1-Deoxynojirimycin attenuates pathological markers of Alzheimer's disease in the in vitro model of neuronal insulin resistance.

Insulin resistance, the hallmark of type 2 diabetes mellitus (T2DM), has emerged as a pathological feature in Alzheimer's disease (AD). Given the shared role of insulin resistance in T2DM and AD, repurposing peripheral insulin sensitizers is a promising strategy to preserve neuronal insulin sensitivity and prevent AD. 1-Deoxynojirimycin (DNJ), a bioactive iminosugar, exhibited insulin-sensitizing effects in metabolic tissues and was detected in brain tissue post-oral intake. However, its impact on brain and neuronal insulin signaling has not been described. Here, we investigated the effect of DNJ treatment on insulin signaling and AD markers in insulin-resistant human SK-N-SH neuroblastoma, a cellular model of neuronal insulin resistance. Our findings show that DNJ increased the expression of insulin signaling genes and the phosphorylation status of key molecules implicated in insulin resistance (Y1146-pIRß, S473-pAKT, S9-GSK3B) while also elevating the expression of glucose transporters Glut3 and Glut4, resulting in higher glucose uptake upon insulin stimuli. DNJ appeared to mitigate the insulin resistance-driven increase in phosphorylated tau and Aß1-42 levels by promoting insulin-induced phosphorylation of GSK3B (a major tau kinase) and enhancing mRNA expression of the insulin-degrading enzyme (IDE) pivotal for insulin and Aß clearance. Overall, our study unveils probable mechanisms underlying the potential benefits of DNJ for AD, wherein DNJ attenuates tau and amyloid pathologies by reversing neuronal insulin resistance. This provides a scientific basis for expanding the use of DNJ-containing products for neuroprotective purposes and prompts further research into compounds with similar mechanisms of action.

A 90-day preclinical toxicological evaluation in rats of a highly purified and concentrated mulberry leaf extract.

Mulberry (genus Morus) leaves have long been used as a human food, especially in Asia, and animal feed. More recently, mulberry leaf extracts have been introduced as a convenient way to consume mulberry for non-nutritional functional effects. Reducose® 5% is an Morus alba leaf extract that has been highly purified and standardized to a content of 5 ± 0.5% 1-deoxynojirimycin, a naturally present polyhydroxylated piperidine alkaloid analog of D-glucose. This extract has previously been evaluated in acute and subacute (28-day) oral toxicity studies in which no adverse effects of the test item were observed in mice or rats, respectively. Due to continued and growing interest in the extract in multinational markets, we have now further investigated potential toxic effects in subchronic (90-day) oral toxicity study in male and female Han:WIST rats. The test item was administered at doses of 850, 1700, and 2550 mg/kg bw/day, and did not cause adverse effects in clinical signs, body weight development, clinical pathology, gross pathology, or histopathology in comparison to the vehicle-control group. The no-observed-adverse-effect-level was determined to be 2550 mg/kg bw/day. These results add to the existing body of both preclinical and clinical work relevant to the safety of the extract and of interest to regulators in various global markets.

Molecular cloning, expression, and functional analysis of copper amine oxidase gene from mulberry (Morus alba L.).

In this study, we investigated a key enzyme encoded by the gene copper amine oxidase (MaCAO), which is involved in the biosynthetic pathway of 1-deoxynojirimycin (DNJ)1, an active ingredient in mulberry leaves. The 1680 bp long MaCAO was successfully cloned (GenBank accession no: MH205733). Subsequently, MaCAO was heterologously expressed using a recombinant plasmid, pET-22b (+)/MaCAO in Escherichia coli BL21 (DE3). A protein with a molecular mass of 62.9 kDa was obtained, whose function was validated through enzymatic reaction. Bioinformatics analysis identified that MaCAO contained the same conserved domain as that of copper amine oxidases ("NYDY"). Furthermore, the tertiary structure of the predicted protein using homology modeling revealed 46% similarity with that of copper amine oxidase (Protein Data Bank ID: 1W2Z). Gas chromatography-mass spectrometry analysis of the enzymatic reaction revealed that MaCAO could catalyze 1,5-pentanediamine to produce 5-aminopentanal. Additionally, levels of mulberry leaf DNJ content were significantly positively correlated with expression levels of MaCAO (P < 0.001). Our results conclude that MaCAO is the key enzyme involved in the biosynthetic pathway of DNJ. The function of MaCAO is validated, providing a foundation for the further analysis of biosynthetic pathways of DNJ in mulberry leaves using tools of synthetic biology.

