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

molecules

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

Kuo Gao 1,†, Chenglong Zheng 1,2,†, Tong Wang 2, Huihui Zhao 1, Juan Wang 1, Zhiyong Wang 1, Xing Zhai 1, Zijun Jia 1, Jianxin Chen 1, Yingwu Zhou 2,* and Wei Wang 1,*

1 Beijing University of Chinese Medicine, Bei San Huan East Road, Beijing 100029, China; [email protected] (K.G.); [email protected] (C.Z.); [email protected] (H.Z.); [email protected] (J.W.); [email protected] (Z.W.); [email protected] (X.Z.); [email protected] (Z.J.); [email protected] (J.C.) 2 Beijing Gulou Hospital of Traditional Chinese Medicine, 13 DouFuChi Hutong, Dongcheng District, Beijing 100009, China; [email protected] * Correspondence: [email protected] (Y.Z.); [email protected] (W.W.); Tel.: +86-10-6401-2858 (Y.Z.); +86-10-6428-7452 (W.W.) † These authors contributed equally to this work.

Academic Editor: Derek J. McPhee Received: 13 October 2016; Accepted: 16 November 2016; Published: 23 November 2016

Abstract: 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.

Keywords: iminosugar; mulberry; Bacillus; fermentation; silkworms; antihyperglycemic; anti-obesity; antiviral; molecular targets

1. Introduction Polyhydroxylated piperidines and their derivatives are famous for their excellent bioactivities [1]. Iminosugars contain an analog of pyranose ring or D-glucose, in which the ring oxygen atom is replaced by a nitrogen atom. 1-Deoxyiminosugars are chemically more stable than normal iminosugars because of absence of a hydroxyl group at the C1 position. Among the iminosugars, naturally occurring 1-deoxyiminosugars such as DNJ are strong glycosidase inhibitors [2]. Originally, reduction of nojirimycin led to the chemical synthesis of DNJ [3]; afterward, DNJ was found from natural source, i.e., mulberry tree root and Bacillus species [4,5]. 1-Deoxynojirimycin (DNJ, C6H13NO4, 163.17 g/mol) (Figure1), an alkaloid azasugar or iminosugar, is a biologically active natural compound [6–8]. The IUPAC name of DNJ is (2R,3R,4R,5S)- 2-(hydroxymethyl)piperidine-3,4,5-triol. It is also known as moranoline [9]. Various derivatives of DNJ are N-azidopropyl-1-deoxynojirimycin, N-nonyl-1-deoxynojirimycin, 1-deoxynojirimycin-6- phosphate, and N-methyl-1-deoxynojiri-mycin-6-phosphate [8]. It exists in mulberry leaves and Commelina communis (dayﬂower) as well as from several bacterial strains such as Bacillus and Streptomyces species [7].

Molecules 2016, 21, 1600; doi:10.3390/molecules21111600 www.mdpi.com/journal/molecules Molecules 2016, 21, 1600 2 of 15 Molecules 2016, 21, 1600 2 of 14

Figure 1. Chemical structure of 1-Deoxynojirimycin [7]. Figure 1. Chemical structure of 1-Deoxynojirimycin [7].

AfterAfter extraction, extraction, DNJDNJ contents contents can can be determin be determineded by HPLC by using HPLC various using detectors various such detectors as suchfluorescent, as fluorescent, evaporative evaporative light-scattering light-scattering detector, pulsed detector,amperometric, pulsed and Mass amperometric, spectrometer [10–12]. and Mass spectrometerSince DNJ [ molecule10–12]. Since does not DNJ contain molecule chromophore, does not the contain quantification chromophore, of DNJ through the quantification spectral analysis of DNJ needs derivatization, i.e., an analytical approach for quantification of nitrogen-containing compounds through spectral analysis needs derivatization, i.e., an analytical approach for quantification of [13–16]. To quantify and resolve structural information of DNJ by GC-MS, Bajpai and Rao used nitrogen-containing compounds [13–16]. To quantify and resolve structural information of DNJ trimethylsilyl (TMS) derivatization [7]. by GC-MS,The Bajpai pharmacokinetic and Rao used of DNJ trimethylsilyl after oral administrati (TMS)on derivatization has been studies [7]. by some researchers [17–19]. TheMoreover pharmacokinetic, DNJ has many of biological DNJ after activities, oral administration including antihyperglycemic has been studies [20–25], by some anti-obesity researchers [26–30],[17 –19]. Moreover,and anti-viral DNJ has [31] many have biological also been activities,reported [32–35]. including antihyperglycemic [20–25], anti-obesity [26–30], and anti-viralCurrently, [31] havethe prediction also been of biological reported targets [32–35 of]. small drug molecules has become very easy owing Currently,to the rapid the growth prediction of bioactivity of biological databases, targets in silico of small target drug fishing molecules approaches has become and accessible very easy web owing to theservices rapid [36]. growth There of are bioactivity many approaches databases, to in in silico silico target target fishing fishing including approaches the structure-based and accessible and web servicesthe ligand-based [36]. There aremethods, many data approaches mining, and to inchemical silico targetsimilarity fishing searching including [37]. Then, the structure-based the output data and of these approaches are validated by adopting useful modality. The structure-based target prediction the ligand-based methods, data mining, and chemical similarity searching [37]. Then, the output data methods are appropriate to the drug-like small organic entities, which induce biological effects but of these approaches are validated by adopting useful modality. The structure-based target prediction have ambiguous macromolecular targets [36]. methodsTherefore, are appropriate the aim to of the this drug-like detailed review small organicarticle is entities,to summarize which the induce existing biological knowledge effects on but haveoccurrence, ambiguous extraction, macromolecular purification, targets determination, [36]. chemistry, and bioactivities of DNJ, so that the Therefore,researchers may the use aim it ofto explore this detailed future perspectives review article of research is tosummarize on DNJ. Moreover, the existing possibleknowledge molecular on occurrence,targets of extraction, DNJ will also purification, be investigated determination, using suitable chemistry, in silico approach. and bioactivities of DNJ, so that the researchers may use it to explore future perspectives of research on DNJ. Moreover, possible molecular targets2. 1-Deoxynojirimycin of DNJ will also be investigated using suitable in silico approach.

2. 1-Deoxynojirimycin2.1. Occurrence 1-Deoxynojirimycin (DNJ), a product of fermentation, exists in mulberry leaves and 2.1. OccurrenceCommelina communis (dayflower) as well as is isolated from several bacterial strains such as Bacillus and Streptomyces species [7,8]. Mulberry, a common deciduous plant, belongs to the genus Morus (Moraceae 1-Deoxynojirimycin (DNJ), a product of fermentation, exists in mulberry leaves and family). The botanical and pharmaceutical names of mulberry tree are Morus alba L. and Folium mori, Commelina communis (dayflower) as well as is isolated from several bacterial strains such as Bacillus respectively [38]. Mulberry tree is found in countries with a subtropical or mild temperate environment, and Streptomycesincluding China,species Japan, [ 7Korea,,8]. Mulberry, India, Pakistan, a common and other deciduous Asian countries plant, [39]. belongs By reason to the of genus folkloreMorus (Moraceaetonic, mulberry family). leaves The botanical have been and used pharmaceutical as an anti-diabetic names tea. The of mulberrygeneral use tree of mulberry are Morus includes alba L. and Foliumsilkworm mori, respectively (Bombyx mori [ 38L.)]. feeding Mulberry (Figures tree 2 isand found 3) [40], in fruit countries production with [41], a subtropical and medicine or preparation mild temperate environment,[42,43]. Mulberry including leaves China, are used Japan, as Korea,a source India, of protein Pakistan, in food and products other Asian[44]. Many countries scientific [39 studies]. By reason of folklorehave reported tonic, mulberry the medicinal leaves importance have been of usedmulberry as an [45]. anti-diabetic Mulberry leaves tea. The are traditionally general use used of mulberry as includesmedicine silkworm for controlling (Bombyx blood mori L.) sugar feeding level [20,46–48]. (Figures2 andStudies3)[ have40], also fruit reported production the effectiveness [41], and medicine of preparationmulberry [ 42leaves,43]. in Mulberry skin aging leaves[49,50] and are neurodegenerative used as a source disorders of protein including in food Alzheimer’s products disease [44]. Many and Parkinson’s disease [51]. Moreover, sedative effect of mulberry fruits and anti-inflammatory, scientific studies have reported the medicinal importance of mulberry [45]. Mulberry leaves are diuretic, antitussive, and antipyretic properties of mulberry root bark have been studies [52]. In addition traditionally used as medicine for controlling blood sugar level [20,46–48]. Studies have also reported to DNJ, many other bioactive compounds including flavonoids, alkaloids, steroids, and coumarins also the effectivenessexist in mulberry of mulberryleaves [53]. leavesDNJ constitutes in skin only aging 0.11% [49 ,(50w/]w and) of mulberry neurodegenerative leaf [20,48], while disorders the synthesis including Alzheimer’sof DNJ is diseasea complex and process Parkinson’s [54]. For diseasethis reason [51, ].DNJ Moreover, is an expensive sedative compound. effect of Thus, mulberry the possible fruits and anti-inflammatory, diuretic, antitussive, and antipyretic properties of mulberry root bark have been studies [52]. In addition to DNJ, many other bioactive compounds including flavonoids, alkaloids, steroids, and coumarins also exist in mulberry leaves [53]. DNJ constitutes only 0.11% (w/w) of mulberry leaf [20,48], while the synthesis of DNJ is a complex process [54]. For this reason, DNJ is an Molecules 2016, 21, 1600 3 of 15 Molecules 2016, 21, 1600 3 of 14

intense demand for DNJ in future has urged exploring other sources of DNJ, including various bacterial expensive compound.Molecules 2016, 21 Thus,, 1600 the possible intense demand for DNJ in future has urged3 of exploring 14 other strains such as Bacillus [7], Streptomyces [8], Actinoplanes [55], and Flavobacterium saccharophilium sourcesspecies of DNJ, [56].intense including demand for various DNJ in future bacterial has urged strains explorin suchg other as sourcesBacillus of DNJ,[7], Streptomycesincluding various[8 bacterial], Actinoplanes [55], and Flavobacteriumstrains such saccharophilium as Bacillus [7], speciesStreptomyces [56 [8],]. Actinoplanes [55], and Flavobacterium saccharophilium species [56].

Figure 2. Mulberry leaf and fruit. Figure 2. Mulberry leaf and fruit. Figure 2. Mulberry leaf and fruit.

Figure 3. Silkworm larvae consuming mulberry leaves.

