Abstract:
This invention relates to extracts rich in N-acids of imino sugars (as opposed to neutral and basic imino sugars) and/or pipecolic acids obtainable from the plant leaves of mulberry ( Morus ). The extracts have been shown to have enzymatic activities making extracts rich in these compounds, and the compounds isolated from these extracts, to be good candidates for use in the treatment of diseases, particularly, but not exclusively, metabolic disorders, such as, for example, diabetes.

Description:
[0001]    This invention relates to extracts rich in N-acids of imino sugars (as opposed to neutral and basic imino sugars) and/or pipecolic acids obtainable from the plant leaves of mulberry ( Morus ) 
         [0002]    The extracts have been shown to have enzymatic activities making extracts rich in these compounds, and the compounds isolated from these extracts, to be good candidates for use in the treatment of diseases, particularly, but not exclusively, metabolic disorders, such as, for example, diabetes. 
       DEFINITIONS 
       [0003]    Imino sugars are a wide spread group of plant and microbial compounds that have the ability to interact with the human glycosidases, other proteins and sugar receptors. In this specification they are considered to be polyhydroxylated secondary and tertiary amines in which the molecules resemble monosaccharide sugars but where the ring oxygen is replaced by nitrogen. There are 5 ring structures fitting this classification that are most common in nature:
       a. pyrrolidine,   b. piperidine,   c. pyrrolizidine,   d. indolizidine, and   e. nor-tropane.       
 
         [0009]    In nature, the presence of these water soluble secondary plant compounds is largely masked by the presence of other water soluble primary compounds which are present in much larger quantities, such as, for example: common sugars, peptides and amino acids, and further the imino sugar acids (ISA) are masked by the prevailing basic imino sugars such as:
       a. 1 deoxynojirmycin (DNJ),   b. Fagomine,   c. 1,4-dideoxy-1,4-imino-D-arabinitol (DAB), and   d. calystegine B2.       
 
         [0014]    The novel extracts of the invention are rich in the N-acids of the aforementioned imino sugars and include, but are not limited to N-alkyloic acids of the imino sugars such as N-ethanoic, propanoic and butanoic DAB and N-ethanoic, propanoic and butanoic DNJ. They are also rich in pipecolic acids. 
         [0015]    By rich is meant that the N-acids of the imino sugars and/or pipecolic acids are present in the extract in an amount, by weight, of greater than 5%, more preferably greater than 10%, and more preferably still greater than 15% (wt/wt). 
         [0016]    Preferably a fraction containing both the N-acids of imino sugars and pipecolic acids comprises greater than 10%, through 20%, and 30% to 40%, 50%, 60%, 70%, 80%, 90% or more (wt/wt). 
         [0017]    Other components present in an extract will likely include an oligosaccharide component and an amino acid component. 
         [0018]    A typical mass balance for an extract will be:
       a. imino sugar acids 5-25%, typically 15%;   b. Pipecolic acids 5-35%, typically 20%;   c. Oligosaccharides 5-25%, typically 15%; and   d. Amino acids (neutral and acidic) 25-75%, typically 50%.       
 
         [0023]    However, it is possible to reduce the oligosaccharide and amino acid components to increase the relative amounts of the imino sugar acids and pipecolic acids so they comprise the primary components of the extract, and/or isolate individual compounds therefrom. 
       BACKGROUND 
       [0024]    In 1976 Yagi et al, studying the anti-diabetic activity of the mulberry plant isolated DNJ and found it to be a potent inhibitor of the enzyme family of α and β glucosidases. 
         [0025]    Medicinal chemistry was subsequently employed to the DNJ scaffold to produce N-Hydroxyethyl DNJ (Miglitol) which was successfully developed as an anti-diabetic (Glyset®). However, it&#39;s broad glycosidase activity causes side effects. 
         [0026]    The present invention is a development of the extract disclosed in WO2011/032502 which describes an extract obtained from the plant leaves of mulberry which inhibits α glucosidase and which can be used to control blood glucose levels. 
         [0027]    The specification teaches that the extracts are rich in imino sugars, containing up to 40% by weight. The extract additionally contains up to 70% amino acids. 
         [0028]    The described extracts are easily water soluble, pale yellow or nearly white in colour, have a pH of 5.5-6.5 in a 1% water solution, and have maximum absorption peaks at 218 and 263 nm. 
         [0029]    The extracts are taught to be useful in controlling blood glucose levels. 
         [0030]    The Examples show the compositions to comprise imino sugars in an amount of from 5.2%-39% (w/w) of the extract with the primary imino sugar DNJ present in an amount of from 1.4%-18.6% (w/w) of the total extract, but in all cases the DNJ content comprised at least 19% (w/w) of the imino acid content (measured by it&#39;s DNJ, N-methyl DNJ and Fagomine content). 
         [0031]    The extracts were prepared by:
       a. Conducting a water or ethanolic extraction;   b. Conducting a column chromatography with a strong acidic cationic exchange resin and collecting the bound fraction eluted with ammonia;   c. Subjecting the eluent to column chromatography using a macroporous resin, collecting the eluent; and   d. Concentrating and drying the extract.       
 
