Metabolism, transformation and dynamic changes of alkaloids in silkworm during feeding mulberry leaves

Abstract Metabolism, transformation and dynamic changes of DNJ, 2-O-α-D-Gal-DNJ, fagomine, isofagomine and 4-O-β-d-Glc-fagomine from mulberry leaves in silkworms at different instars were observed. UPLC-Q/TOF-MS and UPLC-TQ/MS methods were adopted for qualitative and quantitative analysis respectively. Three species mulberry leaves were used to feed the silkworm as controls. By analyzing and comparing the content changes of DNJ, fagomine and their derivatives in silkworms and silkworm excrement at different instar, we revealed the dynamic changes, confirmed the enrichment effect of the polyhydroxy alkaloids by silkworm, and inferred the conversion process behind this effect. The experimental results indicated that DNJ and its derivatives turned into some intermediate substances in the metabolic process, and finally they converted back and the content increased. Fagomine and its derivatives interconverted into each other in the process, 4-O-β-d-Glc-fagomine transformed into fagomine, while fagomine transformed into isofagomine. Graphical Abstract


Introduction
Polyhydroxy alkaloids are the compounds which the oxygen atom of carbohydrate ring is replaced by a nitrogen atom and can be considered as sugarmimic (Zhang et al. 2012), represented by 1-deoxynojirimycin (DNJ), fagomine and so on. As a highly effective a-glucosidase inhibitor (Li et al. 2013;Deng et al. 2017), DNJ and its derivatives have hypoglycemic, antiviral effects and less cytotoxicity, and have applied in the treatment of diabetes and obesity (Kimura et al. 2007;Yatsunami et al. 2008;Li et al. 2011Li et al. , 2015Liu et al. 2015). However, DNJ and its derivatives are difficult to synthesize, the content in natural products is low and difficult to purify. The sources of raw materials has become the key to the development of this industry. DNJ and its derivatives exist in mulberry, silkworm, silkworm excrement and some other plants and microorganisms, while the content is highest in mulberry (Asano et al. 2001). Mulberry leaves are the only source of food for silkworm. Silkworm have a significant enrichment effect of the polyhydroxy alkaloids in mulberry leaves (Yoshiaki and Hivonu 1976;Asano et al. 2000;Yin et al. 2010;Liu et al. 2013;Zhang et al. 2016). Studies have been reported that sugarmimic alkaloids are toxic to many phytophagous insects except silkworm (Konno et al. 2006;Hirayama et al. 2007). It is thought that there are some differences in accumulation and metabolism between mulberry specialists and non-specialists Masatoshi et al. 2014). On the basis of previous research, this study researched the characteristic of metabolism and enrichment effects of the alkaloids by silkworm and changes of components in silkworm excrement by analyzing the content changes of the alkaloids in the different instars silkworms. It lays a foundation for revealing the regulation of the metabolism and enrichment, and able to promote the further development of mulberry and silkworm resources.

The metabolism and transformation of DNJ
The content of DNJ in silkworm at 1$4 instar and the first day of fifth instar was relatively low, 0$0.8 mg approximately, in 2$4 days of fifth instar was highest, 2.8 mg in average, in 5$6 day of fifth instar decreased rapidly, 1.0 mg in average. It is consistent with the report that the content of DNJ in the third day of fifth instar was higher than other instars (Yin et al. 2010). DNJ was not detected in newly hatched silkworm, suggested that silkworms could not synthesize DNJ by themselves (Yin et al. 2010). The content of DNJ in silkworm excrement at the first day of fifth instar was relatively low, 0$1.0 mg approximately, in 2$4 day of fifth instar was highest, 3.08, 4.39 and 2.07 mg respectively, and in 5$6 day of fifth instar decreased gradually, 0.4$1.49 mg. The content in silkworm excrement at second day of fifth instar was lower than mulberry leaves at corresponding instars, while at third day of fifth instar was higher than that, suggested that transformations happened in silkworm which increased the accumulation of DNJ (Figure 1).

The dynamic change in silkworm of 2-O-a-D-Gal-DNJ
The content of 2-O-a-D-Gal-DNJ in silkworm was extremely low and the change was indistinctive. The content in silkworm excrement at first day of fifth instar was relatively low, 0$1.4 mg, while in the 2$4 days of fifth instar increased rapidly, 1.65, 2.20 and 1.50 mg respectively, then decreased rapidly in 5$6 days of fifth instar, 0.3$1.0 mg. The content in silkworm excrement at third day of fifth instar was lower than that in the corresponding instars mulberry leaves, but after the fourth day of fifth instar, the content was higher than that. The characteristics of this changes were basically consistent with DNJ ( Figure 2).

The metabolism and transformation of fagomine
The content of fagomine in mulberry leaves of the three groups was 2.05 mg in average. The content of fagomine in the silkworm at the first day of fifth instar was low, 0$0.01 mg, while in 2$4 days of fifth instar was relatively high, 0.45$0.51 mg approximately. In addition, the change of the content was indistinctive in 2$4 days of  Notes: Values represent the mean ± SD (n = 3). The ordinate represents the total content of 2-O-a-D-Gal-DNJ in sample which amount to 0.5 g mulberry leaves.
fifth instar. As previous studies have reported that DNJ and fagomine concentrations in silkworm larvae decreased with growth (Hori et al. 2014), our study also found the content in 5$6 days of fifth instar decreased gradually, 0.02 mg in average. Fagmine was not detected in silkworm excrement, suggested that fagomine have all transformed into other substances in the body of silkworm so that the total content of fagomine decreased after being metabolized by silkworm (Figure 3).

