Chemical Constituentsand Tumor Associated Activity Study On Blaps Rynchopetera,a Medical Insect Of Yi Minority In Yunnan

In this dissertation,a series research work focuses on Blaps rynchopetera Fairmaire,a medical insect popular in Yi minority area in Yunnan,including the chemical constituents study and antitumor associated cytotoxic,antioxidant activity and immunomodulatory activity research.In the first chapter,the chemical constituents and the tumor cell growth inhibition,antioxidation and immunomodulation activities for different extract and pure compounds were studied.Guided with cytotoxicity screening,76 compounds,including 9 new compounds and 1 new natural product have been isolated and identified from the chloroform,ethyl acetate and n-butanol extract parts of B.rynchopetera,by a variety of chromatography（siliga gel,Sephadex LH-20,RP-18）.The cytotoxicity of 5 new compounds 1,2,5,6,7 and several known ones were tested in vitro against ten common human tumor cell lines A549,AGS,Caco-2,Hela,HepG2,K562,MDA-MB-231,TE-1,U251,and Bel-7402 using the modified MTT method.The results showed that rynchopeterine B（2）exhibited moderate anti-tumor activity against Caco-2 cell and A549 cells with IC₅₀ values of 119.7μg/mL and 108.5μg/mL,respectively.Rynchopeterine C（5）exhibited mild anti-tumor activity against Caco-2cells with an IC₅₀ value of 158.7μg/mL.Our previous studies revealed that the ethyl acetate fraction of B.rynchopetera exhibited strong DPPH radical scavenging activity,with an IC₅₀ value of 23.35μg/mL.Some compounds obtained from this active fraction were measured the DPPH radical-scavenging activity.All the tested compounds,including new compounds 1-7 and other knowm compounds 15,22,23and 25 showed impressive anti-radical activity at the same grade with positive control vitamin C,except for 7.It’s worth noting that compound 15 exhibited significant activity with an IC₅₀ value of 1.02μg/mL.Their effective anti-radical activities could be attributed to the hydroxyl group in the molecules.Furthermore,in vitro immune activity for different parts of B.rynchopetera and in vivo for ethyl acetate fraction was investigated.The total extract,chloroform fraction,ethyl acetate fraction and polysaccharides could enhance the phagocytic function of mice peritoneal macrophages.The ethyl acetate fraction could enhance immuno–activities of normal and immunocompromised mice,increased the immune organ index,the level of hemolysin antibody and peripheral blood immune cells.In the second chapter,the content of polysaccharides and its composition in B.rynchopetera were determined.Polysaccharides were prepared by water extraction,alcohol precipitation and Sevage method to removing protein.The polysaccharides content of B.rynchopetera was determined as 3.97%with phenol sulfuric acid method.PMP pre-column derivatization HPLC method was used to analyse the monosaccharide composition and content of purificatory polysaccharides from B.rynchopetera.Eight kinds of monosaccharide were detected,and glucose was the main constituent with a content of 45.4 mg/g,while other monosaccharides′content varied from 11.47 to 21.45 mg/g,the total content of 8 monosaccharides were 171.7 mg/g.Third chapter,the content of polyphenols,which was the characteristic and active components in B.rynchopetera were determined.Many polyphenols possess good antioxidant activity.Using Folin-phenol colorimetry method,the content of polyphenols was tested using gallic acid as control.The content in dried body,fresh body,defense secretion collected by stimulated with ethanol and with direct touch its tail gland were 4.80±0.02,8.07±0.59,10.91±0.40 and 34.44±0.14 mg.g^-1 respectively.Fresh insect body had higher polyphenols than dried one.In the fourth chapter,the volatile components of the defense secretion and the petroleum ether extract of B.rynchopetera were analyzed by GC/MS,and the activity of tumor cell growth inhibition for the defense secretion and its main substituted benzoquinone compounds were screened.There were 77 main peaks at the total ion chromatogram in the petroleum ether extract,while 56 of them,accounted for 95.27%of the total peak area,were identified by NIST automatic retrieval.The main constituents were long chain fatty acid and fatty acid ester,long chain alkane.The richer compounds（relative amount more than 5%）were（E）methyl 9-octadecenoate,（33.27%）,methyl hexadecanoate（15.17%）,methyl stearate（8.16%）and ethyl oleate（5.69%）.The defense secretion of B.rynchopetera obtained through direct stimulating could be divided into two parts,the supernatant liquid was clear yellow oil and the lower was brown yellow emulsion.The GC/MS analysis and cytotoxic activity detection of the total defense liquid and the two layers were carried out.Results showed that the total defense secretion and defense secretion sublevel had strong cytotoxic activity against testing tumor cell lines,while the upper layer had not.The total defense was comprised with 3 main compounds,2-ethyl benzoquinone（31%）,1-thirteen（28.02%）and 2-methyl allyl benzoquinone（22.86%）in the total ion current of GC/MS,the lower secretion had the same main peaks just with higher content of 2-methyl p-benzoquinone and 2-ethyl benzoquinone,lower 1-tridecylene content,while the relative content of 1-tridecylene reached more than 90%in the upper layer secretion.The separation and purification of defense secretion to obtained 1-tridecylene and the mixture of 1-methyl benzoquinone and 2-ethyl-benzoquinone.The cytotoxic activity screening result suggested that two substituted benzoquinone exhibits stronger activity than the total defense secretion and the lower layer,for same cell lines,which were no significant differences with the positive drug cisplatin.1-tridecylene showed no cytotoxic activity.The result was in good accordance with above three defense secretion groups.The last chapter,research progress of Blaps insects,especially the medical insect,B.rynchopetera was reviewed.