         [0036]    It was observed that the IC50 (inhibiting α glucosidase) in Example 1 (5.8% DNJ, 21% total imino sugars) was 13.6 μg/ml compared to 70 μg/ml for pure DNJ. 
         [0037]    Other imino sugars identified in the extract included 1,4-dideoxy-1,4-imino-D-arabinitol (DAB), 2-Oα-D-galactopyranosyl-DNJ (GAL-DNJ) and calystegine B2. 
         [0038]    Furthermore, it is noted that DAB is a glycogen phosphorylase inhibitor. 
         [0039]    The resulting fraction typically comprised about from 0.04-1.4% (w/w) of the starting material ( Morus  leaves). 
         [0040]    A problem with these and other mulberry extracts, which contain significant amounts of DNJ (typically 5-10% DNJ by weight), is that DNJ is not a selective inhibitor of any one α-glucosidase and, as such, extracts containing significant amounts of DNJ give rise to side effects by inhibiting multiple glucosidases including digestive disaccharidases. 
         [0041]    A further problem with seeking to treat metabolic diseases with a single drug entity is that these typically operate via a single mechanism and most metabolic disorders are characterised by multiple effects which would benefit from treatment through multiple mechanisms and not just reducing glucose uptake from the digestive tract. 
         [0042]    It is an object of the present invention to develop compositions or isolate compounds which can operate on multiple levels, or offer more specificity, such that they are likely to prove more effective and/or safer in treating diseases, such as metabolic disorders and may be prophylactic or therapeutic. 
       BRIEF SUMMARY OF THE DISCLOSURE 
       [0043]    In accordance with a first aspect of the present inventions there is provided a purified extract, obtainable from mulberry leaf, characterised in that it is
       a. water soluble;   b. inhibits α-glucosidase, and   c. comprises greater than 5% (w/w) N-acids of imino sugars and/or pipecolic acids by weight of the extract.       
 
         [0047]    Preferably the N-acids of imino sugars and/or pipecolic acids comprise at least 10%, through 15%, through 20%, through 30% (w/w) or more of the extract. 
         [0048]    Preferably the N-acids of imino sugars comprise C1-C9 alkyloic acids of one or more of DAB, DNJ, Fagomine or calystegine B2. These include the ethanoic, propanoic and butanoic acids. 
         [0049]    Preferably the extract further inhibits glycogen phosphorylase. This activity may be in the absence of DAB. 
         [0050]    Preferably the extract preferentially and selectively inhibits rice α glucosidase. 
         [0051]    A purified extract may further comprise oligosaccharides and amino acids. 
         [0052]    In a preferred embodiment a purified extract has a mass balance in which:
       a. Iminosugar acids comprise by weight 5-25% of the extract;   b. Pipecolic acids comprise by weight 5-35% of the extract;   c. Oligosaccharides comprise from 5-25% by weight of the extract; and   d. Amino acids comprise from 25-75% by weight of the extract       
 