A comprehensive review on the production, pharmacokinetics and health benefits of mulberry leaf iminosugars: Main focus on 1-deoxynojirimycin, d-fagomine, and 2-O-ɑ-d-galactopyranosyl-DNJ.

Mulberry leaves are rich in biologically active compounds, including phenolics, polysaccharides, and alkaloids. Mulberry leaf iminosugars (MLIs; a type of polyhydroxylated alkaloids), in particular, have been gaining increasing attention due to their health-promoting effects, including anti-diabetic, anti-obesity, anti-hyperglycemic, anti-hypercholesterolemic, anti-inflammatory, and gut microbiota-modulatory activities. Knowledge regarding the in vivo bioavailability and bioactivity of MLIs are crucial to understand their role and function and human health. Therefore, this review is aimed to comprehensively summarize the existing studies on the oral pharmacokinetics and the physiological significance of selected MLIs (i.e.,1-deoxynojirimycin, d-fagomine, and 2-O-ɑ-d-galactopyranosyl-DNJ). Evidence have suggested that MLIs possess relatively good uptake and safety profiles, which support their prospective use for oral intake; the therapeutic potential of these compounds against metabolic and chronic disorders and the underlying mechanisms behind these effects have also been studied in in vitro and in vivo models. Also discussed are the biosynthetic pathways of MLIs in plants, as well as the agronomic and processing factors that affect their concentration in mulberry leaves-derived products.

Screening, cloning and functional characterization of key methyltransferase genes involved in the methylation step of 1-deoxynojirimycin alkaloids biosynthesis in mulberry leaves.

MAIN CONCLUSION: The novel C-methyltransferase, MaMT1, could catalyze the conversion of piperidine to 2-methylpiperidine, which may be involved in the methylation step of DNJ biosynthesis in mulberry leaves. Mulberry (Morus alba L.) is a worldwide crop with medicinal, feeding and nutritional value, and 1-deoxynojirimycin ((2R, 3R, 4R, 5S)-2-hydroxymethyl-3, 4, 5-trihydroxypiperidine, DNJ) alkaloid, a potent α-glucosidase inhibitor, is its main active ingredient. Our previous researches clarified the biosynthetic pathway of DNJ from lysine to Δ1-piperideine, but its downstream pathway is unclear. Herein, eight differential methyltransferases (MTs) genes were screened from transcriptome profiles of mulberry leaves with significant differences in DNJ content (P < 0.01). Subsequently, MaMT1 (OM140666) and MaMT2 (OM140667) were hypothesized as candidate genes related to DNJ biosynthesis by correlation analysis of genes expression levels and DNJ content of mulberry leaves at different dates. Functional characterization of MaMT1 and MaMT2 were performed by cloning, prokaryotic expression and enzymatic reaction in vitro, and it showed that MaMT1 protein could catalyze the conversion of piperidine to 2-methylpiperidine. Moreover, molecular docking confirmed the interaction of MaMT1 protein with piperidine and S-adenosyl-L-methionine (SAM), indicating that MaMT1 had C-methyltransferase activity, while MaMT2 did not. The above results suggested that MaMT1 may be involved in the methylation step of DNJ alkaloid biosynthesis in mulberry leaves, which is a breakthrough in the analysis of DNJ alkaloid biosynthetic pathway. It is worth mentioning that the novel MaMT1, annotated as serine hydroxymethyltransferase, could rely on SAM to perform C-methyltransferase function. Therefore, our findings contribute new insights into the research of DNJ alkaloid biosynthesis and C-methyltransferase family.

Genome analysis of 1-deoxynojirimycin (1-DNJ)-producing Bacillus velezensis K26 and distribution of Bacillus sp. harboring a 1-DNJ biosynthetic gene cluster.