Numerous researchers have reported the DJN contents in different parts and various varieties of mulberry [25,57–60].Figure Various 3. Silkworm mulberry larvae varietie consumings contain DNJ mulberry contents leaves. ranging between 0.68 and 2.78 mg/gm [7], whileFigure this 3.fractionSilkworm varies between larvaeconsuming 1.57 and 3.48 mg/gm mulberry in different leaves. Chinese mulberry leaves [25]. Moreover, mulberry shoots contain the highest contents of DNJ, followed by young Numerous researchers have reported the DJN contents in different parts and various varieties mulberry leaves. Mature leaves of mulberry contain the least contents DNJ [6]. Another group of Numerousof mulberryinvestigators researchers[25,57–60]. has reported Various have the reportedmulberry quantity of thevarietie DJN DJN contentss contain contents in mulberry DNJ in contents different leaves fermented ranging parts byandbetween different various 0.68 varietiesand of mulberry2.78 mg/gmmicroorganisms, [25 ,[7],57– while60]. Various this such fraction as Lactobacillus mulberry varies plantarum between varieties ( lactic1.57 contain andacid bacteria 3.48 DNJ mg/gm), Zygosaccharomyces contents in different ranging rouxii Chinese between(yeast mulberry), 0.68 and 2.78 mg/gmleaves [25].Wickerhamomyces [7], Moreover, while this anomalusmulberry fraction (yeast varies shoots), and between Bacilluscontain subtilis 1.57the .highest They and also 3.48 contents examined mg/gm ofthe inDNJ, extent different followed to which Chinese the by young mulberry mulberryfermented leaves. mulberryMature leafleaves powder of mulberryextract (FMLE) cont inhibitedain the the least α-glucosidase contents activity. DNJ [6].All the Another mulberry group of leaves [25].leave Moreover, groups showed mulberry 1–2-fold shoots increase containin DNJ cont theents highestin comparison contents to unfermented of DNJ, mulberry followed leaf by young investigators has reported the quantity of DJN contents in mulberry leaves fermented by different mulberry leaves.powder Mature extract (UFMLE). leaves Additionally, of mulberry FMLE contain exhibited the higher least α-glucosidase contents activity DNJ [compared6]. Another to group of microorganisms, such as Lactobacillus plantarum (lactic acid bacteria), Zygosaccharomyces rouxii (yeast), investigatorsUFMLE has reported [61]. Resultantly, the quantity DNJ-rich food of DJN products contents can be prepared in mulberry by fermenting leaves mulberry fermented leaves by different Wickerhamomycesusing the above-mentioned anomalus (yeast fermenting), and Bacillus agents. subtilis. They also examined the extent to which the microorganisms,fermented mulberry such asleafLactobacillus powder extract plantarum (FMLE) inhibited(lactic acid the bacteria α-glucosidase), Zygosaccharomyces activity. All the mulberry rouxii (yeast ), Wickerhamomycesleave groups2.2. Extraction showed anomalus 1–2-fold(yeast increase), and Bacillus in DNJ cont subtilisents. in They comparison also examined to unfermented the extent mulberry to which leaf the fermentedpowder mulberry extractThere (UFMLE). leafare four powder routes Additionally, to extract produce (FMLE) DNJ:FMLE (i) extractiexhibited inhibitedon from higher the plantsα -glucosidaseα -glucosidasesuch as the mulberry activity. activity trees; compared All (ii) the mulberry to leaveUFMLE groups extraction[61]. showed Resultantly, from 1–2-fold silkworm; DNJ-rich increase (iii) chemical food in DNJproducts synthesis contents followingcan be in prepared comparison different synthetic by fermenting to unfermentedstrategies; mulberry and (iv) mulberry leaves leaf fermentation by various Bacillus or Streptomyces. Currently, the use of mulberry dry tea is flourishing using the above-mentioned fermenting agents. powder extractas functional (UFMLE). food. The Additionally, content of DNJ FMLEis as low exhibitedas approximately higher 100 mg/100α-glucosidase g of dry tea. This activity fraction compared to UFMLE [61].of Resultantly,DNJ is biological DNJ-rich ineffective (Biologically food products effective can dose be of DNJ prepared is 6 mg per by 60 fermenting kg human weight). mulberry leaves 2.2. Extraction using the above-mentionedThus, the possible intense fermenting demand for agents. DNJ in future has urged exploring new strategies for its efficient extraction and purification. In this context, a modality was proposed by Ezure et al. who developed There are four routes to produce DNJ: (i) extraction from plants such as the mulberry trees; (ii) a rapid screening approach (oblate agar plate method) for isolation of DNJ-producing Streptomyces 2.2. Extractionextractionlavendulae from silkworm; GC-148. Its DNJ(iii) chemicalspectra were synthesis identical tofollowing that obtained different from mulberry synthetic [9]. strategies;Afterwards, and (iv) fermentationthey noted by various 27–33 folds Bacillus increase or in Streptomyces the production. Currently, of DNJ through the media use of improvement mulberry dryand mutagenictea is flourishing Thereas functional are four food. routes The content to produce of DNJ is DNJ: as low (i) as extraction approximately from 100 plants mg/100 such g of dry as tea. the This mulberry fraction trees; (ii) extractionof DNJ is biological from silkworm; ineffective (iii) (Biologically chemical effe synthesisctive dose following of DNJ is different6 mg per synthetic60 kg human strategies; weight). and (iv) fermentationThus, the possible by various intenseBacillus demandor forStreptomyces DNJ in future. Currently, has urged exploring the use of new mulberry strategies dry for tea its isefficient flourishing as functionalextraction food. and purification. The content In of this DNJ context, is as low a mo asdality approximately was propos 100ed mg/100by Ezure get of al. dry who tea. developed This fraction of DNJa rapid is biological screening ineffective approach (oblate (Biologically agar plate effective method) dose for isolation of DNJ isof 6DNJ-producing mg per 60 kg Streptomyces human weight). Thus,lavendulae the possible GC-148. intense Its DNJ demand spectra for were DNJ inidentical future to has th urgedat obtained exploring from mulberry new strategies [9]. Afterwards, for its efficient extractionthey noted and 27–33 purification. folds increase In this in context,the production a modality of DNJwas through proposed media improvement by Ezure et al.and who mutagenic developed a rapid screening approach (oblate agar plate method) for isolation of DNJ-producing Streptomyces lavendulae GC-148. Its DNJ spectra were identical to that obtained from mulberry [9]. Afterwards, they noted 27–33 folds increase in the production of DNJ through media improvement and mutagenic Molecules 2016, 21, 1600 4 of 15 treatments (ultraviolet irradiation and N-methyl-N0-nitro-N-nitrosoguanidine treatment). Furthermore, to develop tea with higher DNJ content, Vichasilp et al. studied 35 Thai mulberry varieties to explore the content distribution and α-glucosidase inhibitory activity of DNJ [6]. DNJ content among various mulberry varieties ranged between 30 and 170 mg/100 g of dry leaves. They found that the mulberry shoots contained the highest contents of DNJ (300 mg of DNJ/100 g of dry shoot), followed by young mulberry leaves. Mature leaves of mulberry contained the least contents DNJ [6]. Thus, the shoot is the most suitable part of mulberry tree for the preparation of biological effective tea for the suppression of postprandial blood glucose. A current study has stated the use of a statistical procedure, response surface methodology (RSM), for the optimization of extraction efficiency of DNJ. The optimum extraction conditions at which DNJ yield was maximum (256 mg of DNJ per 100 g of dry mulberry leaves) is given here: ethanol concentration of 55%, extraction temperature of 80 ◦C, extraction time of 1.2 h and ratio of solvent to sample of 12:1. For efficient separation of DNJ from other components in mulberry leaves extracts, a column packed with a selected 732 resin was used. The recovery and purity of DNJ in the end product were >90% and >15%, respectively [62]. Jiang et al. stated that fermentation of mulberry leaf by Ganoderma lucidum produced the highest DNJ content [52]. The optimal condition for mulberry fermentation, obtained from RSM, was the following: pH 6.97, potassium nitrate content 0.81%, and inoculums volume 2 mL. The recovery of DNJ in the end product was 2.74 fold of those in mulberry leaf [52]. Another study described the use of Ultrasound-assisted extraction technique for mulberry DNJ extraction. The extraction efficiency and productivity of DNJ in the end product were 98% and 20%, respectively [63].

2.3. Quantitation of DNJ It is difficult to quantify DNJ using ultraviolet or fluorescence detector because 1-deoxynojirimycin is a polar compound lacking a chromophore. Moreover, it is not retained or quantified by generally used reverse-phase chromatography columns. Instead, DNJ is partially retained or quantified using ligand-exchange and aminopropyl columns [10]. However, many other methods have been developed for quantifying DNJ. For example, hydrophilic interaction liquid chromatographic (HILIC) method using an evaporative light-scattering detector (ELSD) has been developed for determining water-soluble compounds such as DNJ [11]. The HILIC column has shown considerable retention potential of hydrophilic compounds; rather the compounds containing amino groups, the iminosugars such as DNJ, have exhibited good retention in HILIC-ELSD system [64]. Another study has reported the use of 9-fluorenylmethoxycarbonyl chloride for determining DNJ using reverse-phase high-performance liquid chromatography (HPLC) coupled with a fluorescence detector. This method works on the basis of secondary amino groups in DNJ [65]. Besides, many other methods, including HPLC-MS/MS [66], HILIC-MS/MS [67], and high-performance anion-exchange chromatography using pulsed amperometric detector (HPAEC-PAD) [12], have been developed for determining DNJ concentration. Using HPLC-MS/MS, Nuengchamnong et al. reported the isolation of DNJ from the mulberry leaf extract on a TSK gel Amide-80 column using a mobile phase mixture of 0.1% formic acid and acetonitrile. The limits of detection and quantitation were 100 pg and 75 pg, respectively [66]. Since DNJ molecule does not contain chromophore, the quantification of DNJ through spectral analysis needs derivatization, i.e., an analytical approach for quantification of nitrogen-containing compounds [13–16]. To quantify and resolve structural information of DNJ by GC-MS, Bajpai and Rao used trimethylsilyl (TMS) derivatization [7]. The disadvantage of this derivatization is the requirement of water removal from samples for silylation [7,65,67]. Another study has reported the derivatization of mulberry sample by using 9-fluorenylmethyl chloroformate, which reacts with primary and secondary amines under mild conditions producing DNJ derivatives. However, the reaction of 9-fluorenylmethyl chloroformate with tertiary amines requires dealkylation [68,69]. Mulberry-based food products are difficult to be labeled with DNJ contents since the quantification of DNJ through spectral analysis needs derivatization; in order to tackle this problem, HPAEC-PAD method was found useful. The HPAEC-PAD method was coupled with CarboPac MA1 column Molecules 2016, 21, 1600 5 of 15 and sodium hydroxide gradient. As compared to high-performance liquid chromatography (HPLC), HPAEC-PAD was found to be more selective, simple, rapid, and sensitive method of DNJ analysis in mulberry-based food products in terms of good resolution (no interference of DNJ with other contents), simple sample preparation (water extract of mulberry tea sample), and time-consumption (retention time as low as 7.26 min), and sensitivity (limit of detection as low as 5 ng). This method was also found equally effective for mulberry-based products sterilized by heat treatment [7]. Moreover, most of the reported reversed-phase high performance liquid chromatography (HPLC) methods for DNJ determination based on pre-column derivatization involved the use of fluorescence detector, C 18 column, acetonitrile: 0.1% acetic acid (50:50, v/v) as mobile phase with a flow rate of 1.0 mL·min−1, and excitation and emission wavelengths 254 nm and 322 nm, respectively [7,53,70].

2.4. Chemistry Polyhydroxylated piperidines and their derivatives are famous for their excellent bioactivities [1]. The iminosugars contain an analog of pyranose ring or D-glucose, in which the ring oxygen atom is replaced by a nitrogen atom. 1-Deoxyiminosugars are chemically more stable than normal iminosugars because of absence of a hydroxyl group at the C1 position. Among the iminosugars, naturally occurring 1-deoxyiminosugars such as DNJ are strong glycosidase inhibitors [2]. Originally, reduction of nojirimycin led to the chemical synthesis of DNJ [3]; afterward, DNJ was found from natural source, i.e., mulberry tree root [4]. These iminosugars, also known as azasugars, have attracted great attention of chemists and pharmacologists. Chemists have synthesized various DNJ derivatives, for instance N-hydroxyethyl-DNJ 7 (Miglitol) and N-butyl-DNJ 8 (Zavesca). Both are FDA approved drugs for non-insulin-dependent diabetes [5].