         [0057]    The amino acids comprise mainly acidic amino acids and the oligosaccharides comprise unusual tri- and larger saccharides with deoxysugars, aryl glycosides and uronic acids. 
         [0058]    Most of the non-acidic imino sugars, such as DNJ and DAB are removed such that the imino sugars comprise less than 19% (w/w) DNJ through less than 15% (w/w) through 10%(w/w), through 5% (w/w), through 3% (w/w), through 1% (w/w) DNJ of the imino acid content. 
         [0059]    The purified extract is a white semi crystalline or amorphous material and will have been purified by at least a factor of 200 compared with the weight of the starting material and often by more than a factor of 1000. 
         [0060]    The purified extract may also act as an inhibitor of one or more of: a naringinase, glucosidase, or mannosidase; a glucuronidase or hexosaminidase; or an α and β galactosidase or I-d-uronidase. 
         [0061]    The extract is obtained from
       a.  Morus alba  L.   b.  Morus alba  var.  multicaulis  L.   c.  Morus nigra  or   d.  Morus australis  Poir.       
 
         [0066]    The purified extract is obtained using a strongly acidic cation exchange resin and may further utilise a strongly basic anion exchange resin and/or a weakly basic anion exchange resin in the purification process 
         [0067]    The selection of an extract in which DNJ is not the primary imino sugar goes against conventional thinking. 
         [0068]    The use of extracts rich in N-acids of imino sugars (as opposed to basic imino sugars) and/or pipecolic acids, obtainable from the plant leaves of mulberry ( Morus ), has not previously been suggested. 
         [0069]    The extracts of the invention are potent mulberry leaf extracts which are rich in acidic imino sugars and pipecolic acids (and contain very little DNJ or DAB). 
         [0070]    In contrast, most commercially available mulberry leaf extracts have an imino sugar content in which DNJ is the most abundant imino sugar, typically accounting for 50% of the mulberry leaf imino sugars (Asano at al. 2001 J. Agric. Food Chem. 49:4208-4213). 
         [0071]    Extracts of the present invention are enriched in imino sugar acids (ISAs) and/or pipecolic acids which may be present with or without other constituents such as oligosaccharides and amino acids. 
         [0072]    Typical structures of the ISAs present include compounds of the general Formula 1 to 5 below: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    where R is a C1-C9 straight chained alkanoic group, such as, ethanoic, propionic or butanoic. 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    where R is a C1-C9 straight chained alkanoic group, such as, ethanoic, propionic or butanoic. 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    where R is a C1-C9 straight chained alkanoic group, such as, ethanoic, propionic or butanoic. 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    where R is a C1-C9 straight chained alkanoic group, such as, ethanoic, propionic or butanoic, and 
         [0000]    
       
                 
         
             
             
         
       
     
         [0000]    where R is a C1-C9 straight chained alkanoic group, such as, ethanoic, propionic or butanoic. 
         [0073]    Extracts which are not selective for the imino sugar DNJ, and are instead selective for, and are substantially enriched for ISA content and/or pipecolic acids, are very different from any reported mulberry leaf extracts. More particularly they differ in the following ways: 
         [0074]    (1) The extracts of the invention are novel with a completely different chemical profile from that of conventional mulberry leaf extracts. One of the most notable and important chemical features of the extracts of the invention are the provision of a variety of N-substituted imino sugars, which are regarded as a source of promising drug candidates from the imino sugars. Successful examples of substituted imino sugars include N-alkylated imino sugars, which are the only two approved imino sugar based drugs, Miglitol (Glyset)—indicated for type II diabetes, which is a N-2-hydroxyethyl alkylated imino sugar and Miglustat (Zavesca), N-butyl-DNJ, for type I Gaucher&#39;s disease and Niemann Pick type C disease, however the present invention teaches of N-alkyloic substitution. 
         [0075]    (2) The extracts of the invention have a different and probably favourable biological activity profile from that of conventional mulberry leaf extracts, for example, more specificity in enzyme inhibition to enhance therapeutic efficacy and reduce side effects. The GI side effects caused by DNJ are due to its non-selective inhibition of glucosidases and can be minimised by the reduction in DNJ content. In addition, the N-alkyl substituted ISAs could alter the hydrophilic/hydrophobic property of the active molecules to improve their bioavailability and membrane permeability or binding affinity. Also, the ISAs enriched composition could increase the versatility of the stereo-structures (for example, the chirality) of the active compounds, allowing for chaperon-mediated activity mechanism to be involved in the treatment of relevant diseases, such as lysosomal storage disorders. 
         [0076]    (3) The extracts of the invention, owing to their unique features mentioned above, could have an expanded therapeutic spectrum ranging from metabolic syndrome, type I and II diabetes to anti-viral, anti-inflammation or, such rare diseases as type I Gaucher&#39;s disease. 
         [0077]    (4) The extracts of the invention should have a better safety profile. Mulberry leaf is an extremely safe medicinal plant material in its own right and is the sole feed for silkworm and has been used in humans in raw form or as a water decoction for thousands of years in traditional medicine. 
         [0078]    (5) The manufacturing process for the extracts of the invention can be easily scaled-up without the use of organic solvents. In conventional phytochemical research, and most current commercial manufacturing process, plant extracts are usually obtained and concentrated by the use of various organic solvents of different polarities, such as ethanol, methanol, ethyl acetate, acetone, dichloromethane, etc. The water-soluble constituents are either left in the plant material or discarded during the purification process. More than often, the process is difficult, if not impossible to repeat in scale-up and solvent residues in the final extract can become an issue. 
         [0079]    Thus, to produce extracts according to the invention rich in N-acids of imino sugars and pipecolic acids involves fractionation steps over and above those taught in WO2011/032502. Preferred processes include the use of various ion exchange techniques which include the use of:
       a. A strongly acidic cation exchange resin such as Dowex 50 or IR120;   b. A strongly basic anion exchange resin such as Dowex 1, Dowex 2, or Amberlite CG400; and   c. A weakly basic anion exchange resin such as Amberlite IR45.       
 