The metabolism and transformation of isofagomine
As increasing of their instars, the content of isofagomine in silkworm increased gradually. Except the first day of fifth instar, the content in silkworm at the other days of fifth instar were almost consistent, 0.075 mg approximately. The change of the content in silkworm excrement at 1$4 instars were similar to that in mulberry leaves at corresponding instars. The content is highest in the third day of fifth instar, 2 mg approximately, and the content in silkworm excrement was higher than that in mulberry leaves, suggested that some bio-transformation in the body of silkworm increased it. The consistent of the change characteristics in silkworm excrement and silkworm suggested that There may be a relationship between them (Figure 4).

The metabolism and transformation of 4-O-b-D-Glc-fagomine
The content of 4-O-b-D-Glc-fagomine in silkworm excrement at different instar was lower than that in corresponding instars mulberry leaves. The content in the silkworm excrement at first day of fifth instar was low, 0$0.025 mg, while in 2$6 days of fifth instar was relatively high. The content of 4-O-b-D-Glc-fagomine in silkworm was extremely low no matter which instar they were, and the change of the content between each instars was indistinctive, 0.006 mg in average. We speculated that one part of 4-O-b-D-Glc-fagomine in the body of silkworm was metabolized and the other was expelled from the body ( Figure 5).

Analysing the changes of alkaloids in silkworm
The content changes of isofagomine, 4-O-b-D-Glc-fagomine and fagomine were added as Total 1, O-a-D-Gal-DNJ and DNJ were added as Total 2. Based on the following formula, the content change in each instar was calculated. The results are shown in Table 1: the content change in N th instar = the content in mulberry leaves + the content in silkworm at N-1 th instar-the content in silkworm at N th instar-the content in silkworm excrement at N th instar. Table 1 showed that the net changes of DNJ and its derivatives in the whole metabolic process were extremely small, but the change between each instar fluctuated greatly: reduced in 1$4 instars and increased in fifth instar, suggested that some biotransformation may exist in this process. The total content of fagomine and its derivatives in silkworm and silkworm excrement at each instar was higher than that in  mulberry leaves at corresponding instars, suggested that transformation occurred during the metabolic process so that the content of fagomine and its derivatives increased.

Preparation of the sample solution
The samples of mulberry leaves, silkworm and silkworm excrement powder, about 0.5 g, were immersed in 7.5 mL 70% ethanol with 1 h. We extracted them by ultrasonic (Frequency: 90 kHz; Power: 500 W) with 45 min. The extracted solution was transferred to 1.5 mL centrifuge tube and centrifugated 10 min at 13,000 rpm/min. The supernatant filtered with a 0.2 lm filter membrane, then the filtrate we got was the sample solution.

Preparation of reference solution
DNJ, fagomine reference standard were dissolved by 40% methanol as the reference mother liquor at the concentration of 14.5 and 12 lgÁmL À1 respectively. Meanwhile, a series of concentration were prepared for use.

Conditions of chromatography and mass spectrometry
The five alkaloids were detected and quantitative analyzed by UPLC-TQ/MS. As 2-Oa-D-Gal-DNJ, 4-O-b-D-Glc-fagomine and isofagomine difficult to obtain, we analyzed them by UPLC-Q/TOF-MS and confirmed five alkaloids with the existing literature (Jakobsen et al. 2001). Based on the previous research in our laboratory, we took DNJ and fagomine as reference to determinate and quantitative analyze the five alkaloids.

Conclusion
By comparing the trend of change in each instar, we found that the content of DNJ and its derivatives decreased firstly and then increased, began to change in an opposite direction at the first day of fifth instar or so. As DNJ can be transformed by microbes and converted into other substances (Hardick et al. 1992), we boldly guessed that metabolic transformation of DNJ and its derivatives have occurred in the body of silkworm. Some intermediate substances arose and substituted DNJ, then a reverse transformation occurred after first day of fifth instar and reproduced DNJ and its derivatives. It could also explain the phenomenon that the content of DNJ and its derivatives in silkworm at fifth instar was higher than that in mulberry leaves. The possible conversion process was presented as Figure S2. The content of fagomine and its derivatives in each instar has significant differences, suggested that silkworm made some biotransformation during the metabolic process and caused the content increased. Fagomine was not detected in silkworm excrement, and the content of fagomine and 4-O-b-D-Glc-fagomine in mulberry leaves was higher than that in the same instars silkworm; by contrast, the isofagomine was enriched significantly in silkworm. It can be seen that the biotransformation occurred in silkworm not only increased the content of fagomine and its derivatives, but also changed the proportion of these components. The possible reaction of metabolic transformation was shown in Figure S3 and Figure S4.
By analyzing the concentrations of three major mulberry alkaloids in two mulberry specialists and six non-specialists insect species, Hori et al. (2014) discovered that DNJ and fagomine concentrations in the two Bombyx larvae were much higher than those in the other larvae in the case of feeding same mulberry leaves. It shows that the enrichment is unique to silkworm. But there is few in depth reports on the silkworm and silkworm excrement, the accumulation and transformation of DNJ in silkworm are unclear. So far some products made from silkworm have been becoming popular for auxiliary therapy for diabetes mellitus (Gui et al. 2001;Kimura et al. 2007;Ryu et al. 2013). Our study could conclude that it is most effective to utilize silkworm at their 2$4 day of fifth instar. In addition, miglitol, an a-glucosidase inhibitor which can be synthesized by DNJ, is mainly prepared by chemical synthesis combine with microbial transformation (You and Yin 2012). The accumulation of alkaloids in silkworm may be able to create new ideas to explore an appropriate production process to obtain miglitol. However, further studies are needed to determine the mechanism underlying these phenomena. We expect that this study can provide inspiration for the efficient acquisition of polyhydroxy alkaloids.