1-Deoxynojirumycin (1-DNJ) is a representative iminosugar with α-glucosidase inhibition (AGI) activity. In this study, the full genome sequencing of 1-DNJ-producing Bacillus velezensis K26 was performed. The genome consists of a circular chromosome (4,047,350 bps) with two types of putative virulence factors, five antibiotic resistance genes, and seven secondary metabolite biosynthetic gene clusters. Genomic analysis of a wide range of Bacillus species revealed that a 1-DNJ biosynthetic gene cluster was commonly present in four Bacillus species (B. velezensis, B. pseudomycoides, B. amyloliquefaciens, and B. atrophaeus). In vitro experiments revealed that the increased mRNA expression levels of the three 1-DNJ biosynthetic genes were closely related to increased AGI activity. Genomic comparison and alignment of multiple gene sequences indicated the conservation of the 1-DNJ biosynthetic gene cluster in each Bacillus species. This genomic analysis of Bacillus species having a 1-DNJ biosynthetic gene cluster could provide a basis for further research on 1-DNJ-producing bacteria.

Chalcone-1-Deoxynojirimycin Heterozygote Reduced the Blood Glucose Concentration and Alleviated the Adverse Symptoms and Intestinal Flora Disorder of Diabetes Mellitus Rats.

Chalcone-1-deoxynojirimycin heterozygote (DC-5), a novel compound which was designed and synthesized in our laboratory for diabetes treatment, showed an extremely strong in vitro inhibitory activity on α-glucosidase in our previous studies. In the current research, its potential in vivo anti-diabetic effects were further investigated by integration detection and the analysis of blood glucose concentration, blood biochemical parameters, tissue section and gut microbiota of the diabetic rats. The results indicated that oral administration of DC-5 significantly reduced the fasting blood glucose and postprandial blood glucose, both in diabetic and normal rats; meanwhile, it alleviated the adverse symptoms of elevated blood lipid level and lipid metabolism disorder in diabetic rats. Furthermore, DC-5 effectively decreased the organ coefficient and alleviated the pathological changes of the liver, kidney and small intestine of the diabetic rats at the same time. Moreover, the results of 16S rDNA gene sequencing analysis suggested that DC-5 significantly increased the ratio of Firmicutes to Bacteroidetes and improved the disorder of gut microbiota in diabetic rats. In conclusion, DC-5 displayed a good therapeutic effect on the diabetic rats, and therefore had a good application prospect in hypoglycemic drugs and foods.

α-Glucosidase Inhibitory Activity of Fermented Okara Broth Started with the Strain Bacillus amyloliquefaciens SY07.

In this work, a new strain of Bacillus amyloliquefaciens SY07 isolated from a traditional fermented soybean food was reported to possess remarkable α-glucosidase inhibitor-producing ability. Different culture media were applied for the proliferation of B. amyloliquefaciens SY07, and it was found that fermented okara broth presented the highest α-glucosidase inhibitory activity, while Luria-Bertani medium showed a negative effect. The extract from fermented okara broth acted in a dose-dependent manner to inhibit α-glucosidase activity, with an IC50 value of 0.454 mg/mL, and main inhibitors in the fermentation extract presented a reversible, uncompetitive pattern according to Lineweaver-Burk plots. Moreover, 1-deoxynojirimycin, a recognized α-glucosidase inhibitor, was found in the extract. Results indicated that B. amyloliquefaciens SY07 could utilize okara, a by-product from the soy processing industry, to generate α-glucosidase inhibitors effectively, and be regarded as a novel excellent microbial candidate for safe, economical production of potential functional foods or ingredients with hypoglycemic effect.

Chemometric evaluation of functional components and anti-quorum sensing activity of mulberry leaves from Indian cultivars: a potential contribution to the food industry.