2.5. Oral Pharmacokinetic of 1-Deoxynojirimycin The pharmacokinetic of DNJ after oral administration has been studies by some researchers [17–19]. It has been reported that there is a proportional increase in plasma DNJ level with increase in mulberry derived DNJ dose (1.1, 11, and 110 mg/kg of body weight), a dose-dependent phenomenon. An improved bioavailability was observed by pure DNJ versus the mulberry leaf extract administered to rats. It has been narrated that the plasma levels of mulberry derived DNJ after single oral administration (110 mg/kg of body weight) rapidly inclined reaching to a maximum level of 15 µg/mL, followed by quick decline in its level due to its rapid excretion from the body with a Tmax (time to reach maximum plasma drug concentration) value of 30 min [17,18]. On the other hand, the Tmax values of acarbose and miglitol were 1.27 h and 2.5 h, respectively [19,71]. It indicates that absorption and excretion of DNJ is faster than both acarbose and miglitol, which elaborates prolonged therapeutic effect of both acarbose and miglitol as compared to that of pure DNJ. The difference in absorption of acarbose, miglitol, and DNJ may be owing to their slight structural differences. The ethanol hydroxyl group of miglitol may modify the lipo-hydro partition coefficient. It leads to its reduced affinity for glucosyltransferase and glucose transporter, resulting in the slower absorption rate of miglitol as compared to that of DNJ. This information paved the path to development of a hypothesis, i.e., the formulation adjuvant may slow down the absorption rate of DNJ resulting in the improved postprandial hypoglycemic activity in vivo. In 2012, Wang et al. studied the influence of carboxymethylcellulose sodium (CMCNa) as an adjuvant on the absorption rate of DNJ. The results revealed that the absorption rate of DNJ decreased when DNJ and CMCNa were concomitantly ingested to rats through oral route. This change in pharmacokinetics of DNJ leads to an improved antihyperglycemic effect. Conclusively, CMCNa was found to be involved in modifying pharmacokinetics and pharmacodynamics of DNJ in rats [72]. In another pharmacokinetic study of DNJ, Xu et al. (2012) calculated the values of various pharmacokinetic parameters including area under plasma drug concentration curve (AUC), maximum plasma drug concentration (Cmax), Tmax, and Ka and found values were as here: 19.22 ± 1.37 mg·h/L, 12.98 ± 1.92 mg/L, 0.50 ± 0.10 h, and 4.85 ± 0.95 h−1, respectively. Moreover, DNJ is not metabolized in the rat plasma after oral Molecules 2016, 21, 1600 6 of 15 administration [17]. Faber et al. have studied the distribution and elimination of DNJ in the rat after intravenous administration. Plasma protein binding of DNJ was very low. DNJ disappeared from plasma in two phases, with an initial and terminal half life of 3 min and 51 min, respectively. Major mode of DNJ elimination is the renal excretion. DNJ does not undergo tubular reabsorption. Bile and feces also contains some percentage of DNJ dose [73].

2.6. Biological Activities 1-Deoxynojirimycin has many biological activities, including antihyperglycemic [20–25], anti-obesity [26–30], and anti-viral [31] have also been reported [31–35]. (Table1)

Table 1. Biological activities of 1-Deoxynojirimycin.

No. Biological Activity Reference 1 Antihyperglycemic [20–25] 2 Anti-viral [26–30] 3 Anti-obesity [31–35]

2.7. Antihyperglycemic Activity Our diet consists of various essential components including the carbohydrates, for example sucrose, maltose, and starch. During the process of digestion, these are complex molecules, which undergo reactions under the effect of various enzymes [74]. For example, the pancreatic α-amylase plays key role in the conversion of starch into its oligosaccharides including maltose, isomaltose, maltotriose, and α-dextrins in duodenum and jejunum. Further, digestion of these hydrolytic products of starch into the monosaccharides is needed for their intestinal absorption [75]. This conversion is principally modulated by α-glucosidase enzymes, present in the brush-border membrane of the small intestine. Two important enzyme of this family are malto-glucoamylase and sucrose-isomaltase. These enzymes modulate the cleavage of α-1,4 linkages in oligosaccharides and α-1,6 linkages in α-dextrins, respectively [76]. D-glucose, the main product of the α-glucosidase-mediated hydrolysis, can be actively delivered across the mucosal membrane by glucosyltransferase and glucose transporter [77], leading to elevated level of blood glucose. Glucose transport across the intestinal membrane can be hindered by using α-glucosidase inhibitors, which can competitively bind to the catalytic site of α-glucosidase [78]. The important examples of α-glucosidase inhibitors are acarbose, miglitol, and DNJ; the former possess potential to bind to α-amylase, while α-glucosidase is a promising target site of the later two compounds [79–81]. The mode of action of DNJ involves the suppression of intestinal α-1,4-glucosidase as well as α-1,6-glucosidase of hepatic glycogen-debranching enzymes leading to the reduced rate of oligosaccharide breakdown [81]. The antihyperglycemic activity of DNJ has widely been studied [57]. DNJ is capable of binding to and inhibiting α-glycosidase and glucoamylase [22–25], leading to decrease in hepatic glucose metabolism and postprandial hyperglycemia [71]. Mechanistically, DNJ induces the inhibition of intestinal glucose absorption by diminishing the expression of proteins engaged in the transepithelial glucose transport (Figure4). Moreover, the down-regulation of intestinal SGLT1, Na+/K+-ATP and GLUT2 mRNA and protein expression is also provoked by DNJ [82]. The anti-hyperglycemic effect of DNJ is evident from another study that DNJ plays a signiﬁcant role in improving the insulin sensitivity through the activation of insulin signaling PI3K/AKT pathway in skeletal muscle of hyperglycemic model mice [53]. Adiponectin and its receptors in differentiated 3T3-L1 adipocytes have been found to be effective in reducing blood glucose levels and improving insulin sensitivity. In this view, there is an enhancement effect of DNJ (0.5 µM) on following parameters: (i) the levels of adiponectin and its receptors (AdipoR1 and AdipoR2) in differentiated 3T3-L1 adipocytes; (ii) phosphorylation of 50 adenosine monophosphate-activated protein kinase (AMPK); (iii) mRNA expression of glucose transporter 4 (GLUT4); and (iv) an excellent enhancement in glucose uptake into the adipocytes [83]. Consequently, Molecules 2016, 21, 1600 6 of 14

Table 1. Biological activities of 1-Deoxynojirimycin.

No. Biological Activity Reference 1 Antihyperglycemic [20–25] 2 Anti-viral [26–30] 3 Anti-obesity [31–35]

2.7. Antihyperglycemic Activity Our diet consists of various essential components including the carbohydrates, for example sucrose, maltose, and starch. During the process of digestion, these are complex molecules, which undergo reactions under the effect of various enzymes [74]. For example, the pancreatic α-amylase plays key role in the conversion of starch into its oligosaccharides including maltose, isomaltose, maltotriose, and α-dextrins in duodenum and jejunum. Further, digestion of these hydrolytic products of starch into the monosaccharides is needed for their intestinal absorption [75]. This conversion is principally modulated by α-glucosidase enzymes, present in the brush-border membrane of the small intestine. Two important enzyme of this family are malto-glucoamylase and sucrose-isomaltase. These enzymes modulate the cleavage of α-1,4 linkages in oligosaccharides and α-1,6 linkages in α-dextrins, respectively [76]. D-glucose, the main product of the α-glucosidase-mediated hydrolysis, can be actively delivered across the mucosal membrane by glucosyltransferase and glucose transporter [77], leading to elevated level of blood glucose. Glucose transport across the intestinal membrane can be hindered by using α-glucosidase inhibitors, which can competitively bind to the catalytic site of α-glucosidase [78]. The important examples of α-glucosidase inhibitors are acarbose, miglitol, and DNJ; the former possess potential to bind to α-amylase, while α-glucosidase is a promising target site of the later two compounds [79–81]. The mode of action of DNJ involves the suppression of intestinal α-1,4-glucosidase as well as α-1,6-glucosidase of hepatic glycogen-debranching enzymes leading to the reduced rate of oligosaccharide breakdown [81]. The antihyperglycemic activity of DNJ has widely been studied [57]. DNJ is capable of binding to and inhibiting α-glycosidase and glucoamylase [22–25], leading to decrease Molecules 2016in ,hepatic21, 1600 glucose metabolism and postprandial hyperglycemia [71]. Mechanistically, DNJ induces 7 of 15 the inhibition of intestinal glucose absorption by diminishing the expression of proteins engaged in the transepithelial glucose transport (Figure 4). Moreover, the down-regulation of intestinal strong anti-hyperglycemicSGLT1, Na+/K+-ATP and effects GLUT2 of DNJmRNA may and beprotein attained expression by large is also quantity provoked ofby GLUT4DNJ [82]. proteinThe in the plasma membraneanti-hyperglycemic due to effect the of enhanced DNJ is evident transcript from another levels study of thethat GLUT4DNJ plays gene a signi andﬁcant the role activation in of improving the insulin sensitivity through the activation of insulin signaling PI3K/AKT pathway in AMPK. Thus, DNJ can be used to prevent or treat hyperglycemia. skeletal muscle of hyperglycemic model mice [53].

FigureFigure 4. Supposed 4. Supposed mode modeof of DNJ action action in the in thedigestive digestive tract. tract.

2.8. Anti-Obesity Activity The lipoprotein lipase-mediated conversion of serum VLDL (Very low density lipoprotein, known as very bad cholesterol) to LDL (low density lipoprotein) at a rate lower than normal leads to hypertriglyceridemia, the elevated VLDL level [84], which is a risk factor for arteriosclerosis. Arteriosclerosis is a side effect of lipid oxidation, while VLDL is more prone to oxidation. The hypertriglyceridemia can be prevented by mediating the continuous conversion of serum VLDL to LDL at normal rate. In an attempt to explore the effect of DNJ on TG level, DNJ-rich mulberry leaf extract (12 mg) was given to nine human subjects three times daily before meals over a period of 12 weeks. On Day 12, an elevated level of LDL but reduced contents of VLDL was observed [32]. It can be recommended that use of DNJ-rich mulberry leaf extract may be valuable by improving plasma lipoprotein profile. Many tissues and organs in human body play a crucial role in maintaining normal metabolic balance. The metabolic imbalance can result in metabolic disorders, such as diabetes and obesity [85]. Adipose tissue is a loose connective tissue made majorly of adipocytes, which are originated from preadipocytes through a process known as adipogenesis. Adipose tissue not only acts a store-house for lipids, but also behaves as a major endocrine organ. This glandular organ produces hormones such as leptin, estrogen, and resistin. In short, adipose tissue is involved in metabolic regulation and prevention of harmful lipid accumulation in body [86]. It has been documented that DNJ prevents diet-provoked obesity via activation of β-oxidation system and augmented adiponectin levels, which inhibited lipid buildup in the liver and suppressed plasma triacylglycerol level [47]. Another study has stated that mulberry leaf ethanol extract (MLEE) treatment exerts anti-obesity through anti-adipogenic action in differentiated adipocytes [87]. From another study based on adipogenesis, it has been noted that 4 µM DNJ significantly suppresses adipogenesis. The possible mode of adipogenesis suppression by DNJ is extracellular regulated protein kinases 1/2/Peroxisome proliferator-activated receptor signaling pathway in the adipocytes [33]. Another study has reported the anti-obesity feature of DNJ. The investigators studied the influence of Bacillus subtilis-based DNJ on hepatic lipid metabolism and mitochondrial status of model mice (C57BL/6 mice) fed a fat-rich diet for twelve weeks. By Week 12, control (group of mice that received neither fat-rich diet nor DNJ) and DNJ (group of mice that received DNJ in addition to fat-rich diet) group mice did not show weight gain, unlike the HF group (group of mice that received fat-rich diet only), which showed significant weight gain. The hepatic C/EBPα and CD36 mRNA of the HF group also was highly expressed as compared to that of the control and DNJ group, which showed, on the other hand, the higher expression of hepatic p-AMPK/AMPK and PGC-1β mRNA [31]. These results give explanation for the proposed use of DNJ as a dietary supplement to avoid obesity and its consequences. Molecules 2016, 21, 1600 8 of 15

2.9. Anti-Viral Activity The antiviral activity of DNJ has also been studied. There are several studies that describe the mode of action of DNJ against HIV [88–92]. One of the studies has reported that DNJ inhibits the spread of human immunodeficiency virus (HIV). They conducted this study virus-associated oligomeric Env [30]. Later on, the activity of silkworm extract and one of its purified constituents, DNJ, was compared against bovine viral diarrhea virus (BVDV), GB virus-B (GBV-B), woodchuck hepatitis virus (WHV), and hepatitis B virus (HBV). Against all these viruses, the effectiveness of the silkworm extract was significantly greater than that of purified DNJ. It can be assumed that five constituent iminosugars present in the silkworm extract contribute to the antiviral effect in a synergistic manner [93]. Additionally, Kang et al. studied the influence of DNJ (in a concentration of 10 mM DNJ) on the replication of Baculoviruses, Bombyx Mori Nucleopolyhedrovirus (BmNPV) and Autographa Californica Multiple Nucleopolyhedrovirus (AcMNPV). The results revealed that there was no effect of DNJ on the replication of Baculoviruses and BmNPV. However, the replication of AcMNPV was suppressed by 67%, this suppresses replication of AcMNPV was attributed to the higher sensitivity of α-glucosidase activity to DNJ [94]. It can be suggested that use of DNJ may be useful against viral infections, however further studies should be conducted to obtain benefit from this valuable phytochemical.

