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Quantitative Analysis of 1-Deoxynojirimycin Content Using Silkworm Genetic Resources
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1-Deoxynojirimycin(1-DNJ or DNJ), a component in silkworm powder, prevents glucose from being absorbed into the bloodstream from the small intestine by inhibiting α-glucosidase activity. This study compared the functional components of 1-DNJ from various silkworm species using a gene database with those of 1-DNJ produced by silkworms bred through cross-combinations. We utilized comparisons of geographical origins and species of silkworms using a gene database and discovered that 1-DNJ activity was ranked in the following order by species, Japanese (SK-1) > Chinese (C48) > European (Rock191). 1-DNJ constituted varying percentages of silkworm organs in the following order, blood > epithelial tissue > body fat > silk glands. With regard to sex, 1-DNJ, levels were higher in males than females. However, 1-DNJ levels with respect to various genetic traits (blood color, silk color, and egg color) were consistent. In order to study 1-DNJ changes that occurred during cross breeding of the silkworm gene, we bred cross-combinations utilizing SK-1 and C48 silkworms. In conclusion, in order to provide information about the constituents of functional materials contained in silkworm powder, it is imperative that silkworm cross breeding occurs so that the database of functional materials extracted from silkworms will expand.

Bombyx mori L. , 1-deoxynojirimycin (1-DNJ) , α-glucosidase , cross-combination
  • Introduction

    The silkworm, Bombyx mori L. has long been used in China and Korea as a folk remedy for the treatment of diabetes and is an economically important insect that converts mulberry leaf protein into silk.

    The anti-diabetes mechanism of silkworm powder and its extracts was found to effectively inhibit α-glucosidase in the human small intestine. Related to this factor, the major functional component of silkworm powder is 1-deoxynojirimycin (1-DNJ), an intestinal α-glucosidase inhibitor present in mulberry leaves (Asano, 2003) and sericulture products such as silkworm powder (Asano et al., 2001), which produces-lowered blood glucose levels. In particular, 1-DNJ and its derivatives have been extensively investigated for their α-glucosidase inhibitory effects on postprandial hyperglycemia; it also has applications in nutraceutical medicine as a prophylactic that delays the progression of type 2 diabetes (Asai et al., 2011; Kimura et al., 2007; Vichasilp et al., 2012). 1-DNJ is naturally synthesized in some vascular plants, such as the mulberry and dayflower (Yagi et al., 1976, Shibano et al., 2004). In addition, DNJ has long been thought to be produced in several strains of Bacillus (Hardick and Hutchinson, 1993, Stein et al., 1984) and Streptomyces spp. (Ezure et al., 1985; Hardick et al., 1991, Paek et al., 1997). Further, Ryu et al. (1997) first reported that the silkworm larval powder of the 5th instar (prepared by lypophilization) had positive effects on diabetic patients, the blood sugar-lowering effects were further verified by subsequent research (Han et al., 2007).

    According to previous data, the concentration of 1-DNJ in most commercial mulberry and sericulture products is known to be as low as approximately 0.2% (Asano et al., 2001; Kimura et al., 2004). On the other hands, comparisons have shown that DNJ content is 2.7 times higher in silkworm powder than in mulberries (Asano et al., 2001), and thus exhibits more effective results in diabetics. Previously, a direct comparison of 1-DNJ content of various silkworm species through genetic analysis had not been performed.

    In this study, we wanted to provide a basis for understanding 1-DNJ content in various silkworm species. Furthermore, our goal was to a database for silkworm genetic resources utilizing morphological and, genetic characteristics data.

    Materials and methods

      >  Materials

    Silkworm resources were collected from the Sericulture and Apiculture Division for the Department of Agricultural Biology, RDA in Suwon, Republic of Korea. Both spring and autumn reared silkworms 270 varieties were used for the DNJ (Japanese 90; Chinese 88; European 33; Tropicals 9; Korean 2; Commercial Varieties 8; and Miscellaneous species; 40). The freeze-dried 5th instar, 3-day-old silkworms (Ilshin Lab Co., Ltd) were ground and used within 48 h.

      >  Sample preparation procedures

    About 0.1 g of larva powder was mixed with 10 mL H2O, followed by vigorous shaking for 30 min, and then treatment with sonication for 1 h. After extraction with 60oC for 1 h, the sample was centrifuged at 4,000 rpm for 20 min, and then the supernatant was collected. The pellet was treated again by repeating the above steps, and then supernatants were combined and diluted to 100 mL by adding distilled water. This crude DNJ extract was mixed with 10 μL 0.4 M borate buffer, and 100 μL FMOC-CI (9-fluorenylmethyl chloroformate); the mixture was reacted on 40oC for 1 h. Finally, 10 μL 0.1 M glycine was added to this sample, and then adjusted to 1 mL with 0.1% acetic acid.

      >  DNJ measurement

    In order to measure DNJ within the analytes, high performance liquid chromatography(HPLC, SHISEIDO SP3203), fluorescence detector (Em 254, EX 322) and a C18 (100×4.6 mm, ID 3 μm) column were used. The absorbance of the effluent was monitored at 254 nm and flow rate was 1 mL/min. The mobile phase consisted of solvent A (acetonitrile) and solvent B (0.1% aqueous acetic acid). The initial solvent condition was solvent A: solvent B (20:80, v/v) for 16 min. After each analysis, a gradient was used on solvent A: solvent B (40:60, v/v) for 16 min, A:B (80:20, v/v) for 16.1, A:B (80:20, v/v) for 20 min, and A:B (20:80, v/v) for 20.1 min. The standard material was 1-deoxynojirimycin hydrochloride (SIGMA, 10 mg) and the DNJ content was calculated (Fig. 1.).

