Isolation, Purification And Identification Of An Important Pigment Sepiapterin From Integument Of The Lemon Mutant Of Silkworm, Bombyx Mori

Sepiapterin (SP) is a kind of yellow pterin pigment, belongs to the derivativeof pteridine. Previous studies showed that SP plays an important role in many cellmetabolism processes and is one of endogenous pigments to form insect body color. In themetabolic pathway of GTP, SP is an intermediate product to synthesize tetrahydrobiopterin(BH4), a crucial coenzyme for aromatic amino acid hydroxylases and nitric-oxide synthase.Disorder of synthesis and regeneration of BH4would cause some neurological andphysiological diseases. Many animal experiments and clinical researches suggested thatBH4deficiency-dependent diseases could be improved by oral supply of BH4and/or SP.lemon (lem) is a body color mutant of the silkworm, Bombyx mori, which displaysyellow body coloration during larval developmental stages markedly different from that ofwild-type strains. Studies showed that SP was contained in the integument of lem larvae inextremely high concentration so that the lem mutant is an invaluable biological resource toextract SP. However, knowledge about extracting SP from silkworms is limited by far.In this study, in order to set up an effective experimental system for isolation andpurification of SP from lem silkworms, we optimized the homogenization solvent, elutionbuffer and separation chromatographic column. The results showed that ethanol is the mostsuitable solvent to homogenize the integument with a concentration of50%andsolid-liquid ratio at1:20(g/m1). SP was purified successively by column chromatographyof cellulose Ecteola, sephadex G-25-150and cellulose phosphate and identified by paperchromatography, UV-Vis absorption spectrometry and HPLC.In addition, we constructed a stable and accurate HPLC method to identify SP andconduct qualitative and quantitative analyses. ODSC18（250mm×4.6mm，5um）was usedas the stationary phase. The mobile phase was methanol-0.006%acetic acid (20:80,v/v)solution, with the flow rate of1mL／min. The column temperature was25℃,and thedetecting wavelength was420nm．The results suggested that the HPLC method was simple,sensitive and accurate. SP of high purity was achieved and the harvest reached to about40ug per gram integument in our experiments.Together, an effective system for SP isolation, purification and identification from thesilkworm B. mori has been established in this work, which is lower cost, time saving, andhigher output and maybe of more medical security than the chemical synthesis methodused in SP industry. Aiming at utilizing novel B. mori genetic resources, our findings provides scientific basis for industrially extracting natural SP from lem silkworms in largescale in the future. It is of great significance that we are trying out producing BH4in vitroby using the extracted SP as a substrate.