Determination And Analysis Of 1-deoxynojirimycin In Mulberry And Silkworm

1-Deoxynojirimycin (DNJ) and it's derivate is a kind of effective inhibitor ofα-glycosidase inhibitor, and could be used to cure diabetes, adipose and viral infection disease. A few plants and microorganisms have been found synthesizing and accumulating DNJ, among of them the content of mulberry leaves is the highest. Silkworms fed on Mulberry leaves, and DNJ can be accumulated in the body, the content of DNJ in silkworm body much more than in Mulberry leaves. However, the research on DNJ content and accumulation rule in silkworm body and Mulberry leaves is insufficient due to the limitation of testing method. Meanwhile,plant alkaloid is an important secondary metabolite which formed in the co-evolution process between plant and insects, DNJ is a kind of alkaloid in Mulberry, but the researches on the effects to other insects than silkworm are rare, we did some researches on these problems,the main results are as follows:1 DNJ was extracted from silkworm and mulberry leaves powder by using 0.05mol/L HCl. After blending silkworm powder or mulberry leaves powder extraction and borate potassium solution (pH= 8.5), FMOC-Cl was added to derive DNJ. The DNJ-FMOC was produced which can absorb ultraviolet radiation and then was tested with ultraviolet radiation inspector. The results show that the separation chromatogram of the derivation production is very satisfactory. The peak area of DNJ-FMOC is correspond with the concentration of DNJ. The average of recovery rate is 94.98%. All of the precision test, stability test, recurring test and the recovery test show that the method is credible and stable, and it can be used as an effective method in determining the DNJ content in mulberry leaves, silkworm and other medicines.2 The 1-deoxynojirimycin(DNJ) contents in different silkworm larvae, various growth periods and tissues in 5^th instar were investigated in the paper by Reverse-phase High-performance Liquid Chromatography. The results showed that there were obviously DNJ changes in larvae between Bombyx mori and Bombyx mandarina ,as well among various Bombyx strains. The highest DNJ content exists in Bombyx mandarina larvae. The average is 0.4278%. The lowest content exists in silkworm with yellow blood, is 0.2205% of the average. The content of translucent silkworm is extremely higher than the normal one, and the normal is higher than that with yellow blood. The differences of DNJ among different organizations are obvious, the blood has the highest, then decrease in the midgut, fat body ,skin in order. The silkgland almost has no DNJ. The DNJ of larvae varies markedly within the 5^th instar. It reach the peak at the 4^th day with 0.329% and then decreased quickly. From the consideration of using DNJ as a medecine to cure diabetes, it is an ideal to make silkworm power with translucent silkworm larvae at the 3^rd-4^th day of 5^th instar.3 DNJ contents of 3 cultured mulberry species including 9 varieties M.multicaulis Perr., M.alba Linn., M.bombycis Koidz. and 4 wild mulberry species including.6 varieties M.mongolica Schneid., M.mongolica vardiabolica Koidz., M.laevigata Wall, M.cathayana Hemsl.weve determined. The results show the DNJ contents of different species is M.mongolica Schneid.> M.mongolica vardiabolica Koidz > M.laevigata Wall> M.multicaulis Perr. > M.bombycis Koidz. > M.alba Linn.>M.cathayana Hemsl.from higher to lower in order.The DNJ contents of M.mongolica Schneid., M.laevigata Wall.and M.mongolica vardiabolica Koidz. were significantly much more higher than those of M.multicaulis Perr., M.bombycis Koidz., M.alba Linn., M.cathayana Hemsl. Among the cultured species, an variety ' s DNJ content of M.alba Linn from Xinjiang Hetian district is the highest,0.11%.That of An variety with vertical branches of. M.alba Linn is the lowest,0.021%.The DNJ content of M.multicaulis Perr. is 0.075-0.105%.The content of DNJ was significantly higher among different regional varieties. The DNJ in leaf latex was significantly higher than that of all leaf, blade and jein. The results showed that the wild mulberry species were the hopeful materials to derive DNJ and they could be further developed to multi-purpose mulberry varieties for cultivation.4 Using HCL and strongly acidic styrene type caution exchange resin to extract and purify DNJ products from silkworm and mulberry leaves powders. The DNJ content of silkworm and mulberry leaf powder after purification was 1.4789% and 0.4899%.DNJ purity was 62.9269% and 85.7877%.It showed this method was quite feasible. The product DNJ purity was higher than 60%,so it is the quite ideal methodat present.5 The cell line coming form the ovaries of Tobacco moth and the cell line BmN from ovaries of silkworm were used as the experimental objects. Treatments were applied to the 3 kinds of insect cell lines with different concentration of DNJ respectively. After treatment, MMT methods was used to investgate the inhibition rate for the cell proliferation in vitro. After treatment with DNJ and incubation for 36h, the cell proliferation of 3 cell line exhibited a little inhibitory or acceleration without certain trend, indicated that DNJ has no significant effects on the cell proliferation at the moment. Incubation 72h with DNJ , the growth of the BmN cell performed accelerating effects according to the DNJ does, the lower concentration exhibited a slight acceleration,and the higher concentration exhibited much acceleration. The sf21 exposed to different concentration DNJ for 72h ,the proliferation of cell was markedly inhibited correndence to the dose, the high dose showed higher inhibition and the low dose showed lower effects. This means that DNJ has a strong inhibition effects on qrowth of sf21 .The sf9 cell did not appear the effects.