BACKGROUND: Potential use of many native, easily available vegetal materials for human consumption and value addition is not well recognized. Mulberry, being a traditional industrial crop rich in nutrients and nutraceuticals can be of great importance for the food industry. However, mulberry leaves are mainly being utilized in sericulture and are not exploited for their functional components. Thus, the selection of promising mulberry cultivars, rich in bioactive compounds, like resveratrol and 1-deoxynojirimycin, increase their potential use in functional foods. RESULTS: Chlorogenic acid, myricetin and kaempferol were the major polyphenols present in the nine selected cultivars, in the range 0.001-0.086, 0.003-0.079 and 0.003-0.163 g kg-1 fresh weight (FW), respectively. Protocatechuic acid, epicatechin and rutin were predominantly present in cultivars V-1, G-2 and ML (0.103, 0.080 and 0.121 g kg-1 FW, respectively). Similarly, resveratrol and 1-deoxynojirimycin were highest in cultivars ML and K-2 (0.078 and 0.079 g kg-1 FW, respectively). Leaf extracts of cultivars G-2 and ML were able to effectively inhibit the violacein production with 64.08% and 70.04%, respectively at the concentration of 6 mg mL-1 presumably due to a higher content of polyphenols. Chemometric evaluation of chromatographic data showed the intraspecific variability and secondary metabolite co-existence in different cultivars. CONCLUSIONS: Considering phytoconstituents, cultivars G-2, ML, K-2 and V-1 could contribute efficiently to the rational utilization of mulberry in agro-food industries. Furthermore, cultivars G-2 and ML leaves can be a new source of quorum sensing inhibitory agents. © 2021 Society of Chemical Industry.

Cyclisations and Strategies for Stereoselective Synthesis of Piperidine Iminosugars.

This personal account focuses on synthesis of polyhydroxylated piperidines, a subset of compounds within the iminosugar family. Cyclisations to form the piperidine ring include reductive amination, substitution via amines, iminium ions and cyclic nitrones, transamidification (N-acyl transfer), addition to alkenes, ring contraction and expansion, photoinduced electron transfer, multicomponent Ugi reaction and ring closing metathesis. Enantiomerically pure piperidines are obtained from chiral pool precursors (e. g. sugars, amino acids, Garner's aldehyde) or asymmetric reactions (e. g. epoxidation, dihydroxylation, aminohydroxylation, aldol, biotransformation). Our laboratory have contributed cascades based on reductive amination from glycosyl azide precursors as well as Huisgen azide-alkene cycloaddition. The latter's combination with allylic azide rearrangement has given substituted piperidines, including those with quaternary centres adjacent to nitrogen.

Physicochemical Composition and Characterization of Bioactive Compounds of Mulberry (Morus indica L.) Fruit During Ontogeny.

Mulberry fruit is well recognized as one of the richest sources of bioactive compounds. We investigated the physicochemical composition and characterized the bioactive compounds during different ripening stages of mulberry (Morus indica) fruit and evaluated their anti-quorum sensing activity on Chromobacterium violaceum. The proximate components such as carbohydrates, proteins and lipids were found to be high in the ripe fruit compared to unripe and mid-ripe fruit. The ripe fruit contained higher content of total phenolics and flavonoids (336.05 and 282.55 mg/100 g fresh weight (FW), respectively). Epicatechin and resveratrol were the major polyphenols detected in the fruit with the range 5.13-19.46 and 4.07-14.45 mg/100 g FW, respectively. Chlorogenic acid and myricetin were predominant in the unripe and mid-ripe fruit (7.14 and 1.84 mg/100 g FW, respectively). The fruit was found to be an excellent source of anti-diabetic compound 1-deoxynojirimycin. The highest content of 1-deoxynojirimycin was present in the mid-ripe fruit, with a content of 2.91 mg/100 g FW. Furthermore, fruit extracts exhibited anti-quorum sensing activity against Chromobacterium violaceum by effectively inhibiting violacein production. Ripe fruit extracts showed the highest activity of 76.30% at 1 mg/mL and thus, could be used as a potent anti-quorum sensing agent. The results could be promising in the selection of appropriate developmental stages for M. indica fruit commercial exploitation in the food formulations rich in potential health components.

N,O-Dialkyl deoxynojirimycin derivatives as CERT START domain ligands.

