α-glucosidase

α-Glucosidase inhibitory activities [2]
For decades, researchers have shown that rat and human α-glucosidase is strongly inhibited by mulberry leaf extract (Anno et al., 2004, Miyahara et al., 2004, Oku et al., 2006). α-Glucosidase, located in the brush-border surface membranes of intestinal cells, is considered the most important enzyme in digestion of starch and other carbohydrates (Herscovics, 1999). Modification of carbohydrate metabolism by dietary foods and drugs may have therapeutic value. Mulberry 1-Deoxynojirimycin (Duvoglustat)/DNJ binds to the active center of α-glucosidase and is a potent inhibitor of this enzyme in the small intestine (Junge, Matzke, & Stoltefuss, 1996). For commercial development of nutraceutical products, the target compound and its concentration in the product should be known in order to achieve the best therapeutic results. In the case of mulberry dry tea, we think DNJ is the key compound because it strongly inhibits α-glucosidase and mulberry leaves contain high concentrations of it (50% of total imino sugars) (Asano et al., 2001). α-Glucosidase inhibition was highly correlated with both pure DNJ (r = 0.96) (Fig. 4B) and DNJ content of mulberry leaves (r = 0.84) (Fig. 4A). At comparable 1-Deoxynojirimycin (Duvoglustat)/DNJ concentrations, mulberry leaf extract had more α-glucosidase inhibitory activity than the DNJ standard: for example at 5 μg DNJ/ml, α-glucosidase activity was inhibited 27% by mulberry leaf extract and 23% by pure DNJ. The additional inhibition can be explained by presence in mulberry extract of other imino sugars (i.e., N-methyl-DNJ, 2-O-α-d-galactopyanosyl-DNJ and fagomine) and other ingredients such as isoquercitrin, quercetin and rutin.

Duvoglustat hydrochloride (1-Deoxynojirimycin hydrochloride) (20-80 mg/kg; i.v.; daily for four weeks) shows anti-obesity effect [3]. Duvoglustat hydrochloride significantly improves insulin sensitivity via activating insulin signaling PI3K/AKT pathway in skeletal muscle of db/db mice[3].

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1-Deoxynojirimycin (Duvoglustat)/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-β), 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-β 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. [3]

α-Glucosidase inhibition assay [1]
α-Glucosidase inhibitory activity was measured by a modification of the procedure described by Ma, Hattori, Daneshtalab, and Wang (2008). Briefly, rat-intestine acetone powder (1 g) was suspended in 100 mM potassium phosphate buffer (pH 7.0) and the suspension was sonicated for 20 min. After centrifugation at 3000 rpm for 30 min, the supernatant was used as the source of α-glucosidase. Substrate (2 mM 4-nitrophenyl-α-d-glucopyranoside) in 100 mM potassium phosphate buffer (pH 7.0) was pipetted into 96-well plates (40 μl/well). Five μl mulberry sample or control solution (a 50:50 mixture of ethanol and distilled water) was added and the solution was mixed. After addition of enzyme (5 μl), the mixture was incubated at 37 °C for 20 min, and then UV absorbance (405 nm) was measured. The percent α-glucosidase inhibitory activity of mulberry samples and standard 1-Deoxynojirimycin (Duvoglustat)/DNJ was calculated as: (ΔAcontrol-ΔAsample) × 100/ΔAcontrol, where ΔA is absorbance at 405 nm.

Western Blot [3]
In order to investigate the effects of 1-Deoxynojirimycin (Duvoglustat)/DNJ on insulin signaling pathways, western blot analysis was performed as previously described. Briefly, skeletal muscle tissues (0.1 g) were lysed in lysis buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% NP40, 10 μL phosphatase inhibitors, 1 μL protease inhibitor and 5 μL 100 mM PMSF), centrifuged for 15 min at 16,000× g at 4 °C, and protein concentration was quantified by bicinchonininc acid protein assay. Equal amounts of protein (70 μg) were loaded on 10% SDS-PAGE and transferred onto PVDF membranes. After membranes were blocked, they incubated with the primary antibodies against IR-β, p-Tyr1361-IR-β, IRS1, p-Tyr612-IRS1, PI3K, p-p85-PI3K, AKT, p-Ser473-AKT, GLUT4, β-actin or Na+K+-ATPase α1 overnight at 4 °C followed by HRP conjugated secondary antibody for 2 h at room temperature. Protein bands were visualized using an ECL detection kit. Normalization of total protein expression was carried out by using β-actin as control. Normalization of m-GLUT4 expression was carried out using Na+K+-ATPase α1 as control