    Results and discussion

      >  DNJ content of 270 silkworm varieties

    There were many reports on DNJ in Morus, and it was found that DNJ contents in mulberry leaves were affected by many factors, such as varying Morus species and seasons. However, almost no studies were done on the silkworm, and DNJ accumulation and excretion in the larva are still not clear.

    Using data acquired from this study, we built a database of silkworm and mulberry resources with morphological and, genetic characteristics data, moreover, certain bioactive compounds have been receiving increasing attention. We analyzed DNJ content in addition to the base genetic information using 270 varieties of silkworms. All silkworms were the freeze-dried 5th instar species that were 3 days old.

    Results showed that, SK-1 had the highest DNJ content with 0.743% (Table 1). In contrast, the N27 had the lowest DNJ content on 0.146%. Yatsunami et al. (2011) reported that the DNJ contents of silkworm powder (SwP1, Wwp2) ranged from 0.39% to 0.58%. In addition, DNJ content in silkworm powder, was 3-7 times than in mulberry powder (Anno et al., 2004; Kimura et al., 2004; Murata et al., 2005). DNJ content is higher in silkworm powder than in mulberry powder because when mulberry leaves are damaged by insects, a milky sap is secreted, and this sap contains the majority of the DNJ content, ranging from 0.32% to 0.47% (Konno et al., 2006).

    In Table 2, C48 had the highest DNJ content (0.646%) while Shinjoong 103 had the lowest of all Chinese species with 0.159%. Of the European breeds, Rok 191 had the highest DNJ content (Table 3). In addition, there was no discernable difference in DNJ content with regard to blood color, cocoon color, or egg color of the silkworm. DNJ content of the Korean breeds was relatively lower than the other varieties (Table 3). On the other hand, the highest Tropics varieties were HM (0.732%) and PR (0.694%) within the miscellaneous speicies (Table 4). Ryu et al. (2013) reported that Yeonnokjam (5th instar 3rd larvae) larvae of the special silkworm varieties in Korean had the highest DNJ content. As a result, the mean 1-DNJ content of all 270 silkworm strains was 0.365±0.119%. For comparison of geographical origins of the silkworm gene resource, 1-DNJ contents was as follows: Japanese (SK-1) > Chinese (C48) > European (Rock191).

      >  DNJ content about larvae organs and between male and female silkworm larvae

    The results showed that the DNJ content in each of organs was the highest in the silkworm blood, then epithelial tissue, and finally body fat (Table 5). The distribution of DNJ in the larval body was investigated in all organs and, except for the silk glands, all organs and tissues contained DNJ. In addition, among the tissues, there was a significant increase in DNJ content in blood, followed by the intestinal juices and alimentary canals (Yin et al., 2010). The DNJ content in males was relatively higher than of the females (Table 6). As indicated by the results, male larvae absorb and accumulate DNJ from mulberry leaves more efficiently than female larvae. Yin et al. (2010) reported that DNJ content in male larvae were higher than females, and there was a significant increase in DNJ content in both sexes when the larvae were fed with a special mulberry varieties.

      >  DNJ accumulation among silkworm varieties

    The purpose of combining tests was to optimize hybrid combinations that yielded higher DNJ contents than basic silkworm larvae. The DNJ contents among various silkworm varieties were investigated. Results indicated that there was a difference among various silkworm varieties. When the SK-1 and N27 were crossbreed, the DNJ content was a higher than the parent SK-1. In contrast, between C48 and Shinjoong 103 in Chinese breeds, the hybrid was a lower than Shinjoong 103 (Table 7). The variation difference of DNJ content by a crossbreeding test could be utilized in a silkworm breeding database.

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이미지 / 테이블
  • [ Fig. 1. ]  Analysis chromatogram of 1-DNJ standard (A) and silkworm variety SK-1 containing the best 1-DNJ content (B).
    Analysis chromatogram of 1-DNJ standard (A) and silkworm variety SK-1 containing the best 1-DNJ content (B).
  • [ Table 1. ]  1-DNJ contents for Japanese silkworm varieties
    1-DNJ contents for Japanese silkworm varieties
  • [ Table 2. ]  1-DNJ contents for Chinese silkworm varieties
    1-DNJ contents for Chinese silkworm varieties
  • [ Table 3. ]  1-DNJ contents for Europe, Tropics, Korean silkworm varieties
    1-DNJ contents for Europe, Tropics, Korean silkworm varieties
  • [ Table 4. ]  1-DNJ contents for Practical application and ETC silkworm varieties
    1-DNJ contents for Practical application and ETC silkworm varieties
  • [ Table 5. ]  1-DNJ contents for each of larval organs.
    1-DNJ contents for each of larval organs.
  • [ Table 6. ]  1-DNJ contents for female and male larvae.
    1-DNJ contents for female and male larvae.
  • [ Table 7. ]  1-DNJ contents among silkworm varieties.
    1-DNJ contents among silkworm varieties.
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