Dysregulation of the ceramide transport protein CERT is associated to diseases such as cancer. In search for new CERT START domain ligands, N-dodecyl-deoxynojirimycin (N-dodecyl-DNJ) iminosugar was found to display, as a ceramide mimic, significant protein recognition. To reinforce the lipophilic interactions and strengthen this protein binding, a docking study was carried out in order to select the optimal position on which to introduce an additional O-alkyl chain on N-dodecyl-DNJ. Analysis of the calculated poses for three different regioisomers indicated an optimal calculated interaction pattern for N,O3-didodecyl-DNJ. The two most promising regioisomers were prepared by a divergent route and their binding to the CERT START domain was evaluated with fluorescence intensity (FLINT) binding assay. N,O3-didodecyl-DNJ was confirmed to be a new binder prototype with level of protein recognition in the FLINT assay comparable to the best known ligands from the alkylated HPA-12 series. This work opens promising perspectives for the development of new inhibitors of CERT-mediated ceramide trafficking.

Semi-synthesis of C28-modified triterpene acid derivatives from maslinic acid or corosolic acid as potential α-glucosidase inhibitors.

Combining two bioactive moieties by covalent bond into a novel single hybrid biological entity in view of the principle of active splicing, twenty-two C28-modified derivatives of pentacyclic dihydroxytriterpene carboxylic acids with saturated nitrogen heterocycle segments (i.e. 1-deoxynojirimycin or piperazines) have been synthesized. The inhibitory activities of all final target compounds on α-glucosidase were evaluated in vitro. The results of α-glucosidase inhibition assay indicate that some derivatives (e.g. 4b: IC50 = 1468.4 µM; 12b: IC50 = 499.6 µM 12c: IC50 = 768.5 µM, 13c: IC50 = 819.2 µM) show superior inhibitory activity in α-glucosidase than that of the precursor maslinic acid (IC50 = 2540.6 µM) or corosolic acid (IC50 = 1363.7 µM), in which compound 12b (IC50 = 499.6 µM) possesses stronger inhibitory activity than that of acarbose (IC50 = 606 µM). In addition, the result of enzyme kinetics study reveals that the inhibitory mechanism of the compound 12b is non-competitive inhibition and the inhibition constant Ki is 570 µM. The binding interaction between compounds with α-glucosidase are predicted by molecular docking simulation.

Synthesis, in vitro inhibitory activity, kinetic study and molecular docking of novel N-alkyl-deoxynojirimycin derivatives as potential α-glucosidase inhibitors.

A series of novel N-alkyl-1-deoxynojirimycin derivatives 25 ∼ 44 were synthesised and evaluated for their in vitro α-glucosidase inhibitory activity to develop α-glucosidase inhibitors with high activity. All twenty compounds exhibited α-glucosidase inhibitory activity with IC50 values ranging from 30.0 ± 0.6 µM to 2000 µM as compared to standard acarbose (IC50 = 822.0 ± 1.5 µM). The most active compound 43 was ∼27-fold more active than acarbose. Kinetic study revealed that compounds 43, 40, and 34 were all competitive inhibitors on α-glucosidase with Ki of 10 µM, 52 µM, and 150 µM, respectively. Molecular docking demonstrated that the high active inhibitors interacted with α-glucosidase by four types of interactions, including hydrogen bonds, π-π stacking interactions, hydrophobic interactions, and electrostatic interaction. Among all the interactions, the π-π stacking interaction and hydrogen bond played a significant role in a various range of activities of the compounds.

Alpha-glucosidase inhibitor 1-Deoxynojirimycin promotes beige remodeling of 3T3-L1 preadipocytes via activating AMPK.

Obesity is a serious health challenge in the world, and searching effective drugs to cure obesity is of great importance. 1-Deoxynojirimycin (DNJ) is extracted from mulberry leaves and acts as an α-glucosidase inhibitor to lower blood glucose. Recent studies demonstrated that it also has anti-obesity effect, but the mechanisms remain unknown. In our present study, we mainly examined the effects of DNJ on beige remodeling of 3T3-L1 preadipocytes. We observed that DNJ didn't affect the mRNA levels of fatty acid binding protein 4 (aP2), peroxisome proliferator-activated receptor γ (PPARγ), preadipocyte factor-1 (Pref-1) as well as the mitochondrial uncoupling protein 1 (UCP1), PR domain containing protein 16 (PRDM16), transmembrane protein 26 (TMEM26) in undifferentiated preadipocytes. But after inducing 3T3-L1 preadipocytes to differentiation with white or beige adipogenic medium, DNJ significantly reduced aP2, PPARγ and Pref-1 expressions, while up-regulated the expressions of UCP1, PRDM16 and TMEM26, accompanying with decreased lipid deposition. The ratio of p-AMPK/AMPK was up-regulated by DNJ (10 µM) treatment for 10 days, and the effects of DNJ on p-AMPK/AMPK, UCP1 and PRDM16 could be blocked by AMPK inhibitor Compound C. These results demonstrated that hypoglycemic agent DNJ could suppress the adipogenesis during the differentiation of white preadipocytes, and promote the switch of white preadipocytes to beige adipocytes via activating AMPK, which provided new mechanisms for explaining the benefits of DNJ on obesity-related disorders.