db/db mice
20, 40, 80 mg/kg
Intravenously; daily for four weeks

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At the end of ten weeks, wild-type C57BLKS mice, which received intravenously normal saline, served as a normal control (N control) (n = 6). The db/db mice were divided into four groups (n = 6): Group I served as a diabetic control and received intravenously normal saline (D control). Group II, III, and IV were treated intravenously with 1-Deoxynojirimycin (Duvoglustat)/DNJ 20, 40, and 80 mg·kg−1·day−1, respectively. An intravenous injection was selected to avoid the function of DNJ as an α-Glycosidase inhibitor inthe gastrointestinal tract. For DNJ doses selection, in our previous study, we screened a large number of Chinese traditional medicines including mulberry leaves by glucose tolerance test of ICR mice. We found the alkaloids (DNJ 40 mg·kg−1) isolated from mulberry leaves could improve the glucose tolerance test of ICR mice (Figure A1). We then tested doses of 10, 20, and 40 mg·kg−1, but both 10 and 20 mg·kg−1 did not have any effect (Figure A2). Therefore, we selected the 1-Deoxynojirimycin (Duvoglustat)/DNJ doses as 20, 40, and 80 mg·kg−1·day−1. All these doses were given for 4 weeks. The blood glucose, body weight and average food intake, water intake, and urine output were measured every week. At the end of the experimental period, the mice were anesthetized with chloral hydrate after withholding food for 12 h, and blood samples were taken to determine the serum insulin levels. Besides, skeletal muscle were removed after the blood was collected, then rinsed with a physiological saline solution, and immediately stored at −80 °C [3].

Oral LD50 in rats >5 gm/kg. Behavioral effects: lethargy (reduced overall activity); kidneys, ureters, and bladder: increased urine output; skin and its appendages (skin); hair: other. International Journal of Toxicology, 16 (Supplement).

[1]. 1-Deoxynojirimycin: Occurrence, Extraction, Chemistry, Oral Pharmacokinetics, Biological Activities and In Silico Target Fishing. Molecules. 2016 Nov 23;21(11). pii: E1600.

[2]. Development of high 1-deoxynojirimycin (DNJ) content mulberry tea and use of response surface methodology to optimize tea-making conditions for highest DNJ extraction. LWT - Food Science and Technology. Volume 45, Issue 2, March 2012, Pages 226-232.

[3]. 1-Deoxynojirimycin Alleviates Insulin Resistance via Activation of Insulin Signaling PI3K/AKT Pathway in Skeletal Muscle of db/db Mice. Molecules. 2015 Dec 4;20(12):21700-14.

AT2220 is an investigational oral medication for the treatment of Pompe disease, belonging to a class of molecules known as pharmacological chaperones. It is a small molecule designed to act as a pharmacological chaperone, specifically binding to, stabilizing, and promoting the proper folding and transport of α-glucosidase (GAA). GAA is transported to lysosomes, where it performs its normal function. Studies have shown that AT2220 can increase GAA activity in cell lines derived from Pompe disease patients and in transfected cells expressing misfolded GAA. It is also an α-glucosidase inhibitor with antiviral activity. Deoxynojirimycin derivatives may have anti-HIV activity. Drug Indications Pompe disease, also known as glycogen storage disease type II or acid maltase deficiency, is a relatively rare neuromuscular and lysosomal storage disorder caused by a genetic mutation in a key enzyme called α-glucosidase (Gaa).

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Mechanism of Action
AT2220 is designed as a pharmacological chaperone, selectively binding to Gaa, the misfolded enzyme that causes Pompe disease. Upon binding to this enzyme, AT2220 is believed to promote the correct folding, processing, and transport of the enzyme from the endoplasmic reticulum to its final destination—the lysosome, the intracellular region where the enzyme functions. Once at the lysosome, the pharmacological chaperone protein is displaced, allowing the enzyme to perform its normal function of breaking down its natural substrate, glycogen.

