranocoumarins were first found high antifungal activities,there are no reports on the antifungal activities of angular pyranocoumarins to date,so further studies will be necessary to develop these results.The results also showed that 11 kinds of natural coumarins had good antifungal activities with EC50<30 μg·mL-1 against some tested fugi and had reached the biological fungicides level(Osthol against powdery mildew with EC50 30.195 μg·mL-1),which can be used as lead compounds for the development of botanical fungicides.From the results of the insecticidal and antifungal activities of the tested coumarins Murrayone,Pd-D-V and Columbianedin had better insecticidal and insecticidal activity than Osthol,which can be used as lead compounds for the development of botanical pesticides.5.The study on the anti-inflammatory activity of tested compoundsInflammation is the body’s attempt at self-protection to remove harmful stimuli and begin the healing process,Chronic inflammation can eventually cause several diseases and conditions including some cancers,rheumatoid arthritis,atherosclerosis,periodontitis,and hay fever.Looking for anti-inflammatory ingredients from Traditional Chinese Medicine has become a research hotspot.In the present study,we investigated the anti-inflammatory activity of some natural constituents to support its plants’ traditional use and find active compounds.In vitro cytotoxicity and anti-inflammatory against LPS-induced NO levels macrophage RAW264.7 cells of these compounds were tested.The results showed that linear furanocoumarins had weaker anti-inflammatory activity than other tested type of coumarins.In other tested type of coumarins,Isomeranzin(a simple coumarin)had good anti-inflammatory activity and no cytotoxicity.Cniforin B(an angular furanocoumarin)and Anomalin(an angular pyranocoumarin)had good anti-inflammatory activity,but both had cytotoxicity.Imperatorin(a linear furanocoumarin)and isopimpinellin(a linear furanocoumarin)had weak anti-inflammatory activity,but higher anti-inflammatory activity than other tested linear furanocoumarins.Murrayone(a simple coumarin)and Isomeranzin(a simple coumarin)had good anti-inflammatory activity,their structures different from other simple coumarins is the substituents on eight position containing 2’ substituent as carbonyl,which may be its efficacy reactive groups;Murrayone have strong cytotoxicity,which may be the reason for its good insecticidal and antifugal activities,its structure different from other simple coumarins is the substituents on eight position containing conjugated double bond;Murrayone has good anti-inflammatory activity but strong cytotoxicity,so it is not suitable as major efficacy components for Traditional Chinese Medicine M.exotica.Isomeranzin has good anti-inflammatory activity and no cytotoxicity,its structure different from Murrayone is only the substituent on eight position that 3’ position not double bond,which may be the reason for its no cytotoxicity.The anti-inflammatory compositions of M.exotica were not clear,in this study coumarins were not isolated but some flavonoids from M.exotica,and the anti-inflammatory activity of the flavonoids were determined,the results demonstrated that 5,7,3’,4’,5’-pentamethoxyflavanone(PMFA)from M.exotica has good anti-inflammation activity and no cytotoxicity,which provide theoretical-reference for the establishment of major efficacy composition for quality standards of Traditional Chinese Medicine M.exotica used for inflammatory diseases.6.Effect of Pd-D-V on morphological and physiological characteristics of S.sclerotiorumIn this study,the effect of Pd-D-V on morphological and physiological characteristics of S.sclerotiorum has been invistigated.The results indicated that Pd-D-V had good inhibition on mycelia growth of S.sclerotiorum with offshoot of top increasing.After Pd-D-V treatment,cell membrance permeability,the chitinase activity and the N-acetyl glucosamine content increased obviously,but oxalate,EPS content,protein content and glucose content clearly decreased.With chitinase activity increased,more n-acetyl glucosamine was generated by the hydrolysis of chitin the main components of the cell wall,which caused n-acetyl glucosamine content higher,so the structures of cell wall were destroyed thus inhibiting the growth of the fungi.The protective and curative test of Pd-D-V that curative effect was better than protective on leaves.The results showed that Pd-D-V had systemic translocation in leaves.These results will increase our understanding of action mechanism of Pd-D-V against S.sclerotiorum.