Use of a deoxynojirimycin-fluorophore conjugate as a cell-specific imaging probe targeting α-glucosidase on cell membranes.

Molecules designed for cell-specific imaging were studied, taking advantage of an enzyme-inhibitor interaction. 1-Deoxynojirimycin (DNJ) can be actively captured by cells which express the surface membrane protein α-glucosidase. New probes composed of DNJ for recognition linked to a fluorophore signal portion were prepared (DNJ-CF31, DNJ-Dans 2 and DNJ-DEAC 3). Docking simulations revealed that the inhibitors acarbose and miglitol and the inhibitor portion of the probes bind at the same position in the pocket of α-glucosidase (human-derived PDB: 3TON). The ability of probes 1-3 to detect the difference between HeLa cells (from human cervical cancer tissue), Neuro-2a cells (from a mouse neuroblastoma C1300 tumor), N1E-115 cells (from a mouse brain neuroblastoma C1300 tumor), A1 cells (from the astrocyte of a newborn mouse brain), and Caco-2 cells (from a human colon carcinoma) was evaluated, and cell-specific fluorescence imaging was possible for conjugate probes 1 and 2. Caco-2 cells treated with probes 1 and 2 showed blue and green fluorescence, respectively, from the cell membrane, and did not stain the Caco-2 cells inside. These results show that DNJ-CF31 and DNJ-Dans 2 recognize an α-glucosidase protein on the surface of Caco-2 cells. Probes 1 and 2 did not stain any part of the other cells. This cell-specific imaging strategy is applicable for a variety of therapeutic agents for many diseases.

An overview of the biological production of 1-deoxynojirimycin: current status and future perspective.

1-Deoxynojirimycin (DNJ), a representative iminopyranose, is widely used in anti-diabetic, antioxidant, anti-inflammatory, and anti-obesity applications. It naturally exists in plants, insects, and the culture broth of some microbes as a secondary metabolite product. However, the content of DNJ in plants and insects is relatively low, which is an obstacle to investigating DNJ in terms of structure-function relationships and restricts large-scale industrial production. With the development of micro-biotechnology, the production of DNJ during microbial fermentation has potential for industrial applications. In this review, we primarily focus on the microbial production of DNJ. The review covers sources of DNJ, determination methods, and physiological functions and applications. In a discussion of the efficient production of DNJ microorganisms, we summarize the current methods for DNJ screening and how to guide industrialized large-scale production of DNJ through fermentation regulation strategies. In addition, differences in the biosynthetic pathways of DNJ in different sources are also summarized. Finally, a comparison of DNJ content derived from different sources is discussed.

Design, Synthesis, and Activity Evaluation of Novel N-benzyl Deoxynojirimycin Derivatives for Use as α-Glucosidase Inhibitors.