2.10. In Silico Target Fishing After compiling all above data, we used PASS Prediction (Prediction of Activity Spectra for Substances) software to predict various types of biological activity potential of a small organic molecule on the basis of its structure, particularly before their chemical synthesis and biological analysis. The data required for predictingMolecules 2016, 21, 1600 through PASS is “SMILES” or a structural8 of 14 formula of the compound in MOLfile format. Throughpresent in the this silkworm search extract contribute tool, to the antiviral 44 possible effect in a synergistic activities manner [93]. Additionally, of DNJ with a probability of Kang et al. studied the influence of DNJ (in a concentration of 10 mM DNJ) on the replication of Baculoviruses, Bombyx Mori Nucleopolyhedrovirus (BmNPV) and Autographa Californica Multiple Pa > 0.7 (probability to beNucleopolyhedrovirus active) were (AcMNPV). found The results (Table revealed2 ).thatIt there clearly was no effect indicates of DNJ on the that DNJ can be used as replication of Baculoviruses and BmNPV. However, the replication of AcMNPV was suppressed by anti-diabetic drug. 67%, this suppresses replication of AcMNPV was attributed to the higher sensitivity of α-glucosidase activity to DNJ [94]. It can be suggested that use of DNJ may be useful against viral infections, Subsequently, to narrowhowever further down studies the should target be conducted search, to obtain benefit we from used this valuable STITCH phytochemical. for in silico target fishing in order to identify protein2.10. targets In Silico Target of Fishing DNJ as well as to predict the possible interactions of DNJ with other chemicals and proteins.After STITCH,compiling all above a data, database we used PASS of Prediction protein (Prediction interactions, of Activity Spectra for integrates many sources of Substances) software to predict various types of biological activity potential of a small organic molecule information, i.e., text mining,on the basis experimental of its structure, particularly evidences, before their chemical and synthesis other and biological databases analysis. The data including STRING. Targets required for predicting through PASS is “SMILES” or a structural formula of the compound in MOLfile with a confidence score >format. 0.7 wereThrough this opted search tool, for 44 possible construction activities of DNJ with of a probability protein of Pa > interaction 0.7 (probability to network. Predicted DNJ be active) were found (Table 2). It clearly indicates that DNJ can be used as anti-diabetic drug. targets and the probabilisticSubsequently, confidence to narrow scoredown the target are search, presented we used STITCH in for Table in silico target3. The fishing proteinin network of DNJ is order to identify protein targets of DNJ as well as to predict the possible interactions of DNJ with shown Figure5. In this confidenceother chemicals and view, proteins. stronger STITCH, a database interactions of protein interactions, are integrates represented many sources of by thicker lines (since all information, i.e., text mining, experimental evidences, and other databases including STRING. Targets lines are thick, all the interactionswith a confidence arescore > 0.7 strong). were opted for Protein–protein construction of protein interaction interacting network. Predicted couplesDNJ are CALR–GANAB, targets and the probabilistic confidence score are presented in Table 3. The protein network of DNJ MGAM–GAA, MGAM–GLA,is shown Figure and 5. In this GLA–GAA. confidence view, stronger Chemical interactions are represented (DNG)–protein by thicker lines (since associations are found all lines are thick, all the interactions are strong). Protein–protein interacting couples are CALR–GANAB, between following couples:MGAM–GAA, DNJ–CALR, MGAM–GLA, and DNJ–GANAB, GLA–GAA. Chemical (DNG)–protei DNJ–MGAM,n associations are DNJ–GLA, found and DNJ–GAA. between following couples: DNJ–CALR, DNJ–GANAB, DNJ–MGAM, DNJ–GLA, and DNJ–GAA.

Figure 5. The protein network of DNJ. The proteins and their relationships are represented by the Figure 5. The protein networknodes and edges. of DNJ. The proteins and their relationships are represented by the nodes and edges. Molecules 2016, 21, 1600 9 of 15

