Heat Stability and Glucose-Lowering Effect of 1-Deoxynojirimycin from Silkworm (<italic>Bombyx mori</italic>) extract Powder
Heat Stability and Glucose-Lowering Effect of 1-Deoxynojirimycin from Silkworm (Bombyx mori) extract Powder
International Journal of Industrial Entomology. 2013. Dec, 27(2): 277-281
Copyright © 2013, Korean Society of Sericultural Science
  • Received : October 27, 2013
  • Accepted : November 09, 2013
  • Published : December 31, 2013
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Kang-Sun Ryu
Heui-Sam Lee
Kee-Young Kim
Mi-Ja Kim
Pil-Don Kang

Silkworm powder, which contains 1-deoxynojirimycin (DNJ), is a promising complementary and alternative medicine (CAM) in Korea. Silkworm powder was produced from Yeonnokjam pupae at d 3 of the 5th instar at the National Academy of Agricultural Science. The powder was derivatized with 9-fluorenylmethyl chloroformate (FMOC-Cl), and the DNJ-FMOC content was measured by HPLC. We investigated the content of 1-DNJ in the silkworm powder and its glucose-lowering effect when it was treated at different temperatures. The content of 1-DNJ was the lowest at 150℃, while it was constant at other temperatures. The silkworm extract powder was orally administered to diabetic mice (20 mg/kg/d) for 4 wk. Water intake did not significantly change when compared with the control group (T0). The blood glucose levels significantly decreased when mice where administered silkworm powder treated at 60℃ (T60) compared to the control group, but no difference was observed between the groups T100and T150. Moreover, the blood levels of TG significantly decreased compared with the control group. Based on these results, we surmise that the properties of the silkworm extract powder were stable upon heating at 100℃ but not at 150℃.
Silkworm and silkworm droppings have long been used in China and Korea as a folk remedy for the treatment of diabetes.1-Deoxynojirimycin (1-DNJ) is the hydrogenation product of nojirimycine, which was firstly discovered in Streptomyces. Natural DNJ was first isolated from the mulberry tree (Yoshikaki and Hivonu, 1976), and to date, more than 20 polyhydroxy alkaloids have been identified in mulberry and silkworm (Asano et al ., 1994a; 1994b; 2001). As a piperidine alkaloid, DNJ is known to possess highly effective α-glycosidase inhibition activity (Yoshikuni, 1988; Yoshikuni et al ., 1988; Hughes and Rudge, 1994) and is an effective anti-hyperglycemia agent. Currently, DNJ and its analogs have been extracted from a wide range of plants and microbes (Asano et al ., 1998; 2000; Kim et al ., 1999), but its content in the mulberry tree is the highest compared with other plants (Kimura et al ., 2007; Yatsunami et al ., 2008). Mulberry has been used in Chinese medicine against diabetes mellitus for a long time. Ryu et al. (1997) first reported that the silkworm larval powder of the 5th instar (prepared by lyophilization) had a positive effect on diabetic patients (Ryu et al ., 1997; 1999), and the action of lowering the blood sugar level was further proved by subsequent research (Han et al ., 2007). Silkworm powder possesses blood glucose-lowering effects (Ryu et al ., 2002), and mulberry leaves, which form the diet of silkworms, effectively inhibit α-glucosidase in the human small intestine (Oku et al ., 2006). DNJ from silkworm powder is stable upon heating to 121℃ for 15 min (Yatsunami et al ., 2011). In recent times, in Korea, Japan, and China, mulberry and silkworm larva products are becoming a popular auxiliary therapy for diabetes mellitus. In this report, we measured the content of 1-DNJ in the powder according to different heating temperatures and investigated the glucoselowering effect of heated silkworm powder on db/db mice.
Materials and Methods
- Preparation of silkworm powder
Silkworm larvae ( Bombyx mori ) were reared by feeding mulberry leaves during the spring season in 2012 at the National Academy of Agricultural Science. The silkworm varieties used for the experiment was Yeonnokjam. The larvae of 3 rd d of the 5th instar were quickly frozen with in liquid nitrogen and lyophilized.
Heat treated at 60℃, 100℃ and 150℃ for 30 min in the oven.
- Content of 1-DNJ in silkworm extract powders
DNJ content was measured according to the method reported by Kim et al . (2003). DNJ in the silkworm extract powder was extracted with 0.05 mol/L HCl, treated with 9-fluorenylmethyl (FMOC) to produce the DNJ-FMOC complex, and finally analyzed by high-pressure liquid chromatography (HPLC).
- Administration of silkworm extract powders to db/db mice
Male C57BL/KSJ-(db/db) mice (6 wk old) were purchased from Japan SLC Inc. (Japan). Mice were housed in a conventional cage at the appropriate temperature (23℃ ± 3℃) and humidity (55% ± 15%) under a 12-h light/dark cycle, and had free access to food and water. All the 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 dividedinto five groups (n = 10 in each group): G1( control group : no silkworm powder), G2(T0 ; silkworm extract powder noheating), G3(T60 ; 60℃ / 30 min), G4(T100 ; 100℃/30 min), and G5(T150 ; 150℃/30 min), the silkworm powder was remade with diet.
