Effect of 1-Deoxynojirimycin on insulin resistance in prediabetic mice based on next-generation sequencing and intestinal microbiota study.

ETHNOPHARMACOLOGICAL RELEVANCE: 1-Deoxynojirimycin (DNJ), the major alkaloid in Morus alba L., is the main effective constituent in "Mulberry twig Alkaloids Tablets" launched in China in 2020. Prediabetes, characterized by insulin resistance, is regarded as the key period for reversing Type 2 diabetes mellitus (T2DM) through lifestyle intervention and glucose-lowering drugs. Besides the excellent activity as an α-glucosidase inhibitor, DNJ also improves insulin sensitivity in T2DM murine models, yet the mechanism is still unclear. Besides, the pharmaceutical effect of DNJ on prediabetes is also undocumented. AIM OF THE STUDY: The aim of this study was to investigate the pharmaceutical effect of DNJ on high-fat and streptozotocin (STZ)-induced prediabetes mice, and to elucidate the mechanism of insulin resistance ameliorated by DNJ. MATERIALS AND METHODS: Oral glucose tolerance test (OGTT) and insulin tolerance test (ITT) were performed to detect blood glucose level and insulin sensitivity in mice. The levels of circulating lipopolysaccharide (LPS), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) in the plasma of mice were measured by limulus reagent and enzyme-linked immunosorbent assay (ELISA), respectively. Next-generation sequencing (NGS) and intestinal microbiota sequencing were used to screen the alterations in the transcriptome of liver tissues and to assess the differences in intestinal flora composition, respectively. Expression of cytokine signaling pathway inhibitor 3 (SOCS3), insulin receptor substrate (IRS1), p-IRS1 (Tyr896), occludin, and toll like receptor 4 (TLR4)/NF-κB signaling pathway were confirmed by western blotting. RESULTS: Our study revealed that DNJ decreased the blood glucose level and improve insulin sensitivity in prediabetic mice. DNJ significantly reduced the relative risk of T2DM in prediabetic mice by approximately 83.7%. Mechanistically, DNJ treatment suppressed the circulating levels of LPS, IL-6, and TNF-α in plasma and decreased the inflammatory infiltration in liver and colon tissues. DNJ-treatment increased the abundance of Akkermansia, Bifidobacterium, and Lactobacillus, and decreased the abundance of Enterococcaceae and Lachnospiraceae. Moreover, DNJ suppressed the expression of SOCS3 and the activity of TLR4/NF-κB signaling pathway, meanwhile improving the expression of occludin and the ratio of p-IRS1 (Tyr896)/IRS1. CONCLUSIONS: DNJ effectively ameliorates glucose and lipid metabolism in prediabetic mice, and decreased the relative risk of progression into T2DM from prediabetes. The suppressed immune responses play essential roles in the improvement of insulin resistance by DNJ treatment. In conclusion, DNJ from Morus alba L. is a promising alternative agent in T2DM prevention.

Advances in the Extraction, Purification and Detection of the Natural Product 1-Deoxynojirimycin.

1-Deoxynojirimycin (1-DNJ), a polyhydroxylated alkaloid, is a highly selective and potent glycosidase inhibitor that has garnered great interest as a tool to study cellular recognition and as a potential therapeutic agent. The development of analytical methods for the quantification polyhydroxylated alkaloids in natural products requires a multifaceted approach. Many publications over the past five decades have described analytical methods for this compound. However, recently more advanced techniques have come to prominence for sample extraction, purification, detection, and identification. This review provides an updated, extensive overview of the available methods for the extraction, purification, identification or detection of 1-DNJ. The review highlights different strategies for the design of 1-DNJ detection methods, which we analyzed in light of recent detection data. Finally, we conclude with perspectives on possible strategies for increasing the efficiency of identification and quantification of 1-DNJ in the future.

Effect of 1-Deoxynojirimycin Isolated from Mulberry Leaves on Glucose Metabolism and Gut Microbiota in a Streptozotocin-Induced Diabetic Mouse Model.