Table 2. List of activities with a probability at Pa > 0.7 (probability to be active). Molecules 2016, 21, 1600 9 of 14 Molecules 2016, 21, 1600 9 of 14 No. Pa Activity No. Pa Activity Molecules 2016, 21, 1600 9 of 14 Molecules 2016, 21, 1600 Table 2. List of activities with a probability at Pa > 0.7 (probability to be active). 9 of 14 1 0.953 Phosphatidylcholine-sterol O-acyltransferase inhibitor 23 0.829 CDP-glycerol glycerophosphotransferase inhibitor No. Pa TableActivity 2. List of activities with a probabilityNo. at Pa > 0.7 Pa (probability to be active). Activity 2No. Pa0.937 GlucanTable 1,3-Activity 2.β List-glucosidase of activities inhibitor with a probability No. at Pa 24> 0.7 Pa (probability 0.802 to be active). Activity β-glucosidase inhibitor 3No.1 0.953 Pa0.924 Phosphatidylcholine-sterolTableL-iduronidaseActivity 2. ListO-acyltransferase of activities inhibitor withinhibitor a probability No. 23at Pa 25> 0.8290.7 Pa (probability 0.801 CDP-glycerol to be active). glycerophosphotransferase Activity Fucosterol-epoxide inhibitor lyase inhibitor No.2 0.937 Pa Glucan 1,3-β-glucosidaseActivity inhibitor No. 24 0.802 Pa β-glucosidaseActivity inhibitor 4No.12 0.9530.937 Pa0.916 Phosphatidylcholine-sterol Mannosyl-oligosaccharide Glucan 1,3-β-glucosidaseActivity O-acyltransferase 1,2- inhibitorα-mannosidase inhibitor inhibitor No. 2324 26 0.8290.802 Pa 0.792 CDP-glycerol βglycerophosphotransferase-glucosidaseActivity Ceramide inhibitor glucosyltransferase inhibitor inhibitor 213 0.9370.9530.924 Phosphatidylcholine-sterol GlucanL-iduronidase 1,3-β-glucosidase O-acyltransferase inhibitor inhibitor inhibitor 242325 0.8020.8290.801 CDP-glycerol Fucosterol-epoxide βglycerophosphotransferase-glucosidase lyaseinhibitor inhibitor inhibitor 531 0.9240.953 0.905 Phosphatidylcholine-sterolL Glycosylceramidase-iduronidase O-acyltransferase inhibitor inhibitor inhibitor 2523 27 0.8010.829 0.798 CDP-glycerol Fucosterol-epoxide glycerophosphotransferase Glucan lyase inhibitor endo-1,6- inhibitor β-glucosidase inhibitor 324 0.9240.9370.916 Mannosyl-oligosaccharide GlucanL-iduronidase 1,3-β-glucosidase 1,2- inhibitorα-mannosidase inhibitor inhibitor 252426 0.8010.8020.792 Ceramide Fucosterol-epoxideβ-glucosidase glucosyltransferase lyaseinhibitor inhibitor inhibitor 642 0.9160.937 0.903 Mannosyl-oligosaccharideGlucan Oligo-1,6-glucosidase 1,3-β-glucosidase 1,2-α-mannosidase inhibitor inhibitor inhibitor 2624 28 0.7920.802 0.783 Ceramideβ-glucosidase glucosyltransferase Mucinaminylserine inhibitor inhibitor mucinaminidase inhibitor 345 0.9240.916 0.905 Mannosyl-oligosaccharide GlycosylceramidaseL-iduronidase 1,2- inhibitorα-mannosidase inhibitor inhibitor 262527 0.7920.8010.798 Glucan Ceramide Fucosterol-epoxide endo-1,6- glucosyltransferaseβ-glucosidase lyase inhibitor inhibitor inhibitor 73 0.924 0.886 SucroseL-iduronidaseα-glucosidase inhibitor inhibitor25 29 0.801 0.776Fucosterol-epoxide lyase Fructan inhibitorβ-fructosidase inhibitor 546 0.9050.9160.903 Mannosyl-oligosaccharide Oligo-1,6-glucosidase Glycosylceramidase 1,2-α-mannosidase inhibitor inhibitor inhibitor 262728 0.7920.7980.783 Mucinaminylserine Glucan Ceramide endo-1,6- glucosyltransferase βmucinaminidase-glucosidase inhibitorinhibitor inhibitor 864 0.9030.916 0.880 Mannosyl-oligosaccharide Oligo-1,6-glucosidase Mannosidase 1,2-α-mannosidase inhibitor inhibitor inhibitor 2826 30 0.7830.792 0.773 MucinaminylserineCeramide glucosyltransferase mucinaminidase Manganese inhibitor inhibitor peroxidase inhibitor 657 0.9030.9050.886 Sucrose Oligo-1,6-glucosidase Glycosylceramidase α-glucosidase inhibitor inhibitorinhibitor 282729 0.7830.7980.776 Mucinaminylserine Glucan Fructan endo-1,6- β-fructosidase βmucinaminidase-glucosidase inhibitor inhibitor inhibitor 975 0.886 0.905 0.879 SucroseGlycosylceramidase Glucan α-glucosidase 1,3-α-glucosidase inhibitor inhibitor inhibitor 2927 31 0.7760.798 0.784Glucan Fructan endo-1,6- β Alkenylglycerophosphocholine-fructosidaseβ-glucosidase inhibitor inhibitor hydrolase inhibitor 768 0.886 0.9030.880 Sucrose Oligo-1,6-glucosidase Mannosidase α-glucosidase inhibitor inhibitorinhibitor 292830 0.7760.7830.773 MucinaminylserineManganese Fructan β-fructosidase peroxidase mucinaminidase inhibitorinhibitor inhibitor 1086 0.8800.903 0.880 Oligo-1,6-glucosidase Mannosidaseβ-mannosidase inhibitor inhibitor inhibitor 3028 32 0.7730.783 0.788Mucinaminylserine Manganese Testosterone peroxidase mucinaminidase inhibitor 17β-dehydrogenase inhibitor (NADP+) inhibitor 879 0.8800.8860.879 Glucan Sucrose Mannosidase 1,3- α-glucosidaseα-glucosidase inhibitor inhibitor inhibitor 302931 0.7730.7760.784 Alkenylglycerophosphocholine Manganese Fructan β-fructosidase peroxidase hydrolase inhibitorinhibitor inhibitor 1197 0.8790.886 0.869 GlucanSucrose Sugar-phosphatase 1,3- α-glucosidaseα-glucosidase inhibitor inhibitor inhibitor 3129 33 0.7840.776 0.750 AlkenylglycerophosphocholineFructan β-fructosidase hydrolase inhibitorβ-glucosidase inhibitor inhibitor 1098 0.879 0.880 Glucanβ Mannosidase-mannosidase 1,3-α-glucosidase inhibitor inhibitor inhibitor 313032 0.7840.7730.788 Testosterone Alkenylglycerophosphocholine Manganese 17β-dehydrogenase peroxidase hydrolase inhibitor(NADP+) inhibitorinhibitor 12108 0.880 0.856 βMannosidase-mannosidaseα-mannosidase inhibitor inhibitor inhibitor 3230 34 0.7880.773 0.753 TestosteroneManganese 17β-dehydrogenase peroxidase Glucan 1,4-(NADP+)inhibitorα-maltotriohydrolase inhibitor inhibitor 10119 0.8800.8790.869 Glucan Sugar-phosphataseβ-mannosidase 1,3-α-glucosidase inhibitor inhibitor inhibitor 323133 0.7880.7840.750 Testosterone Alkenylglycerophosphocholine 17ββ-glucosidase-dehydrogenase inhibitor hydrolase (NADP+) inhibitorinhibitor 13119 0.8690.879 0.850 Mannosyl-oligosaccharideGlucan Sugar-phosphatase 1,3-α-glucosidase inhibitor inhibitor glucosidase inhibitor 3331 35 0.7500.784 0.755Alkenylglycerophosphocholineβ-glucosidase Ribulose-phosphate inhibitor hydrolase inhibitor 3-epimerase inhibitor 111012 0.8690.8800.856 Sugar-phosphataseαβ-mannosidase inhibitor inhibitor 333234 0.7500.7880.753 Testosterone Glucan 1,4-17ββ-glucosidaseα-dehydrogenase-maltotriohydrolase inhibitor (NADP+) inhibitor inhibitor 141210 0.8560.880 0.853 Nicotinicαβ-mannosidaseα6β3β4α inhibitor5 receptor antagonist 3432 36 0.7530.788 0.741 Testosterone Glucan 1,4-17βα-dehydrogenase-maltotriohydrolase (NADP+)α-glucosidase inhibitor inhibitor inhibitor 121113 0.8560.869 0.850 Mannosyl-oligosaccharide Sugar-phosphataseα-mannosidase glucosidaseinhibitor inhibitor inhibitor 343335 0.7530.7500.755 GlucanRibulose-phosphate 1,4-β-glucosidaseα-maltotriohydrolase 3-epimerase inhibitor inhibitor 151311 0.8500.869 0.842 Mannosyl-oligosaccharideSugar-phosphatase Amylo-α-1,6-glucosidase glucosidase inhibitor inhibitorinhibitor 3533 37 0.7550.750 0.756 Ribulose-phosphateβ-glucosidase 3-epimerase inhibitor Benzoate-CoA inhibitor ligase inhibitor 131214 0.8500.8560.853 Mannosyl-oligosaccharide Nicotinicα-mannosidase α6β3β4α5 receptor glucosidase inhibitor antagonist inhibitor 343536 0.7550.7530.741 GlucanRibulose-phosphate 1,4-α-glucosidaseα-maltotriohydrolase 3-epimerase inhibitor inhibitor 161412 0.8530.856 0.838 UDP- NicotinicN-acetylglucosamineα-mannosidase α6β3β4α5 receptor inhibitor 4-epimeraseantagonist inhibitor 3634 38 0.7410.753 0.730Glucan 1,4-α-glucosidaseα-maltotriohydrolase inhibitor Glucan 1,4- inhibitorα-glucosidase inhibitor 141315 0.853 0.8500.842 Mannosyl-oligosaccharide Nicotinic Amylo- αα6-1,6-glucosidaseβ3β4α5 receptor glucosidase inhibitor antagonist inhibitor 363735 0.7410.7560.755 Ribulose-phosphate Benzoate-CoAα-glucosidase 3-epimeraseligase inhibitor inhibitor inhibitor 1713 0.850 0.833 Mannosyl-oligosaccharideα-L-fucosidase glucosidase inhibitor inhibitor 35 39 0.755 0.727 Ribulose-phosphate 3-epimeraseα,α-trehalose inhibitor phosphorylase inhibitor 151416 0.8420.8530.838 UDP- NicotinicN Amylo--acetylglucosamine αα6-1,6-glucosidaseβ3β4α5 receptor 4-epimerase inhibitor antagonist inhibitor 373638 0.7560.7410.730 Glucan Benzoate-CoAα-glucosidase 1,4-α-glucosidase ligase inhibitor inhibitor inhibitor 1614 0.8380.853 UDP-NicotinicN-acetylglucosamine α6β3β4α5 receptor 4-epimerase antagonist inhibitor 3836 0.7300.741 Glucanα-glucosidase 1,4-α-glucosidase inhibitor inhibitor 1817 0.833 0.828 α- ExoribonucleaseL-fucosidase inhibitor II inhibitor 39 40 0.727 0.721α,α-trehalose phosphorylase Endo-1,3(4)- inhibitorβ-glucanase inhibitor 161517 0.8380.8420.833 UDP-N Amylo--acetylglucosamineα-Lα-fucosidase-1,6-glucosidase 4-epimeraseinhibitor inhibitor inhibitor 393738 0.7270.7560.730 α, Glucanα-trehalose Benzoate-CoA 1,4-α phosphorylase-glucosidase ligase inhibitor inhibitor inhibitor 1915 0.842 0.827 Amylo- Nicotinicα-1,6-glucosidaseα2β2 receptor inhibitor antagonist 37 41 0.756 0.708Benzoate-CoA Nucleoside ligase inhibitor oxidase (H2O2-forming) inhibitor 171618 0.8330.838 0.828 UDP-N-acetylglucosamine Exoribonucleaseα-L-fucosidase 4-epimeraseIIinhibitor inhibitor inhibitor 393840 0.7270.7300.721 α, Glucanα Endo-1,3(4)--trehalose 1,4-α phosphorylase-glucosidaseβ-glucanase inhibitorinhibitor inhibitor 1816 0.8280.838 UDP-N-acetylglucosamine Exoribonuclease 4-epimeraseII inhibitor inhibitor 4038 0.7210.730 Glucan Endo-1,3(4)- 1,4-αβ-glucosidase-glucanase inhibitor inhibitor 2019 0.827 0.829 Glutamate-5-semialdehyde Nicotinic α2β2 receptor antagonist dehydrogenase inhibitor 41 42 0.708 0.717 Nucleoside oxidase (H2O2-forming) Acylcarnitine inhibitor hydrolase inhibitor 181917 0.8280.8270.833 Nicotinic Exoribonucleaseα-L-fucosidase α2β2 receptor IIinhibitor inhibitor antagonist 403941 0.7210.7270.708 Nucleosideα,α Endo-1,3(4)--trehalose oxidase phosphorylaseβ-glucanase (H2O2-forming) inhibitor inhibitor inhibitor 211720 0.833 0.829 0.812 Glutamate-5-semialdehydeα-L-fucosidase Interleukin dehydrogenase inhibitor 4 antagonist inhibitor 39 42 43 0.7270.717 0.704 α,α Acylcarnitine-trehaloseMannotetraose phosphorylase hydrolase 2-α inhibitor-N inhibitor-acetylglucosaminyl transferase inhibitor 191820 0.827 0.8280.829 Glutamate-5-semialdehyde Nicotinic Exoribonuclease α2β2 receptor dehydrogenase II inhibitor antagonist inhibitor 414042 0.7080.7210.717 Nucleoside Endo-1,3(4)-Acylcarnitine oxidaseβ-glucanase hydrolase(H2O2-forming) inhibitor inhibitor 221821 0.8280.812 0.808 Exoribonuclease Interleukinβ-galactosidase 4 antagonist II inhibitor inhibitor 40 43 44 0.7210.704 Mannotetraose 0.723Endo-1,3(4)- 2-α-N-acetylglucosaminylβ-glucanase Membrane inhibitor transferase integrity inhibitor agonist 192021 0.827 0.8290.812 Glutamate-5-semialdehyde Nicotinic Interleukin α2β2 receptor 4 antagonistdehydrogenase antagonist inhibitor 414243 0.7080.7170.704 Mannotetraose Nucleoside Acylcarnitine 2-α-N oxidase-acetylglucosaminyl hydrolase(H2O2-forming) inhibitor transferase inhibitor inhibitor 19 0.827 Nicotinic α2β2 receptor antagonist 41 0.708 Nucleoside oxidase (H2O2-forming) inhibitor 212022 0.8120.8290.808 Glutamate-5-semialdehydeβ Interleukin-galactosidase 4 antagonistdehydrogenase inhibitor inhibitor 424344 0.7170.7040.723 Mannotetraose Acylcarnitine 2- Membraneα-N-acetylglucosaminyl hydrolaseintegrity agonist inhibitor transferase inhibitor 2022 0.8290.808 Glutamate-5-semialdehydeβ-galactosidase dehydrogenase inhibitor inhibitor 4244 0.7170.723 Acylcarnitine Membrane hydrolaseintegrity agonist inhibitor 2221 0.808 0.812 β Interleukin-galactosidase 4 antagonist inhibitor 4443 0.7230.704 Mannotetraose 2- Membraneα-N-acetylglucosaminyl integrity agonist transferase inhibitor 21 0.812 Interleukin 4 antagonist Table 3. Predicted 43 0.704 DNJ targets. Mannotetraose 2-α-N-acetylglucosaminyl transferase inhibitor 22 0.808 β-galactosidase inhibitor 44 0.723 Membrane integrity agonist 22 0.808 β-galactosidase inhibitor Table 3. Predicted DNJ 44 targets. 0.723 Membrane integrity agonist Table 3. Predicted DNJ targets. NodeNode Color Abbreviations Abbreviations Table 3. Predicted Protein DNJTargets Protein targets. Targets UniProt ID ID Score Score Table 3. Predicted DNJ targets. Node Color AbbreviationsGLA Galactosidase, Protein Targets α (429 aa) UniProt P10253 ID Score 0.921 GLA GLAGalactosidase, Galactosidase, α (429 aa)α (429 aa) P10253 P10253 0.921 0.921 Node Color Abbreviations Protein Targets UniProt ID Score Node Color AbbreviationsGLA Glucosidase, α; neutral AB; Cleaves sequentially the 2 Galactosidase,innermostProtein α Targets-1,3-linked α (429 aa) glucose residues from the Glc(2)Man(9)GlcNAc(2) UniProt P10253 ID Score 0.921 GANAB Glucosidase, α; neutral AB; Cleavesα sequentially the 2 innermost α-1,3-linked glucose residuesα from the Glc(2)Man(9)GlcNAc(2) Q14697 0.854 GANAB Glucosidase, ; neutral AB; Cleaves sequentially the 2 innermost -1,3-linked glucose residues from Q14697 0.854 GLA oligosaccharide precursor of immature glycoproteins (966Galactosidase, aa) α (429 aa) P10253 0.921 GANABGlucosidase,oligosaccharide α; neutralprecursor AB; of Cleaves immature sequentially glycoproteins the 2 (966 innermost aa) α-1,3-linked glucose residues from the Glc(2)Man(9)GlcNAc(2) Q14697 0.854 GANABGLA the Glc(2)Man(9)GlcNAc(2) oligosaccharideGalactosidase, precursorα (429 aa) of immature glycoproteins (966 aa) Q14697P10253 0.854 0.921 oligosaccharideGlucosidase,Calreticulin; Molecularα; neutralprecursor AB;calcium of Cleaves immature binding sequentially glycoproteins chaperone the promoting 2 (966 innermost aa) foldin α-1,3-linkedg, oligomeric glucose assembly residues and from quality the Glc(2)Man(9)GlcNAc(2)control in the ER via the GANAB Calreticulin;Glucosidase, Molecularα; neutral AB;calcium Cleaves binding sequentially chaperone the promoting 2 innermost foldin α-1,3-linkedg, oligomeric glucose assembly residues and from quality the Glc(2)Man(9)GlcNAc(2)control in the ER via the Q14697 0.854 CALR calreticulin/calnexinCalreticulin; cycle. This lectin Molecular interacts calcium transiently binding with almost chaperone all of the promoting monoglucosylated folding, oligomericglycoproteins assembly that are and P27797 0.843 GANABCALR Calreticulin;calreticulin/calnexinoligosaccharide Molecular precursor cycle. calcium ofThis immature lectinbinding interacts glycoproteins chaperone transiently promoting (966 wiaa)th foldin almostg, alloligomeric of the monoglucosylated assembly and quality glycoproteins control in that the areER via the Q14697P27797 0.8430.854 oligosaccharide precursor of immature glycoproteins (966 aa) CALR calreticulin/calnexinCalreticulin;synthesized inMolecular the ER.quality cycle. Interacts calcium This control lectinwithbinding in the interacts the chaperoneDNA-binding ER transiently via thepromoting calreticulin/calnexindomain with foldin almostof NR3C1g, alloligomeric ofand the mediatescycle. monoglucosylated assembly This its nuclear lectinand quality interactsexportglycoproteins control (417 transiently aa) in that the areER withvia the P27797 0.843 CALRsynthesizedCalreticulin; inMolecular the ER. Interacts calcium withbinding the chaperoneDNA-binding promoting domain foldin of NR3C1g, oligomeric and mediates assembly its nuclear and quality export control (417 aa) in the ER via the P27797 0.843 CALR calreticulin/calnexinMaltase-glucoamylasealmost cycle. (α-glucosidase); This all of lectin the monoglucosylatedinteracts May serve transiently as an alternate wi glycoproteinsth almost pathway all of for that the starch monoglucosylated are synthesized digestion when in glycoproteins theluminal ER. α Interacts-amylase that are withactivity the is P27797 0.843 synthesized in the ER. Interacts with the DNA-binding domain of NR3C1 and mediates its nuclear export (417 aa) CALR Maltase-glucoamylasecalreticulin/calnexin cycle. (α-glucosidase); This lectin interacts May serve transiently as an alternate with almost pathway all of for the starch monoglucosylated digestion when glycoproteins luminal α-amylase that are activity is P27797 0.843 MGAM reduced because ofDNA-binding immaturity or malnutrition. domain ofNR3C1 May play and a unique mediates role itsin the nuclear digestion export of malted (417aa) dietary oligosaccharides used in O43451 0.833 MGAM Maltase-glucoamylasereducedsynthesized because in the of ER. immaturity Interacts(α-glucosidase); orwith malnutrition. the May DNA-binding serve May as anplay domain alternate a unique of pathway NR3C1 role in and thefor starchdigestionmediates digestion itsof maltednuclear when dietary export luminal (417oligosaccharides α -amylaseaa) activity used isin O43451 0.833 synthesized in the ER. Interacts with the DNA-binding domain of NR3C1 and mediates its nuclear export (417 aa) MGAM reducedMaltase-glucoamylasefood manufacturing because of immaturity (1857 (α-glucosidase); aa) or malnutrition. May serve May as anplay alternate a unique pathway role in thefor starchdigestion digestion of malted when dietary luminal oligosaccharides α-amylase activity used isin O43451 0.833 foodMaltase-glucoamylase manufacturingMaltase-glucoamylase (1857 (α-glucosidase); aa) May serve (α-glucosidase); as an alternate May pathway serve for as starch an alternate digestion pathway when luminal for starch α-amylase digestion activity when is GAA Glucosidase, α; acid; Essential for the degradation of glygogen to glucose in lysosomes (952 aa) P06280 0.833 MGAM reduced because of immaturity or malnutrition. May play a unique role in the digestion of malted dietary oligosaccharides used in O43451 0.833 GAA Glucosidase,food manufacturing α; acid; (1857Essential aa) for the degradation of glygogen to glucose in lysosomes (952 aa) P06280 0.833 MGAM MGAMreduced because ofluminal immaturityα-amylase or malnutrition. activity May is reduced play a unique because role of in immaturitythe digestion orof malnutrition.malted dietary oligosaccharides May play a unique used role in O43451O43451 0.833 0.833 GAA Glucosidase,food manufacturing α; acid; (1857Essential aa) for the degradation of glygogen to glucose in lysosomes (952 aa) P06280 0.833 food manufacturingin (1857 the digestionaa) of malted dietary oligosaccharides used in food manufacturing (1857 aa) GAA Glucosidase, α; acid; Essential for the degradation of glygogen to glucose in lysosomes (952 aa) P06280 0.833 GAA GAAGlucosidase, α; acid; Glucosidase, Essential for αthe; acid; degradation Essential of glygogen for the degradation to glucose in oflysosomes glygogen (952 to aa) glucose in lysosomes (952 aa) P06280 P06280 0.833 0.833