- Measurement of body fat weight and blood biochemical analysis
The mice were fasted for 3 h, and then blood samples were taken after autopsy. The biochemical analysis of blood included the measurement of the levels of TG(Triglyceride), TCHO(Total cholesterol), LDL(Low density lipoprotein), GLU(Glutamic acid), AST(Aspartate aminotransferase), and ALT(Alanine aminotransferase), which was performed with a blood biochemical analyzer (AU680, Beckman Coulter, Japan). The fat weight was estimated by measuring the circumference of the perirenal area and of the epididymis at autopsy.
Results and Discussion
- 1-DNJ contents in silkworm powder
The content of 1-DNJ in silkworm powder of Yeonnokjam pupae at d 3 of the 5 th instar heated at different temperatures was determined. The content of 1-DNJ in the powders heated at 60℃ and 100℃ for 30 min was similar to that of the powder without heat treatment. However, in the powder treated at 150℃ for 30 min, the 1-DNJ content was approximately half of that in the other samples (6.83 mg/dL). The amount of 1-DNJ remainedthe same when the powder was heated to 121℃ for 15 min (Yatsunami et al ., 2011). Therefore, 1-DNJ in the silkworm powder was relatively stable at up to 100℃ for 30 min and 121℃ for 15 min ( Table 1 ). The mice were fed these diets for 3–4 wk.
- Changes in body weight
Our analysis focused on the comparison between the changesin body weight of the treatment groups and the control group. In
1-Deoxynojirimycin content in silkworm powder as afunction of the heating temperature
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1-Deoxynojirimycin content in silkworm powder as afunction of the heating temperature
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Change in body weight of db/db mice. * G1 (control), G2 (non-heating), G3 (60℃/30 min), G4 (100℃/30 min), G5 (150℃/30 min)
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Change in body weight of db/db mice administered silkworm powder. * G1 (control), G2 (non-heating), G3 (60℃/30 min), G4 (100℃/30 min), G5 (150℃/30 min)
the T60 group, a statistically significant weight gain inhibition was observed compared to the T0 group ( Fig. 1 ).
- Changes in feed intake
The weekly diet used in the experiments consisted of a fixed amount. The feed intake was observed once per week for 4 wk after silkworm powder administration. Mice in the T0, T60, T100, and T150 groups showed statistically significant reduction in feed intake compared to the control group (no silkworm powder administration) at 3–4 wk after administration ( Fig. 2 ).
- Changes in water intake
We prepared a fixed amount of water the day before water intake, for determining water intake quantity. The water that remained unused in the period of 24 h was subtracted from the total amount of water that was offered daily, and the difference was considered as the water intake quantity (g/mouse/d).
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Change in water consumption in db/db mice administered silkworm powder. * G1 (control), G2 (non-heating), G3 (60℃/30 min), G4 (100℃/30 min), G5 (150℃/30 min)
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Blood glucose-lowering effect of silkworm powder in db/db mice. * G1 (control), G2 (non-heating), G3 (60℃/30 min), G4 (100℃/30 min), G5 (150℃/30 min)
The water intake quantity of each cage was the sum of each individual water intake value in each cage. The result showed no significant difference among the different groups ( Fig. 3 ).
- Blood glucose-lowering effect of silkworm extracts in the db/db mice
In the T0 and T60 groups, a statistically significant reduction in the blood glucose level was observed compared to the control group ( P < 0.05 and P < 0.01, respectively). However, in the T100 and T150 group, no significant reduction in the blood glucose level occurred. This result indicates that the silkworm powder as a supplement raw material has to be sterilized at temperatures below 100℃ ( Fig. 4 ). Heat-treated silkworm powder also had a decreased effect in blood glucose levels, but the non-heat-treated silkworm powder was 6–52% better than the heat-treated silkworm powder in reducing blood glucose level.
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Clinical biochemical analysis of the blood of db/db mice administered silkworm powder. * G1 (control), G2 (non-heating), G3 (60℃/30 min), G4 (100℃/30 min), G5 (150℃/30 min)
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Change in body fat in db/db mice administered silkworm powder. * G1 (control), G2 (non-heating), G3 (60℃/30 min), G4 (100℃/30 min), G5 (150℃/30 min)
- Blood biochemical analysis and change in body fat
Blood biochemical analysis indicated that in the T150 group, the level of AST was lower than that in the other groups. The levels of TCHO in the T100 group significantly decreased compared to the control groups. The levels of TG in the T0 and T60 mice were significantly decreased compared to the control groups. For GLU level, no treatment showed significant changes compared to the control groups, but GLU decreased by approximately 20% compared to control group without silkworm powder treatment ( Fig. 5 ).
The epididymal fat weight of mice in the T0, T60, and T150 groups significantly decreased compared to the control groups, while the perirenal fat weight of mice in the T60 and T150 groups significantly decreased compared to the control groups ( Fig. 6 ).