1-Deoxynojirimycin (DNJ) exerts hypoglycemic effects. However, the traditional method for DNJ extraction is inefficient, and the hypoglycemic mechanism of DNJ remains unclear. In this study, the mixed fermentation by Lactobacillus fermentum and Saccharomyces cerevisiae was used to enhance DNJ extraction efficiency. It was found that this strategy was more efficient than the traditional method as the yield improved from the original 3.24 mg/g to 5.97 mg/g. The purified DNJ significantly decreased serum glucose (P < 0.01) and insulin levels (P < 0.05), improved serum lipid levels (P < 0.05), and reversed insulin resistance (P < 0.05) in diabetic mice. These changes were caused by up-regulating the protein expression of insulin receptor and glycolysis enzymes (GK, PK, and PFK) (P < 0.05) and down-regulating the protein expression of insulin receptor substrate-1 and gluconeogenesis enzymes (PCB, PEPCK, FBPase, and G-6-Pase) (P < 0.05), thus alleviating glucose tolerance. Additionally, DNJ treatment relieved gut dysbiosis in diabetic mice by promoting the growth of Lactobacillus, Lachnospiraceae NK4A136 group, Oscillibacter, norank Lachnospiraceae, Alistipes, and Bifidobacterium (P < 0.05) and suppressing the growth of Ruminococcaceae UCG-014, Weissella, Ruminococcus, Prevotellaceae Ga6A1 group, Anaerostipes, Klebsiella, Prevotellaceae UCG-001, and Bacteroidales S24-7 group (P < 0.05).

Preparation of carbon-functionalized magnetic graphene/mesoporous silica composites for selective extraction of miglitol and voglibose in rat plasma.

In this work, magnetic graphene/mesoporous silica composites with carbon-functionalized pore-walls (denoted as MG@mSiO2-C composites) were synthesized and applied as restricted access matrix solid phase extraction (RAM-SPE) adsorbents for the determination of miglitol and voglibose in rat plasma by LC-MS/MS. The MG@mSiO2-C composites were synthesized by using the template (Cetyltrimethyl Ammonium Bromide, CTAB) as carbon source with sulfuric acid pretreated. The obtained nano-composites were proven to have many unique properties such as large specific surface area of 277.1 cm2 g-1, uniform mesopores with average pore size of 3.35 nm, and carbon-functionalized pore-walls. Taking advantage of the hydrophilic interaction between carbon and glycans, α-glucosidase inhibitors (miglitol and voglibose) could be directly extracted from rat plasma with no need of other pre-treatment procedures. The SPE conditions such as the adsorbent amount, elution solvent type, adsorption time and elution time were optimized. For both miglitol and voglibose, good linearities of 10-2000 ng mL-1 were obtained with determination coefficients (R2) > 0.99. The intra-day and inter-day RSDs were 3.3-6.9% (n = 6) and 6.0-8.0% (n = 6), respectively. The recoveries were in the range of 99.9-100.4% and the sensitivities were as low as 2-2.5 ng mL-1 (LOD). This MG@mSiO2-C composites-based RAM-SPE method offers high extraction efficiency for the determination of α-glucosidase inhibitor in plasma.

1-Deoxynojirimycin: Occurrence, Extraction, Chemistry, Oral Pharmacokinetics, Biological Activities and In Silico Target Fishing.

1-Deoxynojirimycin (DNJ, C6H13NO4, 163.17 g/mol), an alkaloid azasugar or iminosugar, is a biologically active natural compound that exists in mulberry leaves and Commelina communis (dayflower) as well as from several bacterial strains such as Bacillus and Streptomyces species. Deoxynojirimycin possesses antihyperglycemic, anti-obesity, and antiviral features. Therefore, the aim of this detailed review article is to summarize the existing knowledge on occurrence, extraction, purification, determination, chemistry, and bioactivities of DNJ, so that researchers may use it to explore future perspectives of research on DNJ. Moreover, possible molecular targets of DNJ will also be investigated using suitable in silico approach.

Identification and Determination of the Polyhydroxylated Alkaloids Compounds with α-Glucosidase Inhibitor Activity in Mulberry Leaves of Different Origins.

