1-Deoxynojirimycin Content and Blood Glucose-Lowering Effect of Silkworm (Bombyx mori) Extract Powder

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  • ABSTRACT

    We investigated the 1-deoxynojirimycin (1-DNJ) content of extracts from silkworm larvae at each developmental stage within three silkworm varieties. We also compared the content of the following polyhydroxylated alkaloids in the silkworm extracts: 1-DNJ, fagomine, and 1,4-dideoxy-1,4-imino-d-arabinitol (DAB). In addition, we evaluated the glucose-lowering effects of silkworm extract powder in db/db mice. The 1-DNJ content was the highest in Yeonnokjam 5th instar 3rd d larvae and Hansaengjam 5th instar 3rd d larvae, which contained 18.4 mg/100 g dry weight and 18.3 mg/100 g dry weight, respectively. The 5th instar 3rd d larvae exhibited a higher content of 1-DNJ than that of 5th instar 5th d larvae among all varieties. The glucose-lowering effects of silkworm extracts and Yeonnokjam powder were tested on db/db mice, and the blood glucose levels were found to decrease significantly in the YR70 group. Silkworm extracts (180 mg/kg, 90 mg/kg, 45 mg/kg, and 22.5 mg/kg) and acarbose (50 mg/kg) were administered orally for 4 wk. Changes in water intake were not statistically significant between control and silkworm extract-treated groups. Compared to the control group, blood glucose levels in the silkworm extract powder-treated group decreased in the 22.5 mg/kg/d group after being administered for 4 wk. This decrease was statistically significant. Furthermore, biochemical changes in the AST(Aspartate aminotransferase), ALT(Alanine aminotransferase), TCHO(Total Cholesterol), TG(Triglyceride), LDL(Low density lipoprotein), and HDL(High density lipoprotein) levels in blood were not observed. However, statistically significant decreases in blood GLU in the 22.5 mg/kg/d group compared to that of the control group occurred. In addition, the epididymal fat weight of the silkworm extract powder-treated group decreased significantly in both the 22.5 mg/kg/d group and 180 mg/kg/d group compared to that of the control group, but there were no statistically significant changes in perirenal fat weight. These results demonstrate that silkworm extracts inhibit changes in blood glucose levels in model diabetic mice.


  • KEYWORD

    Silkworm , Silkworm extract powder , Blood glucose lowering

  • Introduction

    Diabetes mellitus is a serious chronic metabolic disorder that has a significant impact on the health, quality of life, and life expectancy of patients, as well as the healthcare system. Nojirimycine was first discovered from a streptomycete, and its naturally occurring hydrogenated product, 1-deoxynojirimycin (1-DNJ) was first isolated from the mulberry tree (Yoshikaki and Hivonu, 1976). Today, more than 20 polyhydroxy alkaloids have been identified from mulberry and silkworm (Asano et al., 1994a; 1994b; 2001). 1-DNJ is a piperidine alkaloid that is a highly effective alpha-glycosidase inhibitor (Yoshikuni, 1988; Yoshikuni et al., 1988; Hughes and Rudge, 1994) and an effective chemical for the treatment of hyperglycemia. Presently, 1-DNJ and its analogs have been isolated from a wide range of plants and microbes (Asano et al., 1998; 2000; Kim et al., 1999), but the 1-DNJ content in the mulberry tree is the highest among plants (Kimura et al., 2007; Yatsunami et al., 2008). Mulberry has been utilized as a Chinese medicine against diabetes mellitus for years. Ryu et al. (1997) first reported that the silkworm larval powder of the 5th instar (prepared by lyophilization) had beneficial effects on diabetic patients (Ryu et al., 1997; 1999), and the lowering of blood sugar levels was further elucidated by subsequent research (Han et al., 2007). Silkworm powder has blood glucose-lowering effects (Ryu et al., 2002), and mulberry leaves the diet of silkworms effectively inhibit alpha-glucosidase in the small intestine of humans (Oku et al., 2006). In Korea, Japan, and China, products made from mulberry and silkworm larvae are becoming popular for auxiliary therapy for diabetes mellitus. In this report, we compared the 1-DNJ content among silkworm varieties and investigated the antidiabetic effects of silkworm extracts on the db/db mouse.