This study was carried out with the support of the “Cooperative Research Program for Agricultural Science & Technology Development (Project No. PJ0091252013)”, Rural Development Administration, Republic of Korea.
Asano N , Tomioka E , Kizu H , Oseki K , Matsui K (1994a) Sugars with nitrogen in the ring isolated from the leaves of Morus bombycis. Carbohydr. Res. 253 (3) 235 - 245    DOI : 10.1016/0008-6215(94)80068-5
Asano N , Kato A , Miyauchi M , Kizu H , Kameda Y , Watson AA , Nash RJ , Fleet GW (1998) Nitrogen containing furanose and pyranose analogues from Hyacinthus orientalis. J. Nat. Prod. 61 (5) 625 - 628    DOI : 10.1021/np9705726
Asano N , Yamashita T , Yasuda K , Ikeda K , Kizu H , Kameda Y , Kata A , Nash RJ , Lee HS , Ryu KS (2001) Polyhydroxylated alkaloids isolated from mulberry trees (Morus alba L.) and silkworms (Bombyx mori L.). J. Agric. Food Chem. 49 (9) 4208 - 4213    DOI : 10.1021/jf010567e
Han J , Inoue S , Isoda H (2007) Effects of silkworm powder on glucose absorption by human intestinal epithelial cell line Caco-2. J. Nat. Med. 61 (4) 387 - 390    DOI : 10.1007/s11418-007-0164-5
Hughes AB , Rudge AJ (1994) Deoxynojirimycin: synthesis and biological activity. Nat. Prod. Rep. 11 (2) 135 - 162    DOI : 10.1039/np9941100135
Kim HS , Kim YH , Hong YS , Paek NS , Lee HS , Kim TH , Kim KW , Lee JJ (1999) Alpha-glucosidase inhibitors from commelina communis. Planta Med. 65 (5) 437 - 439    DOI : 10.1055/s-2006-960803
Kim JW , Kim SU , Lee HS , Kim I , Ahn MY , Ryu KS (2003) Determination of 1-deoxynojirimycin in Morus alba L. leaves by derivatization with 9-fluorenylmethyl chloroformate followed by reversed phase high-performance liquid chromatography. J Chromatogr. A 1002 (1-2) 93 - 99    DOI : 10.1016/S0021-9673(03)00728-3
Kimura T , Nakagawa K , Kubota H , Kojima Y , Yamaqishi K , Oita S , Oikawa S , Miyazawa T (2007) Food- grade mulberry powder enriched with 1-deoxynojirimycin suppresses the elevation of postprandial blood glucose in humans. J. Agric. Food Chem. 55 (14) 5869 - 5874    DOI : 10.1021/jf062680g
Oku T , Yamada M , Nakamura M , Sadamori N , Nakamura S (2006) Inhibitory effects of extractives from leaves of Morus Alba on human and rat small intestinal disaccharidase activity. British J. of Nutrition 95 933 - 938    DOI : 10.1079/BJN20061746
Ryu KS , Lee HS , Chung SH , Kang PD (1997) An activity of lowering blood-glucose levels according to preparative conditions of silkworm powder. Korean J. Seric. Sci. 39 79 - 85
Ryu KS , Lee HS , Kim SY (1999) Pharmacodynamic study of silkworm powder in mice administered to maltose, sucrose and lactose. Korean J. Sric. Sci. 41 9 - 13
Ryu KS , Lee HS , Kim I (2002) Effects and mechanism of silkworm powder as a blood glucose lowering agent. J. Indust. Entomol. 4 93 - 100
Yatsunami K , Ichida M , Onodera S (2008) The relationship between 1-deoxynojirimycin content and alphaglucosidase inhibitory activity in leaves of 276 mulberry cultivars (Morus spp.) in Kyoto. Jpn Nat. Med. (Tokyo) 62 (1) 63 - 66    DOI : 10.1007/s11418-007-0185-0
Yatsunami K , Murata K , Kamei T (2011) 1-Deoxynojirimycin Content and Alfa-Glucosidase inhibitory Activity and Heat Stability of 1-Deoxynojirimycin in Silkworm Powder. Food and Nutrition science. 2 87 - 89    DOI : 10.4236/fns.2011.22011
Yoshikaki A , Hivonu M (1976) The structure of moranoline, a piperidine alkaloid form Morus species. Nippon Nogei Kagaku 50 (11) 571 - 572    DOI : 10.1271/nogeikagaku1924.50.11_571
Yoshikuni Y (1988) Inhibition of intestinal alpha-glucosidase activity and postprandial hyperglycemia by moranoline and its N-alkyl derivatives. Agric. Biol. Chem. 52 (1) 121 - 128    DOI : 10.1271/bbb1961.52.121
Yoshikuni Y , Ezure Y , Aoyagi Y , Enomoto H (1988) Inhibition of intestinal alpha- glucosidase and postprandial hyperglycemia by N-substituted moranoline derivatives. J. Pharmacobiodyn. 11 (5) 356 - 362    DOI : 10.1248/bpb1978.11.356