Molecules 2016, 21, 1600 10 of 15

3. Conclusions 1-Deoxynojirimycin is biologically active with promising health effects. Its sources include mulberry leaves, Commelina communis (dayflower), and several bacterial strains such as Bacillus and Streptomyces species. Moreover, DNJ concentration is found to be different in different parts of mulberry tree. DNJ constitutes only 0.11% (w/w) of mulberry leaf. 1-Deoxynojirimycin possesses antihyperglycemic, anti-obesity, and antiviral features. Most importantly, pre-meal intake of DNJ in therapeutic concentration has resulted in the inhibition of postprandial hyperglycemia and hyperinsulinemia. Thus, DNJ seems to be a potential treatment for checking or setting back the inception of diabetes. No study is reported on toxicity of DNJ despite its long term use. Additional safety assessment on the pharmacokinetics, i.e., absorption, distribution, metabolism, and excretion of DNJ is needed prior to be utilized as a useful food. In silico target fishing has depicted 44 potential targets of DNJ in addition to α-glycosidase. However, further in silico and experimental validation is required to verify these findings, which may open new doors for drug development. In this regard, docking studies will first be carried in future to explore the binding mechanism of DNJ with these enzymes to elucidate the possible orientation and strength of binding affinity between DNG and recently identified target proteins. Following docking, MD simulation will be carried out to gain insight into the structural dynamics and stability of DNG in complex with these proteins.

Acknowledgments: This work was supported by the National Natural Science Foundation of China (no. 81530100, 81573832, 81470191, 81473521, 81473552 and 81503382), Postgraduate Project of Beijing University of Chinese Medicine (no. 2015-JYB-XS044 and 2016-JYB-XS007), Innovation Team of Wei Wang in Beijing University of Chinese Medicine and Combined Traditional Chinese and Western Medicine Pharmacology (13 Common Construction) Hold by Wei Wang. Author Contributions: Kuo Gao and Chenglong Zheng collected the references, analyzed data, and generated figures and paper. Tong Wang, Xing Zhai, Huihui Zhao, Juan Wang, Zhiyong Wang, Zijun Jia and Jianxin Chen collected the references. Yingwu Zhou and Wei Wang designed and generated paper. All authors have read and approved the final version of the paper. Conflicts of Interest: The authors declare no conflicts of interest.

References

1. Pearson, M.S.M.; Mathe-Allainmat, M.; Fargeas, V.; Lebreton, J. Recent Advances in the Total Synthesis of Piperidine Azasugars. Annalen Der Chemie Und Pharmacie 2005, 36, 2159–2191. 2. Zechel, D.L.; Withers, S.G. Glycosidase mechanisms: Anatomy of a ﬁnely tuned catalyst. Acc. Chem. Res. 2000, 33, 11–18. [PubMed] 3. Inouye, S.; Tsuruoka, T.; Ito, T.; Niida, T. Structure and synthesis of nojirimycin. Tetrahedron 1968, 24, 2125–2144. [CrossRef] 4. Yagi, M.; Kouno, T.; Aoyagi, Y.; Murai, H. The structure of moranoline, a piperidine alkaloid from Morus species. J. Agric. Chem. Soc. Jpn. 1976, 50, 571–572. 5. Butters, T.D.; Dwek, R.A.; Platt, F.M. Therapeutic applications of imino sugars in lysosomal storage disorders. Curr. Top. Med. Chem. 2003, 3, 561–574. [CrossRef][PubMed] 6. Vichasilp, C.; Nakagawa, K.; Sookwong, P.; Higuchi, O.; Luemunkong, S.; Miyazawa, T. 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 Sci. Technol. 2012, 45, 226–232. [CrossRef] 7. Bajpai, S.; Rao, A.V.B. Quantitative determination of 1-Deoxynojirimycin in different Mulberry Varieties of India. J. Pharm. Phytochem. 2014, 3, 17–22. 8. Onose, S.; Ikeda, R.; Nakagawa, K.; Kimura, T.; Yamagishi, K.; Higuchi, O.; Miyazawa, T. Production of the α-glycosidase inhibitor 1-deoxynojirimycin from Bacillus species. Food Chem. 2013, 138, 516–523. [CrossRef] [PubMed] 9. Ezure, Y.; Maruo, S.; Miyazaki, K.; Kawamata, M. Moranoline (1-deoxynojirimycin) fermentation and its improvement. Agric. Biol. Chem. 1985, 49, 1119–1125. 10. Sharpless, K.E.; Margolis, S.; Thomas, J.B. Determination of vitamins in food-matrix Standard Reference Materials. J. Chromatogr. A 2000, 881, 171–181. [CrossRef] Molecules 2016, 21, 1600 11 of 15