    Materials and Methods

      >  Preparation of silkworm extracts

    Silkworm larvae were reared by feeding mulberry leaves in the spring season of 2012 at the National Academy Agricultural Science. The silkworm varieties used for the experiment were Yeonnokjam, Yangwonjam, and Hansaengjam. The 5th instar 3rd d larvae were quickly frozen with liquid nitrogen and lyophilized, and the resulting dried silkworm powder was extracted with ethanol.

      >  High pressure liquid chromatography chromatogram of extracts

    1-DNJ content was measured according to the method reported by Kim et al. (2003). 1-DNJ in the silkworm larva or mulberry leaf powder was extracted with 0.05 mol/L HCl, treated with 9-fluorenylmethyl (FMOC) to produce a 1-DNJ-FMOC complex compound, and finally detected by high pressure liquid chromatography (HPLC).

      >  Measurement of fasting blood glucose levels

    Fasting blood glucose levels were measured after the animals fasted for 3 h for 4 wk. The db/db mice were obtained from Japan SLC, Inc (Japan), and animals at 6 wk of age were used. Blood glucose levels were determined in mouse blood samples from the tail vein using a glucose analyzer (Accu-Chek Active; Roche Diagnostics GmbH, Germany). Blood glucose levels were determined in blood samples from the tail vein at 0 min (prior to maltose administration), 30 min, 60 min, 90 min, and 120 min after maltose administration for glucose tolerance tests.

      >  Administration of silkworm extracts to db/db mice

    Male C57BL/KSJ-(db/db) mice (6 wk old) were purchased from Japan SLC, Inc (Japan). They were housed in a conventional state with the appropriate temperature (23℃ ± 3℃) and humidity (55% ± 15%) under a 12-h light/12-h dark cycle with free access to food and water. All groups were fed a standard diet (certified irradiated global 18% protein rodent diet). After a 1-wk adaptation period, the 7-wk-old mice were divided into the following 6 groups (n = 10 in each group): diabetic control group, low silkworm extract group (db/db low dose, 22.5 mg/kg), high silkworm extract group (db/db high dose, 180 mg/kg), and acarbose groups.

      >  Measurement of body weight and blood biochemical analysis

    The mice fasted for 3 h, and then blood samples were taken after their autopsy. Biochemical analysis of blood TG, TCHO, LDL, GLU, AST, and ALT was performed using a blood biochemical analyzer (AU680, Beckman Coulter, Japan). Fat was taken from the circumference of the perirenal and epididymis during the autopsy, and the weight was measured.

    Results and Discussion

       1-DNJ content in silkworm varieties

    We compared the 1-DNJ content among silkworm extracts of 5th instar 3rd d larvae and 5th instar 5th d larvae of the Yeonnokjam, Yangwonjam, and Hansaengjam. The Yeonnokjam and Hansaengjam larvae contained higher 1-DNJ than those of Yangwonjam. Furthermore, the 1-DNJ content of 5th instar 5th d larva powder was lower than that of the 5th instar 3rd d larvae. The best new candidate among these extracts was YR53. Yeonnokjam is one of the special silkworm varieties in Korea (Table 1).

      >  HPLC analysis of highly polyhydroxylated alkaloids

    We established a profile pattern of highly polyhydroxylated alkaloids, such as 1-deoxynojirimycin (1-DNJ), fagomine, and 1,4-dideoxy-1,4-imino-D-arabinitol (DAB), of Yeonnokjam, a silkworm variety. We standardized the manufacturing process with this profile pattern, and now its development, including the establishment of standards as a natural medicine, is in progress (Fig. 1).

      >  Effects of silkworm extracts and powder on blood glucose levels

    In order to determine the optimal silkworm powder extraction, we selected conditions based on how they lowered glucose levels with varying ethanol concentrations. All groups, with the exception of control and YR30 groups, exhibited glucose-lowering effects. Among these, the YR70 group showed the highest glucose-lowering effects for 4 wk. In conclusion, the optimal extraction condition was 70% ethanol (Fig. 2).