Mulberry leaves have commonly been utilized in China as a herbal medicine for the treatment of diabetes for thousands of years. To evaluate the quality, an ultra-high performance liquid chromatography coupled with quadrupole time of flight mass spectrometry (UPLC-Q-TOF/MS) method was developed for identification of polyhydroxylated alkaloids with α-glucosidase inhibitor activity in mulberry leaf. As a result, five alkaloid compounds were identified or tentatively characterized. Among them, the compound 1-deoxynojirimycin (DNJ) was selected as the most typical and active chemical marker and quantified using an improved high performance liquid chromatography (HPLC) normal phase coupled with evaporative light scattering detector (ELSD) method. The developed method was fully validated in terms of linearity, sensitivity, precision and repeatability, as well as recovery, and subsequently applied to evaluate twenty-nine batches of mulberry leaves from different collections. From the analytical data it was discovered that the average content of DNJ is 1.53 mg/g, while the total contents of DNJ in the 29 mulberry leaf sample ranged from 0.20 to 3.88 mg/g, which suggested remarkable differences, although it reached the highest levels in early August. These data may provide an important reference for the quality of mulberry leaves used as herbal medicine for the treatment of diabetes or as a material to obtain the DNJ of α-glucosidase inhibitor or as a functional food.

Effect of solar radiation on the functional components of mulberry (Morus alba L.) leaves.

BACKGROUND: The functional components of mulberry leaves have attracted the attention of the health food industry, and increasing their concentrations is an industry goal. This study investigated the effects of solar radiation, which may influence the production of flavonol and 1-deoxynojirimycin (DNJ) functional components in mulberry leaves, by comparing a greenhouse (poor solar radiation) and outdoor (rich solar radiation) setting. RESULTS: The level of flavonol in leaves cultivated in the greenhouse was markedly decreased when compared with those cultivated outdoors. In contrast, the DNJ content in greenhouse-cultivated plants was increased only slightly when compared with those cultivated outdoors. Interestingly, the flavonol content was markedly increased in the upper leaves of mulberry trees that were transferred from a greenhouse to the outdoors compared with those cultivated only in the outdoors. CONCLUSION: Solar radiation conditions influence the synthesis of flavonol and DNJ, the functional components of mulberry leaves. Under high solar radiation, the flavonol level becomes very high but the DNJ level becomes slightly lower, suggesting that the impact of solar radiation is great on flavonol but small on DNJ synthesis. © 2016 Society of Chemical Industry.

1-Deoxynojirimycin Alleviates Insulin Resistance via Activation of Insulin Signaling PI3K/AKT Pathway in Skeletal Muscle of db/db Mice.

1-Deoxynojirimycin (DNJ) is widely used for the treatment of diabetes mellitus as an inhibitor of intestinal α-glucosidase. However, there are few reports about its effect on insulin sensitivity improvement. The aim of the present study was to investigate whether DNJ decreased hyperglycemia by improving insulin sensitivity. An economical method was established to prepare large amounts of DNJ. Then, db/db mice were treated with DNJ intravenously (20, 40 and 80 mg·kg(-1)·day(-1)) for four weeks. Blood glucose and biochemical analyses were conducted to evaluate the therapeutic effects on hyperglycemia and the related molecular mechanisms in skeletal muscle were explored. DNJ significantly reduced body weight, blood glucose and serum insulin levels. DNJ treatment also improved glucose tolerance and insulin tolerance. Moreover, although expressions of total protein kinase B (AKT), phosphatidylinositol 3 kinase (PI3K), insulin receptor beta (IR-ß), insulin receptor substrate-1 (IRS1) and glucose transporter 4 (GLUT4) in skeletal muscle were not affected, GLUT4 translocation and phosphorylation of Ser473-AKT, p85-PI3K, Tyr1361-IR-ß and Tyr612-IRS1 were significantly increased by DNJ treatment. These results indicate that DNJ significantly improved insulin sensitivity via activating insulin signaling PI3K/AKT pathway in skeletal muscle of db/db mice.

Improved 1-Deoxynojirimycin (DNJ) production in mulberry leaves fermented by microorganism.