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Duvoglucosamine is the optically active form of 2-(hydroxymethyl)piperidine-3,4,5-triol with a 2R,3R,4R,5S configuration. It is an EC 3.2.1.20 (α-glucosidase) inhibitor, an anti-HIV drug, an anti-obesity drug, a bacterial metabolite, a hypoglycemic agent, a hepatoprotective agent, and a plant metabolite. It is a 2-(hydroxymethyl)piperidine-3,4,5-triol and a piperidine alkaloid.
An α-glucosidase inhibitor with antiviral activity. Derivatives of deoxynojirimycin may have anti-HIV activity.
1-Deoxynojirimycin has been reported in Candida austrosinense, Candida praesorediosum, and other organisms with relevant data.
An α-glucosidase inhibitor with antiviral activity. Derivatives of deoxynojirimycin may have anti-HIV activity.
See also: Fagomin (note moved here).

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1-Deoxynojirimycin (DNJ, C₆H13NO₄, 163.17 g/mol) is an alkaloid azosaccharide or iminosaccharide, a biologically active natural compound found in mulberry leaves, Commelina communis, and various bacterial strains (such as Bacillus and Streptomyces). Deoxynojirimycin has hypoglycemic, anti-obesity and antiviral properties. Therefore, this article aims to review in detail the existing knowledge on the sources, extraction, purification, determination, chemical properties and biological activities of deoxynojirimycin (DNJ) so that researchers can use this knowledge to explore future research directions of DNJ. In addition, this article will also use appropriate computer simulation methods to study the possible molecular targets of DNJ. [1]

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Mulberry 1-deoxynojirimycin (DNJ) is a potent α-glucosidase inhibitor that can inhibit postprandial blood glucose and thus may prevent diabetes. At present, dried mulberry tea is commercially sold as a functional food in many countries, but due to its low DNJ content (about 100 mg/100 g dry weight), these products may not provide an effective dose (6 mg DNJ/60 kg body weight). Therefore, it is necessary to develop tea with higher DNJ content. In order to study the distribution of DNJ and its inhibitory activity on α-glucosidase, we investigated the DNJ content in 35 Thai mulberry varieties. The DNJ content in young leaves varied among different mulberry varieties, ranging from 30 to 170 mg/100 g dry leaves. The varieties with the highest DNJ content were Kam, Burirum 60, and Burirum 51. Leaf position significantly affected DNJ content: young shoots > young leaves > mature leaves. DNJ concentration was highly correlated with α-glucosidase inhibitory activity (r = 0.84), indicating that the α-glucosidase inhibitory activity in mulberry leaves mainly originates from DNJ. Therefore, we used young shoots from varieties such as Burirum 60 to produce mulberry leaf tea with high DNJ content, reaching up to 300 mg/100 g dry leaves. We optimized the tea-making conditions using response surface methodology to obtain the highest DNJ extraction rate. Holding at 98℃ for 400 seconds resulted in the extraction of approximately 95% of the total DNJ from the high-DNJ-content dry tea; these conditions are suitable for preparing commercially available products with high DNJ content. A cup (230 ml, standard serving) of DNJ-rich mulberry leaf tea contains sufficient DNJ (6.5 mg) to effectively suppress postprandial blood sugar. [2]

C6H14CLNO4

199.631

199.061

C, 36.10; H, 7.07; Cl, 17.76; N, 7.02; O, 32.06

73285-50-4

1-Deoxynojirimycin;19130-96-2

13018787

White to off-white solid powder

1.456 g/cm3

361.1ºC at 760 mmHg

195-196ºC

197.3ºC

6

5

1

12

132

4

Cl.OC[C@H]1NC[C@H](O)[C@@H](O)[C@@H]1O

ZJIHMALTJRDNQI-VFQQELCFSA-N

InChI=1S/C6H13NO4.ClH/c8-2-3-5(10)6(11)4(9)1-7-3/h3-11H,1-2H21H/t3-,4+,5-,6-/m1./s1

(2R,3R,4R,5S)-2-(hydroxymethyl)piperidine-3,4,5-triol hydrochloride

Duvoglustat; BAY-h 5595; AT2220; AT-2220; AT 2220; DNJ; 1DNJ; NOJ; Moranoline; Moranolin; Duvoglustat hydrochloride; deoxynojirimycin; 1-Deoxynojirimycin; 1-Deoxy-Nojirimycin 1-Deoxynojirimycin hydrochloride; 73285-50-4; Duvoglustat HCl; Moranoline hydrochloride; Moranoline HCl; (2R,3R,4R,5S)-2-(Hydroxymethyl)piperidine-3,4,5-triol hydrochloride

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O : ~250 mg/mL (~1252.32 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)