      >  Blood glucose-lowering effects of silkworm extracts in db/db mice

    Based on blood glucose measurements, there was no statistically significant reduction in blood glucose levels in the G5 (180 mg/kg/d) group compared to that of the control group (P < 0.05 or P < 0.01). The blood glucose levels from wk 2 to 4 of the G3 (45 mg/kg/d) group did not decrease significantly. In contrast, a statistically significant reduction was observed for the G2 (22.5 mg/kg/d) group for 4 wk after administration (P < 0.05). The positive control (G6) group also exhibited a statistically significant reduction for 4 wk after administration (P < 0.01). The dosage test of silkworm extract powder needed an additional dosage lower than 22.5 mg/kg/d. Nevertheless, it is unclear why the effects of silkworm extract powder on blood glucose levels are dose independent (Fig. 3).

      >  Changes in water intake

    The silkworm extract group did not show statistically significant changes in water intake compared to that of the control group. However, the 22.5 mg/kg/d and 45 mg/kg/d groups reduced their water intake at 3 and 4 wk after administration by approximately 47% and 33% and 41% and 20%, respectively. The water intake of the positive control group was reduced by approximately 70–75% compared to that of the control group over the course of 4 wk (Fig. 4).

      >  Changes in body fat and blood biochemical analysis

    The two groups of G2 (22.5 mg/kg/d) and G5 (180 mg/kg/d) showed statistically significant decreases in epididymal fat weight (P < 0.05) compared to those of the control groups. For perirenal fat weights, the silkworm extract group did not show statistically significant decreases compared to those of control groups (Fig. 5).

    The positive control group did not show statistically significant decreases in epididymal fat weight, whereas its perirenal fat weight increased (P < 0.05 or P < 0.01).

    The blood biochemical analysis indicated that the silkworm extract (180 mg) showed similar decreases in AST and ALT levels. Similar decreases were found in the positive control group as well. G2 (22.5 mg/kg/d) showed statistically significant decreases in GLU levels compared to that of the control groups. The positive control group (G6) also exhibited statistically significant decreases in GLU levels compared to the control groups (P < 0.05 or P < 0.01). The silkworm extract groups did not show statistically significant changes in TCHO, LDL, or HDL levels compared to those of the control groups, whereas the positive control group (G6) showed a statistically significant decrease compared to those of control groups (P < 0.05 or P < 0.01). G3 (45 mg/kg/d) showed statistically significant decreases in TCHO, LDL, and HDL levels compared to those of control groups (P < 0.05). The positive control group (G6) did not show statistically significant changes compared to those of control groups (P < 0.05 or P < 0.01) (Table 2).

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  • [Table 1.] 1-Deoxynojirimycin content in silkworm varieties and its ratio in lyophilized material and extracts
    1-Deoxynojirimycin content in silkworm varieties and its ratio in lyophilized material and extracts
  • [Fig. 1.] HPLC chromatogram of 1-deoxynojirimycin (1-DNJ), fagomine, and DAB of silkworm (Yeonnokjam) extract.
    HPLC chromatogram of 1-deoxynojirimycin (1-DNJ), fagomine, and DAB of silkworm (Yeonnokjam) extract.
  • [Fig. 2.] Effects of silkworm (Yeonnokjam) extracts on blood glucose levels in db/db mice
    Effects of silkworm (Yeonnokjam) extracts on blood glucose levels in db/db mice
  • [Fig. 3.] Effects of Silkworm (Yeonnokjam) extracts on change of blood glucose in db/db mice.
    Effects of Silkworm (Yeonnokjam) extracts on change of blood glucose in db/db mice.
  • [Fig. 4.] Effects of silkworm (Yeonnokjam) extracts on db/db mouse water consumption *
    Effects of silkworm (Yeonnokjam) extracts on db/db mouse water consumption *
  • [Fig. 5.] Effects of silkworm (Yeonnokjam) extracts on the weight of epididymal and perirenal fat weights
    Effects of silkworm (Yeonnokjam) extracts on the weight of epididymal and perirenal fat weights
  • [Table 2.] Blood biochemical analysis in db/db mice.
    Blood biochemical analysis in db/db mice.