DNJ, an inhibitor of α-glucosidase, is used to suppress the elevation of postprandial hyperglycemia. In this study, we focus on screening an appropriate microorganism for performing fermentation to improve DNJ content in mulberry leaf. Results showed that Ganoderma lucidum was selected from 8 species and shown to be the most effective in improvement of DNJ production from mulberry leaves through fermentation. Based on single factor and three factor influence level tests by following the Plackett-Burman design, the optimum extraction yield was analyzed by response surface methodology (RSM). The extracted DNJ was determined by reverse-phase high performance liquid chromatograph equipped with fluorescence detector (HPLC-FD). The results of RSM showed that the optimal condition for mulberry fermentation was defined as pH 6.97, potassium nitrate content 0.81% and inoculums volume 2 mL. The extraction efficiency reached to 0.548% in maximum which is 2.74 fold of those in mulberry leaf.

Response surface optimized extraction of 1-deoxynojirimycin from mulberry leaves (Morus alba L.) and preparative separation with resins.

In the present study, the extraction technology and preparative separation of 1-deoxynojirimycin from mulberry leaves were systematically investigated. Four extraction parameters (ethanol concentration, extraction temperature, extraction time and ratio of solvent to sample) were explored by response surface methodology (RSM). The results indicated that the maximal yield of 1-deoxynojirimycin was achieved with an ethanol concentration of 55%, extraction temperature of 80 °C, extraction time of 1.2 h and ratio of solvent to sample of 12:1. The extraction yield under these optimum conditions was found to be 256 mg/100 g dry mulberry leaves. A column packed with a selected resin was used to perform dynamic adsorption and desorption tests to optimize the separation process. The results show that the preparative separation of 1-deoxynojirimycin from mulberry leaves can be easily and effectively done by adopting 732 resin. In conclusion, 732 resin is the most appropriate for the separation of 1-deoxynojirimycin from other components in mulberry leaves extracts, and its adsorption behavior can be described with Langmuir isotherms and a two-step adsorption kinetics model. The recovery and purity of 1-deoxynojirimycin in the final product were 90.51% and 15.3%, respectively.

Improved 1-Deoxynojirimycin (DNJ) production in mulberry leaves fermented by microorganism

DNJ, an inhibitor of α-glucosidase, is used to suppress the elevation of postprandial hyperglycemia. In this study, we focus on screening an appropriate microorganism for performing fermentation to improve DNJ content in mulberry leaf. Results showed that Ganoderma lucidum was selected from 8 species and shown to be the most effective in improvement of DNJ production from mulberry leaves through fermentation. Based on single factor and three factor influence level tests by following the Plackett-Burman design, the optimum extraction yield was analyzed by response surface methodology (RSM). The extracted DNJ was determined by reverse-phase high performance liquid chromatograph equipped with fluorescence detector (HPLC-FD). The results of RSM showed that the optimal condition for mulberry fermentation was defined as pH 6.97, potassium nitrate content 0.81% and inoculums volume 2 mL. The extraction efficiency reached to 0.548% in maximum which is 2.74 fold of those in mulberry leaf.

1-Deoxynojirimycin isolated from a Bacillus subtilis stimulates adiponectin and GLUT4 expressions in 3T3-L1 adipocytes.

We have demonstrated that 1-deoxynojirimycin (DNJ) isolated from Bacillus subtilis MORI could enhance the levels of adiponectin and its receptors in differentiated 3T3-L1 adipocytes, which has been shown to be effective in lowering blood glucose levels and enhancing insulin sensitivity. DNJ was not toxic to differentiated 3T3-L1 adipocytes for up to a concentration of 5 microM. In terms of expression levels of adiponectin and its receptors (AdipoR1 and AdipoR2), DNJ in concentrations as low as 0.5 microM elevated both mRNA and protein levels of adiponectin and transcript levels of AdipoR1 and AdipoR2. In addition, DNJ increased phosphorylation of 5' adenosine monophosphateactivated protein kinase (AMPK) in a statistically significant manner. Finally, treatment with DNJ resulted in increased mRNA expression of glucose transporter 4 (GLUT4), which encodes for a glucose transporter, along with a significant increase in glucose uptake into the adipocytes based on results of a 2-deoxy-D-[3H] glucose uptake assay. Our findings indicate that DNJ may greatly facilitate glucose uptake into adipose tissues by increasing the action of adiponectin via its up-regulated expression as well as its receptor genes. In addition, the glucose-lowering effects of DNJ may be achieved by an increased abundance of GLUT4 protein in the plasma membrane, as a consequence of the increased transcript levels of the GLUT4 gene and the activation of AMPK.