11. Kimura, T.; Nakagawa, K.; Saito, Y.; Yamagishi, K.; Suzuki, M.; Yamaki, K.; Shinmoto, H.; Miyazawa, T. Determination of 1-deoxynojirimycin in mulberry leaves using hydrophilic interaction chromatography with evaporative light scattering detection. J. Agric. Food Chem. 2004, 52, 1415–1418. [CrossRef][PubMed] 12. Yoshihashi, T.; Do, H.T.T.; Tungtrakul, P.; Boonbumrung, S.; Yamaki, K. Simple, Selective, and Rapid Quantification of 1-Deoxynojirimycin in Mulberry Leaf Products by High-Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection. J. Food Sci. 2010, 75, C246–C250. [CrossRef] [PubMed] 13. Einarsson, S.; Josefsson, B.; Lagerkvist, S. Determination of amino acids with 9-fluorenylmethyl chloroformate and reversed-phase high-performance liquid chromatography. J. Chromatogr. A 1983, 282, 609–618. [CrossRef] 14. Lewis, J.; Morley, J.; Venn, R. Analysis of human β-endorphin 28–31 (melanotropin potentiating factor) and analogues by high-performance liquid chromatography of their 9-fluorenylmethoxy-carbonyl derivatives. J. Chromatogr. B Biomed. Sci. Appl. 1993, 615, 37–45. [CrossRef] 15. Stead, D.; Richards, R. Sensitive fluorimetric determination of gentamicin sulfate in biological matrices using solid-phase extraction, pre-column derivatization with 9-fluorenylmethyl chloroformate and reversed-phase high-performance liquid chromatography. J. Chromatogr. B Biomed. Sci. Appl. 1996, 675, 295–302. [CrossRef] 16. Shangguan, D.; Zhao, Y.; Han, H.; Zhao, R.; Liu, G. Derivatization and fluorescence detection of amino acids and peptides with 9-fluorenylmethyl chloroformate on the surface of a solid adsorbent. Anal. Chem. 2001, 73, 2054–2057. [CrossRef][PubMed] 17. Nakagawa, K.; Kubota, H.; Kimura, T.; Yamashita, S.; Tsuzuki, T.; Oikawa, S.; Miyazawa, T. Occurrence of orally administered mulberry 1-deoxynojirimycin in rat plasma. J. Agric. Food Chem. 2007, 55, 8928–8933. [CrossRef][PubMed] 18. Kim, J.Y.; Kwon, H.J.; Jung, J.Y.; Kwon, H.Y.; Baek, J.G.; Kim, Y.-S.; Kwon, O. Comparison of absorption of 1-deoxynojirimycin from mulberry water extract in rats. J. Agric. Food Chem. 2010, 58, 6666–6671. [CrossRef] [PubMed] 19. Ahr, H.; Boberg, M.; Krause, H.; Maul, W.; Müller, F.; Ploschke, H.; Weber, H.; Wünsche, C. Pharmacokinetics of acarbose. Part I: Absorption, concentration in plasma, metabolism and excretion after single administration of [14C] acarbose to rats, dogs and man. Arzneimittel-Forschung 1989, 39, 1254–1260. [PubMed] 20. Asano, N.; Yamashita, T.; Yasuda, K.; Ikeda, K.; Kizu, H.; Kameda, Y.; Kato, A.; Nash, R.J.; Lee, H.S.; Ryu, K.S. Polyhydroxylated alkaloids isolated from mulberry trees (Morus alba L.) and silkworms (Bombyx mori L.). J. Agric. Food Chem. 2001, 49, 4208–4213. [CrossRef][PubMed] 21. Gui, Z.; Zhuang, D.; Chen, J.; Chen, W. Effect of silkworm powder (SP) lowering blood-glucose levels in mice and its mechanism. Acta Sericol. Sin. 2000, 27, 114–118. 22. Bembi, B.; Deegan, P. Gaucher disease: Improving management. Acta Paediatr. 2008, 97, 81–82. [CrossRef] [PubMed] 23. Kuriyama, C.; Kamiyama, O.; Ikeda, K.; Sanae, F.; Kato, A.; Adachi, I.; Imahori, T.; Takahata, H.; Okamoto, T.; Asano, N. In vitro inhibition of glycogen-degrading enzymes and glycosidases by six-membered sugar mimics and their evaluation in cell cultures. Bioorg. Med. Chem. 2008, 16, 7330–7336. [CrossRef][PubMed] 24. Newbrun, E.; Hoover, C.; Walker, G.J. Inhibition by acarbose, nojirimycin and 1-deoxynojirimycin of glucosyltransferase produced by oral streptococci. Arch. Oral Biol. 1983, 28, 531–536. [CrossRef] 25. Yatsunami, K.; Ichida, M.; Onodera, S. The relationship between 1-deoxynojirimycin content and α-glucosidase inhibitory activity in leaves of 276 mulberry cultivars (Morus spp.) in Kyoto, Japan. J. Nat. Med. 2008, 62, 63–66. [CrossRef][PubMed] 26. Chang, J.; Wang, L.; Ma, D.; Qu, X.; Guo, H.; Xu, X.; Mason, P.M.; Bourne, N.; Moriarty, R.; Gu, B. Novel imino sugar derivatives demonstrate potent antiviral activity against flaviviruses. Antimicrob. Agents Chemother. 2009, 53, 1501–1508. [CrossRef][PubMed] 27. Tanaka, Y.; Kato, J.; Kohara, M.; Galinski, M.S. Antiviral effects of glycosylation and glucose trimming inhibitors on human parainfluenza virus type 3. Antivir. Res. 2006, 72, 1–9. [CrossRef][PubMed] 28. Durantel, D.; Branza-Nichita, N.; Carrouée-Durantel, S.; Butters, T.D.; Dwek, R.A.; Zitzmann, N. Study of the mechanism of antiviral action of iminosugar derivatives against bovine viral diarrhea virus. J. Virol. 2001, 75, 8987–8998. [CrossRef][PubMed] 29. Lazar, C.; Durantel, D.; Macovei, A.; Zitzmann, N.; Zoulim, F.; Dwek, R.A.; Branza-Nichita, N. Treatment of hepatitis B virus-infected cells with α-glucosidase inhibitors results in production of virions with altered molecular composition and infectivity. Antivir. Res. 2007, 76, 30–37. [CrossRef][PubMed] Molecules 2016, 21, 1600 12 of 15

30. Papandréou, M.-J.; Barbouche, R.; Guieu, R.; Kieny, M.P.; Fenouillet, E. The α-glucosidase inhibitor 1-deoxynojirimycin blocks human immunodeficiency virus envelope glycoprotein-mediated membrane fusion at the CXCR4 binding step. Mol. Pharmacol. 2002, 61, 186–193. [CrossRef][PubMed] 31. Do, H.J.; Chung, J.H.; Hwang, J.W.; Kim, O.Y.; Lee, J.-Y.; Shin, M.-J. 1-Deoxynojirimycin isolated from Bacillus subtilis improves hepatic lipid metabolism and mitochondrial function in high-fat–fed mice. Food Chem. Toxicol. 2015, 75, 1–7. [CrossRef][PubMed] 32. Kojima, Y.; Kimura, T.; Nakagawa, K.; Asai, A.; Hasumi, K.; Oikawa, S.; Miyazawa, T. Effects of mulberry leaf extract rich in 1-deoxynojirimycin on blood lipid profiles in humans. J. Clin. Biochem. Nutr. 2010, 47, 155–161. [CrossRef][PubMed] 33. Wang, G.-Q.; Zhu, L.; Ma, M.-L.; Chen, X.-C.; Gao, Y.; Yu, T.-Y.; Yang, G.-S.; Pang, W.-J. Mulberry 1-Deoxynojirimycin Inhibits Adipogenesis by Repression of the ERK/PPARγ Signaling Pathway in Porcine Intramuscular Adipocytes. J. Agric. Food Chem. 2015, 63, 6212–6220. [CrossRef][PubMed] 34. Kong, W.-H.; Oh, S.-H.; Ahn, Y.-R.; Kim, K.-W.; Kim, J.-H.; Seo, S.-W. Antiobesity effects and improvement of insulin sensitivity by 1-deoxynojirimycin in animal models. J. Agric. Food Chem. 2008, 56, 2613–2619. [CrossRef][PubMed] 35. Monte, S.V.; Schentag, J.J.; Adelman, M.H.; Paladino, J.A. Glucose supply and insulin demand dynamics of antidiabetic agents. J. Diabetes sci. Technol. 2010, 4, 365–381. [CrossRef][PubMed] 36. Koutsoukas, A.; Simms, B.; Kirchmair, J.; Bond, P.J.; Whitmore, A.V.; Zimmer, S.; Young, M.P.; Jenkins, J.L.; Glick, M.; Glen, R.C. From in silico target prediction to multi-target drug design: Current databases, methods and applications. J. Proteom. 2011, 74, 2554–2574. [CrossRef][PubMed] 37. Jenkins, J.L.; Bender, A.; Davies, J.W. In silico target fishing: Predicting biological targets from chemical structure. Drug Discov. Today Technol. 2007, 3, 413–421. [CrossRef] 38. Chen, F.; Nakashima, N.; Kimura, I.; Kimura, M. [Hypoglycemic activity and mechanisms of extracts from mulberry leaves (Folium mori) and cortex mori radicis in streptozotocin-induced diabetic mice]. Yakugaku Zasshi J. Pharm. Soc. Jpn. 1995, 115, 476–482. 39. Hocking, D. Trees For Drylands; International Science Publisher: New York, NY, USA, 1993. 40. Arabshahi-Delouee, S.; Urooj, A. Antioxidant properties of various solvent extracts of mulberry (Morus indica L.) leaves. Food Chem. 2007, 102, 1233–1240. [CrossRef] 41. Ercisli, S. A short review of the fruit germplasm resources of Turkey. Genet. Resour. Crop Evol. 2004, 51, 419–435. [CrossRef] 42. Halliwell, B. Dietary polyphenols: Good, bad, or indifferent for your health? Cardiovasc. Res. 2007, 73, 341–347. [CrossRef][PubMed] 43. Manach, C.; Mazur, A.; Scalbert, A. Polyphenols and prevention of cardiovascular diseases. Curr. Opin. Lipidol. 2005, 16, 77–84. [CrossRef][PubMed] 44. Ercisli, S.; Orhan, E. Chemical composition of white (Morus alba), red (Morus rubra) and black (Morus nigra) mulberry fruits. Food Chem. 2007, 103, 1380–1384. [CrossRef] 45. Butt, M.S.; Nazir, A.; Sultan, M.T.; Schroën, K. Morus alba L. nature’s functional tonic. Trends Food Sci. Technol. 2008, 19, 505–512. [CrossRef] 46. Kooij, R.; Branderhorst, H.M.; Bonte, S.; Wieclawska, S.; Martin, N.I.; Pieters, R.J. Glycosidase inhibition by novel guanidinium and urea iminosugar derivatives. MedChemComm 2013, 4, 387–393. [CrossRef] 47. Tsuduki, T.; Kikuchi, I.; Kimura, T.; Nakagawa, K.; Miyazawa, T. Intake of mulberry 1-deoxynojirimycin prevents diet-induced obesity through increases in adiponectin in mice. Food Chem. 2013, 139, 16–23. [CrossRef][PubMed] 48. Kimuar, M.; Chen, F.; Nakashima, N. Antihyperglycemic effects of N-containing sugars derived from mulberry leaves is streptozocin-in-duced diabetic mice. Wakan Iyakugaku Zasshi 1995, 12, 214–219. 49. Lee, S.H.; Choi, S.Y.; Kim, H.; Hwang, J.S.; Lee, B.G.; Gao, J.J.; Kim, S.Y. Mulberroside F isolated from the leaves of Morus alba inhibits melanin biosynthesis. Biol. Pharm. Bull. 2002, 25, 1045–1048. [CrossRef] [PubMed] 50. Fang, S.-H.; Hou, Y.-C.; Chao, P.-D.L. Pharmacokinetic and pharmacodynamic interactions of morin and cyclosporin. Toxicol. Appl. Pharmacol. 2005, 205, 65–70. [CrossRef][PubMed] 51. Niidome, T.; Takahashi, K.; Goto, Y.; Goh, S.; Tanaka, N.; Kamei, K.; Ichida, M.; Hara, S.; Akaike, A.; Kihara, T. Mulberry leaf extract prevents amyloid β-peptide fibril formation and neurotoxicity. Neuroreport 2007, 18, 813–816. [CrossRef][PubMed] Molecules 2016, 21, 1600 13 of 15