To obtain α-glucosidase inhibitors with high activity, 19 NB-DNJDs (N-benzyl-deoxynojirimycin derivatives) were designed and synthesized. The results indicated that the 19 NB-DNJDs displayed different inhibitory activities towards α-glucosidase in vitro. Compound 18a (1-(4-hydroxy-3-methoxybenzyl)-2-(hydroxymethyl) piperidine-3,4,5-triol) showed the highest activity, with an IC50 value of 0.207 ± 0.11 mM, followed by 18b (1-(3-bromo-4-hydroxy-5-methoxybenzyl)-2-(hydroxymethyl) piperidine-3,4,5-triol, IC50: 0.276 ± 0.13 mM). Both IC50 values of 18a and 18b were significantly lower than that of acarbose (IC50: 0.353 ± 0.09 mM). According to the structure-activity analysis, substitution of the benzyl and bromine groups on the benzene ring decreased the inhibition activity, while methoxy and hydroxyl group substitution increased the activity, especially with the hydroxyl group substitution. Molecular docking results showed that three hydrogen bonds were formed between compound 18a and amino acids in the active site of α-glucosidase. Additionally, an arene‒arene interaction was also modelled between the phenyl ring of compound 18a and Arg 315. The three hydrogen bonds and the arene‒arene interaction resulted in a low binding energy (-5.8 kcal/mol) and gave 18a a higher inhibition activity. Consequently, compound 18a is a promising candidate as a new α-glucosidase inhibitor for the treatment of type Ⅱ diabetes.

Cloning, expression, and functional analysis of lysine decarboxylase in mulberry (Morus alba L.).

1-Deoxynojirimycin (DNJ) is the main bioactive compound of Morus alba L.. DNJ has pharmacological effects, including blood sugar level regulation and antiviral activity. In this study, the mulberry lysine decarboxylase gene (MaLDC), which is involved in the biosynthesis of DNJ alkaloids, was cloned, expressed, and functionally verified. MaLDC was induced and expressed in Escherichia coli BL21 (DE3). The recombinant soluble MaLDC protein had a relative molecular mass of 24.0 kDa. The protein was purified by Ni-NTA separation. The results showed that MaLDC protein could catalyze lysine decarboxylation to produce cadaverine. The Km and Vmax values were 19.2 µM and 3.31 µM/min, respectively. Quantitative real-time reverse transcription polymerase chain reaction revealed that MaLDC expression was positively correlated with DNJ content (P < 0.001), indicating that the MaLDC could encode a functional protein involved in the biosynthesis of DNJ alkaloid in mulberry. Our results provided a foundation for further studies of the enzymatic properties of LDC and established a basis for the analysis of key enzymes involved in the biosynthetic pathway of mulberry DNJ alkaloid.

A toxicological, metabonomic and transcriptional analysis to investigate the property of mulberry 1-deoxynojirimycin against the growth of Samia cynthia ricini.

1-Deoxynojirimycin (DNJ) is a natural d-glucose analogue from mulberry with promising physiological activity in vivo. Up to the present, the antidiabetic effects of DNJ on lowering blood sugar and accelerating lipid metabolism in mammals were broadly reported, but the specific character of DNJ against insects was vastly ignored. In this study, a toxicological test of DNJ againgst eri-silkworm, Samia cynthia ricini was carried out to investigate the potential of DNJ in insect management. Further, a method of nuclear magnetic resonance (NMR) metabonomics and real-time qPCR (RT-qPCR) were performed to analyze the alteration in midgut of eri-silkworm caused by DNJ. The result of toxicology showed that 5% and 10% DNJ could significantly inhibit the development of third-instar larvae on day 1-5, and mass deaths happened in DNJ groups on day 3-5. The quantitative analysis of 1H NMR in fifth-instar larvae showed that trehalose level increased in midgut of 0, 6 and 12 h DNJ groups, while the concentrations of glucose, lactate, alanine, pyruvate, α-ketoglutarate and fumarate were reduced in varying degrees. Meanwhile, principal component analysis (PCA) indicated that there were significant differences in the metabolic profiles among 12 h DNJ groups and the control group. In addition, RT-qPCR results displayed that four genes coding α-glucosidase, trehalase (THL) and lactate dehydrogenase (LDH) were lowered in expression of 12 h DNJ groups. Simultaneously, THL activity was significantly lowerd in 12 h DNJ groups. These mutually corroborated results indicated that the backbone pathways of energy metabolism, including hydrolysis of trehalose and glycogens, glycolysis and tricarboxylic acid (TCA) cycle were significantly inhibited by DNJ. Thus, the specific mechanism of DNJ efficiently suppressing the growth and energy metabolism of eri-silkworm was explored in this study, providing the potential of DNJ as to the production of botanical insecticide.