Homonojirimycin, an alkaloid from dayflower inhibits the growth of influenza A virus in vitro.

We have previously examined the antiviral effects of total alkaloids from Commelina communis L. (TAC). Here we investigated the active constituents of TAC, responsible for the antiviral effect. Harman, homonojirimycin (HNJ) and 2,5-dihydroxymethyl-3,4-dihydroxypyrrolidine were isolated from TAC by HPLC. Only HNJ showed strong antiviral activity against influenza A/PR/8/34 virus (H1N1) as measured by cytopathic effect reduction assay. The results suggest that HNJ is one of the active components of TAC.

Simple, selective, and rapid quantification of 1-deoxynojirimycin in mulberry leaf products by high-performance anion-exchange chromatography with pulsed amperometric detection.

1-Deoxynojirimycin (DNJ) occurs in mulberry and other plants and is a highly potent glycosidase inhibitor reported to suppress blood glucose levels, thus preventing diabetes. Derivatization is required for quantification of DNJ upon use of spectral detection methods. Because of this difficulty, the DNJ contents of mulberry-based food products are rarely stated, even if DNJ is their active component. A simple, selective, and rapid method of high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) to quantify DNJ in mulberry-based food products was developed. Stability testing of DNJ under heat treatment was also performed. A water extract of mulberry tea sample was subjected to HPAEC-PAD in a CarboPac MA1 column with a sodium hydroxide gradient. DNJ was clearly separated at a retention time of 7.26 min without interference and was selectively detected in the water extract. The detection limit was 5 ng. Heat stability studies suggested that DNJ was heat stable. HPAEC-PAD was not subject to interference, was highly selective for DNJ, and was superior to other high-performance liquid chromatography (HPLC) techniques in terms of sample preparation, resolution, and sensitivity. The method allowed simple, selective, and rapid analysis of DNJ in food matrices and might be useful for development of mulberry-based food products. Heat treatment could be an option for sterilizing mulberry-based products.

Purification and identification of 1-deoxynojirimycin (DNJ) in okara fermented by Bacillus subtilis B2 from Chinese traditional food (Meitaoza).

This study was to purify an alpha-glucosidase inhibitor from okara (soy pulp) fermented by Bacillus subtilis B2 and to identify its chemical structure. Membrane dialysis, active charcoal, CM-Sepharose chromatography, and preparative thin-layer chromatography (TLC) were used in the purification, while positive mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectrometry were used in the identification. The MS and NMR data showed that the purified alpha-glucosidase inhibitor was 1-deoxynojirimycin (DNJ) with a molecular weight of 163 Da. This is the first time that DNJ was isolated from foods fermented with Bacillus species. Okara fermentation with B. subtilis B2 might be used to produce a food-derived DNJ product as a functional food for diabetic patients.

Optimization of 1-deoxynojirimycin extraction from mulberry leaves by using response surface methodology.

Mulberry 1-deoxynojirimycin (DNJ, a potent alpha-glycosidase inhibitor) has therapeutic potency against diabetes mellitus. However, the amount of DNJ in mulberry leaves is low (about 0.1%), and a more effective extraction method is needed. Ultrasound-assisted extraction (UAE) was applied in this study for mulberry DNJ extraction, and five factors, the percentage of ethanol in the extraction solvent (x(1)), ratio of the extraction solvent to mulberry sample (x(2)), ultrasonic power (x(3)), extraction temperature (x(4)) and extraction time (x(5)), were investigated by fractional factorial 2((5-1)) design (FFD) to obtain the optimum extraction efficiency (DNJ yield, Y(1)) and extraction productivity (total yield, Y(2)). The results showed that x(2), x(3) and x(5) had significant impact on Y(1) and Y(2), and were further optimized by response surface methodology (RSM). Under the optimized conditions (x(2), x(3) and x(5) of 7 ml/g, 180 W and 260 s, respectively), DNJ-enriched powder (0.8%) was produced with high extraction efficiency (98%) and productivity (20%), enabling this product to be used for nutraceutical purposes.