52. Jiang, Y.-G.; Wang, C.-Y.; Jin, C.; Jia, J.-Q.; Guo, X.; Zhang, G.-Z.; Gui, Z.-Z. Improved 1-Deoxynojirimycin (DNJ) production in mulberry leaves fermented by microorganism. Braz. J. Microbiol. 2014, 45, 721–729. [CrossRef][PubMed] 53. Liu, Q.; Li, X.; Li, C.; Zheng, Y.; Peng, G. 1-Deoxynojirimycin Alleviates Insulin Resistance via Activation of Insulin Signaling PI3K/AKT Pathway in Skeletal Muscle of db/db Mice. Molecules 2015, 20, 21700–21714. [CrossRef][PubMed] 54. Asai, A.; Nakagawa, K.; Higuchi, O.; Kimura, T.; Kojima, Y.; Kariya, J.; Miyazawa, T.; Oikawa, S. Effect of mulberry leaf extract with enriched 1-deoxynojirimycin content on postprandial glycemic control in subjects with impaired glucose metabolism. J. Diabetes Investig. 2011, 2, 318–323. [CrossRef][PubMed] 55. Schmidt, D.; Frommer, W.; Junge, B.; Müller, L.; Wingender, W.; Truscheit, E.; Schäfer, D. α-Glucosidase inhibitors. Naturwissenschaften 1977, 64, 535–536. [CrossRef][PubMed] 56. Kanieda, Y.; Asano, N.; Teranishi, M.; Matsui, K. New cyclitols, degradation of validamycin a by Flavobacterium saccharophilum. J. Antibiot. 1980, 33, 1573–1574. [CrossRef] 57. Kimura, T.; Nakagawa, K.; Kubota, H.; Kojima, Y.; Goto, Y.; Yamagishi, K.; Oita, S.; Oikawa, S.; Miyazawa, T. Food-grade mulberry powder enriched with 1-deoxynojirimycin suppresses the elevation of postprandial blood glucose in humans. J. Agric. Food Chem. 2007, 55, 5869–5874. [CrossRef][PubMed] 58. Song, W.; Wang, H.-J.; Bucheli, P.; Zhang, P.-F.; Wei, D.-Z.; Lu, Y.-H. Phytochemical profiles of different mulberry (Morus sp.) species from China. J. Agric. Food Chem. 2009, 57, 9133–9140. [CrossRef][PubMed] 59. Hu, K.; Li, Y.; Du, Y.; Su, B.; Lu, D. Analysis of 1-deoxynojirimycin component correlation between medicinal parasitic loranthus from loranthaceae and their mulberry host trees. J. Med. Plants Res. 2011, 5, 4326–4331. 60. Hu, X.-Q.; Jiang, L.; Zhang, J.-G.; Deng, W.; Wang, H.-L.; Wei, Z.-J. Quantitative determination of 1-deoxynojirimycin in mulberry leaves from 132 varieties. Ind. Crop. Prod. 2013, 49, 782–784. [CrossRef] 61. Jeong, J.H.; Lee, N.K.; Cho, S.H.; Jeong, Y.-S. Enhancement of 1-deoxynojirimycin content and α-glucosidase inhibitory activity in mulberry leaf using various fermenting microorganisms isolated from Korean traditional fermented food. Biotechnol. Bioprocess Eng. 2014, 19, 1114–1118. [CrossRef] 62. Wang, T.; Li, C.-Q.; Zhang, H.; Li, J.-W. Response surface optimized extraction of 1-deoxynojirimycin from mulberry leaves (Morus alba L.) and preparative separation with resins. Molecules 2014, 19, 7040–7056. [CrossRef][PubMed] 63. Vichasilp, C.; Nakagawa, K.; Sookwong, P.; Suzuki, Y.; Kimura, F.; Higuchi, O.; Miyazawa, T. Optimization of 1-deoxynojirimycin extraction from mulberry leaves by using response surface methodology. Biosci. Biotechnol. Biochem. 2009, 73, 2684–2689. [CrossRef][PubMed] 64. Tolstikov, V.V.; Fiehn, O. Analysis of highly polar compounds of plant origin: Combination of hydrophilic interaction chromatography and electrospray ion trap mass spectrometry. Anal. Biochem. 2002, 301, 298–307. [CrossRef][PubMed] 65. Kim, J.-W.; Kim, S.-U.; Lee, H.S.; Kim, I.; Ahn, M.Y.; Ryu, K.S. 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 2003, 1002, 93–99. [CrossRef] 66. Nuengchamnong, N.; Ingkaninan, K.; Kaewruang, W.; Wongareonwanakij, S.; Hongthongdaeng, B. Quantitative determination of 1-deoxynojirimycin in mulberry leaves using liquid chromatography–tandem mass spectrometry. J. Pharm. Biomed. Anal. 2007, 44, 853–858. [CrossRef][PubMed] 67. Keiner, R.; Dräger, B. Calystegine distribution in potato (Solanum tuberosum) tubers and plants. Plant Sci. 2000, 150, 171–179. [CrossRef] 68. Nakagawa, K.; Ogawa, K.; Higuchi, O.; Kimura, T.; Miyazawa, T.; Hori, M. Determination of iminosugars in mulberry leaves and silkworms using hydrophilic interaction chromatography–tandem mass spectrometry. Anal. Biochem. 2010, 404, 217–222. [CrossRef][PubMed] 69. Herraez-Hernandez, R.; Campins-Falco, P. Derivatization of tertiary amphetamines with 9-fluorenylmethyl chloroformate for liquid chromatography: Determination of N-methylephedrine. Analyst 2000, 125, 1071–1076. [CrossRef][PubMed] 70. Feng, Z.; Zhe, S.; Chu, Q.; Lin, T.; Xin, L.; Feng, X.; Li, Z.; Yang, S.; Xianghui, L.; Jingxian, Z. Therapeutic target database update 2012: A resource for facilitating target-oriented drug discovery. Nucleic Acids Res. 2012, 40, D1128–D1136. Molecules 2016, 21, 1600 14 of 15

71. Wang, L.; Peng, J.; Wang, X.; Zhu, X.; Cheng, B.; Gao, J.; Jiang, M.; Bai, G.; Hou, Y. Carboxymethylcellulose sodium improves the pharmacodynamics of 1-deoxynojirimycin by changing its absorption characteristics and pharmacokinetics in rats. Pharm. Int. J. Pharm. Sci. 2012, 67, 168–173. 72. Nirogi, R.V.; Kandikere, V.N.; Shukla, M.; Mudigonda, K.; Maurya, S.; Boosi, R.; Yerramilli, A. Liquid chromatographic tandem mass spectrometry method for the quantification of miglitol in human plasma. Arzneimittelforschung 2006, 56, 328–336. [CrossRef][PubMed] 73. Faber, E.D.; Oosting, R.; Neefjes, J.J.; Ploegh, H.L.; Meijer, D.K. Distribution and elimination of the glycosidase inhibitors 1-deoxymannojirimycin and N-methyl-1-deoxynojirimycin in the rat in vivo. Pharm. Res. 1992, 9, 1442–1450. [CrossRef][PubMed] 74. Dyer, J.; Merediz, E.F.-C.; Salmon, K.; Proudman, C.; Edwards, G.; Shirazi-Beechey, S. Molecular characterisation of carbohydrate digestion and absorption in equine small intestine. Equine Vet. J. 2002, 34, 349–359. [CrossRef][PubMed] 75. Gray, G.M. Starch digestion and absorption in nonruminants. J. Nutr. 1992, 122, 172. [PubMed] 76. Koh, L.W.; Wong, L.L.; Loo, Y.Y.; Kasapis, S.; Huang, D. Evaluation of different teas against starch digestibility by mammalian glycosidases. J. Agric. Food Chem. 2009, 58, 148–154. [CrossRef][PubMed] 77. Drozdowski, L.; Thomson, A.B. Citation of This Article. World J. Gastroenterol. 2006, 12, 1657–1670. [CrossRef] [PubMed] 78. Bischoff, H. The mechanism of α-glucosidase inhibition in the management of diabetes. Clin. Investig. Med. 1995, 18, 303–311. 79. Krentz, A.J. Comparative safety of newer oral antidiabetic drugs. Expert Opin. Drug Saf. 2006, 5, 827–834. [CrossRef][PubMed] 80. Sels, J.-P.J.; Huijberts, M.S.; Wolffenbuttel, B.H. Miglitol, a new α-glucosidase inhibitor. Expert Opin. Pharmacother. 1999, 1, 149–156. [CrossRef][PubMed] 81. Martin, O. Iminosugars: Current and future therapeutic applications. Ann. Pharm. Francaises 2007, 65, 5–13. [CrossRef] 82. Li, Y.-G.; Ji, D.-F.; Zhong, S.; Lin, T.-B.; Lv, Z.-Q.; Hu, G.-Y.; Wang, X. 1-deoxynojirimycin inhibits glucose absorption and accelerates glucose metabolism in streptozotocin-induced diabetic mice. Sci. Rep. 2013, 3. [CrossRef][PubMed] 83. Lee, S.M.; Shin, M.J.; Hwang, K.Y.; Kwon, O.; Chung, J.H. 1-Deoxynojirimycin isolated from a Bacillus subtilis stimulates adiponectin and GLUT4 expressions in 3T3-L1 adipocytes. J. Microbiol. Biotechnol. 2013, 23, 637–643. [CrossRef][PubMed] 84. Oikawa, T.; Mukoyama, S.; Soda, K. Chemo-enzymatic D-enantiomerization of DL-lactate. Biotechnol. Bioeng. 2001, 73, 80–82. [CrossRef] 85. Farmer, S.; Auwerx, J. Adipose tissue: New therapeutic targets from molecular and genetic studies–IASO Stock Conference 2003 report. Obes. Rev. 2004, 5, 189–196. [CrossRef][PubMed] 86. Kersten, S. Mechanisms of nutritional and hormonal regulation of lipogenesis. EMBO Rep. 2001, 2, 282–286. [CrossRef][PubMed] 87. Yang, S.J.; Park, N.-Y.; Lim, Y. Anti-adipogenic effect of mulberry leaf ethanol extract in 3T3-L1 adipocytes. Nutr. Res. Pract. 2014, 8, 613–617. [CrossRef][PubMed] 88. Dedera, D.; Vander Heyden, N.; Ratner, L. Attenuation of HIV-1 infectivity by an inhibitor of oligosaccharide processing. AIDS Res. Hum. Retrovir. 1990, 6, 785–794. [CrossRef][PubMed] 89. Fenouillet, E.; Gluckman, J.-C. Effect of a glucosidase inhibitor on the bioactivity and immunoreactivity of human immunodeficiency virus type 1 envelope glycoprotein. J. Gen. Virol. 1991, 72, 1919–1926. [CrossRef] [PubMed] 90. Jones, I.M.; Jacob, G.S. Anti-HIV drug mechanism. Nature 1991, 352, 198. [CrossRef][PubMed] 91. Ratner, L. Glucosidase inhibitors for treatment of HIV-1 infection. AIDS Res. Hum. Retrovir. 1992, 8, 165–173. [CrossRef][PubMed] 92. Fischer, P.; Collin, M.; Karlsson, G.; James, W.; Butters, T.; Davis, S.; Gordon, S.; Dwek, R.; Platt, F. The α-glucosidase inhibitor N-butyldeoxynojirimycin inhibits human immunodeficiency virus entry at the level of post-CD4 binding. J. Virol. 1995, 69, 5791–5797. [PubMed] Molecules 2016, 21, 1600 15 of 15

93. Jacob, J.R.; Mansﬁeld, K.; You, J.E.; Tennant, B.C.; Kim, Y.H. Natural iminosugar derivatives of 1-deoxynojirimycin inhibit glycosylation of hepatitis viral envelope proteins. J. Microbiol. 2007, 45, 431–440. [PubMed] 94. Kang, K.-D.; Park, J.-S.; Cho, Y.-S.; Park, Y.-S.; Lee, J.-Y.; Hwang, K.-Y.; Yuk, W.-J.; Kamita, S.G.; Suzuki, K.; Seong, S.-I. Effect of 1-deoxynojirimycin on the replication of baculoviruses, Bombyx mori Nucleopolyhedrovirus and Autographa californica multiple nucleopolyhedrovirus. Int. J. Ind. Entomol. 2011, 23, 123–128. [CrossRef]

© 2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/).