1-Deoxynojirimycin (DNJ), an alpha-glucosidase inhibitor, has been used to prevent or treat type 2 diabetes. Low amounts of DNJ are found in mulberry leaf; however, a methodology is necessary to enhance the DNJ content of mulberry leaf extract (MLE) since increasing the amounts of DNJ is required for the prevention and treatment of type 2 diabetes. In this study, the DNJ content of MLE was increased through the optimization of the conditions for MLE fermentation by Lactobacillus plantarum SG-053, using response surface methodology (RSM). By single factor testing, the optimal conditions were identified as an inoculum concentration of 1%(v/v), an MLE concentration of 3°Bx, and no agitation. Based on a Plackett-Burman design, the following factors were considered to majorly affect the DNJ content in the MLE fermentation product: the initial pH, fermentation temperature, and fermentation time. By response surface methodology, the optimal conditions for MLE fermentation was found to have an initial pH of 5.5, a fermentation temperature of 31.9oC, and a fermentation time of 34 h. Under these optimal fermentation conditions, the DNJ content in MLE increased 3.59 times, or from 23.85 to 85.54 μg/mL.

1-deoxynojirimycin (1-DNJ), a potent a-glycosidase inhibitor, has therapeutic applications in treatments of HIV, Gaucher’s disease, and diabetes. 1-DNJ has been extracted from natural sources (mulberry leaves) for therapeutic purposes; however, 1-DNJ ingredients are in limited supply and are costly to obtain on a large scale. Since certain strains of Bacillus and Streptomyces species reportedly produce 1-DNJ, they may serve as potential sources for high-yield 1-DNJ production. In this study, we obtained evidence for four bacteria that produce 1-DNJ in large quantities by high performance liquid chromatography and thin layer chromatography. Investigation of the effect of mulberry leaves powder concentration(1~5%), using the 1-DNJ high-production bacteria, provided evidence for microbial mass production of 1-DNJ.

누에분말중에 들어있는 성분인 1-deoxynojirimysin(1-DNJ)은 α-glucosidase의 활성을 억제하여 소장에서 혈액으로 포도당이 흡수되는 것을 억제하는 기능이 있 다. 이러한 기능 때문에 1-DNJ는 인슐린 비의존형 당뇨환자들에게 혈당강하제로 이용되고 있다. 또한 1-DNJ는 간염바이러스 등 외막형성 바이러스의 증식을 억제 하므로 항바이러스성 소재에 대한 가능성도 연구되고 있다. 본 실험에서는 혈당강 하 및 항바이러스성 기능을 갖는 1-DNJ의 누에 유전자원 중의 계통별 함량, 그리고 누에 교배조합에 의한 1-DNJ 함량 변화를 비교하였다. 1-DNJ의 함량은 누에 유전자원의 지리적 및 계통별로 차이를 보였다. 일본종은 JF가 0.536%로 가장 높았으며, 잠303이 0.249%로 가장 낮았다. 중국종은 C108이 0.519%로 가장 높았으며, 중27이 0.242%로 가장 낮았다. 유럽종은 록191이 0.595%로 가장 높았으며, AP가 0.254%로 가장 낮았다. 그리고 미분류종의 CRE 가 0.653%로 가장 높았으며, J037은 0.297%로 가장 낮았다. 그리고 유전형질인 혈 색, 견색, 그리고 난색에 따른 1-DNJ 함량 변화는 큰 차이가 없는 것으로 나타났다. 한편, 일본종 누에를 이용하여 교배조합(F1)을 작성한 후의 1-DNJ의 함량 변화를 비교한 결과, 교배조합에 따른 1-DNJ의 함량 변화가 확인되었다. 따라서 이러한 누에 유전자원의 계통별 및 교배조합에 따른 1-DNJ 함량 변화는 누에 품종육성 및 누에분말을 이용한 기능성 소재 연구의 기초자료로 활용될 것으 로 생각된다.

1-Deoxynojirimycin(1-DNJ) was hardly detected by general UV detector. Therefore, in this study, we analyzed 1-DNJ, a effective functional material in which was contained the leaves of Cudrania tricuspidata and its tea by gas-chromatography. Synthesized a TMS derivative by attaching trimethylsilyl group to 1-DNJ, analyzed this by GC, and could detect a good peak. The leaves of Cudrania tricuspidata contains 1154.83±56.67 ug/g.d.w of 1-DNJ and tea of Cudrania tricuspidata leaves does 8.01±0.61 ug/g.d.w. This means the contents of 1-DNJ was nearly identical to each other. The larger size of the leaves had the more contents of 1-DNJ and the middle region of collection was the highest than any other collective regions of leaves.

Streptomyces exofoliatus SID9135 균주의 발효배양 여액으로부터 anion(Dowex 1X2-100 Cl^-) 및 cation(Dowex 50X4-100 H^+) 이온교환수지, Sephadex LH20 컬럼, TSK 겔 컬럼크로마토그래피 등으로 저해물질을 정제한 후, 최종적으로 HPLC를 사용하여 α-glucosidase 저해물질인 1-deoxynojirimycin을 순수 분리정제 하였다. IRC 마우스에서의 1-deoxynojirimycin의 급성독성 조사에서 200㎎/㎏의 dose로 경구 투여하여 10일간 관찰하였을 때 치사동물이 전혀 없었으며 체중증가는 대조군과 동일한 양상을 보였다. Agar 희석법으로 20종의 병원균에 대한 1-deoxynojirimycin과 AO-128 및 acarbose의 MIC한 결과 1-deoxynojirimycin과 대조약물인 AO-128 및 acarbose의 농도가 100㎍/ml 이상에서도 20종의 시험균주 모두에 대해 항균효과를 나타내지 않았다.

Background : 1-Deoxynojirimycin (DNJ) is a representative compound of the antidiabetic constituent in mulberry leaves (Morus alba L.). The hot water extracts of mulberry leaves were fermented with lactic acid bacteria in order to analysis the changes of the DNJ contents and α-glucosidase inhibition. Methods and Results : The mulberry leaves were extracted with hot water (121℃, 3 hr). The extracts were fermented with nine strains of lactic acid bacteria such as Lactobacillus acidophilus, in order to increase the contents of DNJ and α-glucosidase inhibition. DNJ contents in the fermented extracts were determinated by HPLC analysis. The α-glucosidase inhibition was mesured by comparing dose-inhibition curves of α-glucosidase and IC50 value. The DNJ contents after fermentation have increased in the almost fermented extracts. Especially, DNJ of the extracts fermented with L. acidophilus was increased from 8.38 ㎍ ㎖ -1 to 21.77 ㎍ ㎖-1. IC50 values of the α-glucosidase inhibition were shown to be decreased in the fermented extracts. The extracts fermented with L. casei KCTC 3109 was determined at 290.04 ㎍ ㎖-1, resulting it is lower about 140 ㎍ ㎖-1 than 429.76 ㎍ ㎖-1 of the control. Conclusion : From the above results, we may suggest that the lactic acid fermentation of mulberry leaves extracts can more enhance the hypoglycemic activities such as DNJ contents.