[Quantitative determination of 1-deoxynojirimycin in mulberry leaves by high-performance liquid chromatographic-tandem mass/mass spectrometry].

OBJECTIVE: To develop a high-performance liquid chromatographic-tandem mass/mass spectrometric method to determine the concentration of 1-deoxynojirimycin (1-DNJ) in mulberry leaves. METHODS: 1-deoxynojirimycin was separated on an SHIMADZU HRC-NH2 column with the mobile phase consisting of acetonitrile and 0.1% formic acid. The mass spectrometric system equipped with a atmospheric pressure chemical ionization (APCI) interface was operated in Multiple Reaction Monitoring (MRM) mode. RESULTS: The retention time of 1-deoxynojirimycin was 2.87 min, and the calibration curve was linear over a concentration range from 482 microg/L to 2410 microg/L, the average recovery was 95.8%. The detection limit was 53.6 microg/L. CONCLUSION: The method is selective and sensitive for determining 1-deoxynojirimycin in mulberry leaves.

[Chemical constituents from Polygala telephioides].

OBJECTIVE: To study the chemical constituents of Polygala telephioides. METHOD: The compounds were isolated and purified on macroporous resin, silica gel, Sephadex LH-20, Chromatorex ODS column chromatograph and the structures were determined based on the spectral and chemical evidences. RESULT: Four compounds were obtained and characterized as telephiose G, telephiose D, isomangiferin, quescetin 3-O-beta-D-glucopyranoside, respectively. CONCLUSION: Compounds 2-4 were obtained from this plant for the first time and the compound 2 (telephiose G) was a new compound.

[Contrast of 1-deoxynojirimycin content in mulberry leaves from different habitats].

OBJECTIVE: To Contrast the content of 1-deoxynojirimycin in Mulberry Leaves from different habitats. METHODS: The samples were analyzed by high performance liquid chromatography equipped with fluorescence detector and separated on HiQSiLC18 column at 25 degrees C. Mobile phase consisted of acetonitrile-0.1% aqueous acetic acid (55: 45) with a flow rate of 1.0 ml x min(-1). The fluorescence detector was operated at lambdaEX = 254 nm, lambdaEM = 322 nm. RESULTS: The contents of 1-deoxynojirimycin from different habitats of Mulberry Leaves were remarkably different, while those from the same habitats but different species or varieties were different too. CONCLUSION: The result can offer the proof for exploiting the resource of Mulberry Leaves preferably.

Novel anti-adherence activity of mulberry leaves: inhibition of Streptococcus mutans biofilm by 1-deoxynojirimycin isolated from Morus alba.

OBJECTIVES: The present study focused on isolation, characterization and evaluation of purified compounds from Morus alba against Streptococcus mutans biofilm formation. METHODS: The effect of crude extract from M. alba leaves was evaluated against oral pathogens, chiefly S. mutans. MICs were determined by the microdilution method. The compound was purified by employing silica gel chromatography and critically analysed with GC-MS, NMR and IR spectroscopy. The S. mutans traits of adherence and biofilm formation were assessed at sub-MIC concentrations of the crude extract and purified compound. Both water-soluble and alkali-soluble polysaccharide were estimated to determine the effect of the purified compound on the extracellular polysaccharide secretion of S. mutans. Its effect on biofilm architecture was also investigated with the help of confocal microscopy. RESULTS: The purified compound of M. alba showed an 8-fold greater reduction of MIC against S. mutans than the crude extract (MICs, 15.6 and 125 mg/L, respectively). The extract strongly inhibited biofilm formation of S. mutans at its active accumulation and plateau phases. The purified compound led to a 22% greater reduction in alkali-soluble polysaccharide than in water-soluble polysaccharide. The purified compound was found to be 1-deoxynojirimycin (DNJ). Confocal microscopy revealed that DNJ distorts the biofilm architecture of S. mutans. CONCLUSIONS The whole study reflects a prospective role of DNJ as a therapeutic agent by controlling the overgrowth and biofilm formation of S. mutans.