         [0083]    In accordance with a further aspect of the present invention there is provided a pharmaceutical preparation, dietary supplement or food ingredient comprising the extract of the invention. 
         [0084]    In accordance with yet a further aspect of the present invention there is provided the use of a purified extract of the invention for the treatment of metabolic disorders. 
         [0085]    The extract may provide one or more of:
       a. Reduced insulin resistance;   b. Weight loss;   c. Blood lipid management; and   d. Beta cell protection.       
 
         [0090]    In a preferred embodiment the extract is used in the treatment of diabetes. 
         [0091]    In accordance with yet a further aspect of the present invention there is provided a method of producing a purified extract of the invention comprising the steps of:
       a. Obtaining a water soluble extract from mulberry leaves;   b. Selecting an imino rich fraction using a strongly acidic cation exchange resin to bind the desired fraction, washing with water, and then eluting the desired fraction with ammonia; and   c. Selecting for an acidic fraction using one or more of:
           i. A second strongly acidic cation exchange resin;   ii. A strongly basic anion exchange resin; and   iii. A weakly basic ion exchange resin.   
               
 
         [0098]    Preferably in step ci) an un-retained fraction is selected which fraction is further purified using one or more of cii) and ciii). 
         [0099]    In accordance with yet a further aspect of the present invention there is provided a method of screening for compounds for use in the treatment of metabolic disorders comprising isolating compounds from an extract of the invention and screening them for enzymatic activity. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0100]    The invention is further described, by way of example only, with reference to the following Examples and the accompanying drawings relating to Examples 4 and 5, in which: 
           [0101]      FIG. 1  is a GC MS chromatogram of PhynoRadiance crude extract; 
           [0102]      FIG. 2  is GC-MS Chromatogram of IminoNorm crude extract; 
           [0103]      FIG. 3  is a Mass spectra (tms) of iminosugar at 9.5 minutes; 
           [0104]      FIG. 4  is a GC-MS Chromatogram of PhynoRadiance IR120 Retained (iminosugar fraction) 1.38 g; 
           [0105]      FIG. 5  is a GC-MS Chromatogram of IminoNorm IR120 Retained (iminosugar fraction 1) 6.14 g; 
           [0106]      FIG. 6   a  and  b  are the Mass spectra of two iminosugars not yet identified from IminoNorm; 
           [0107]      FIG. 7   a  and  b  are a GC-MS Chromatogram of IminoNorm IR120 Retained (iminosugar fraction 2) 23 g and a Mass spectra for Fagomine; 
           [0108]      FIG. 8   a, b  and  c  are a GC-MS Chromatogram of IminoNorm IR120 Retained (iminosugar fraction 3) 25.5 g and two Mass spectra of hydroxylated pipecolic acids; 
           [0109]      FIG. 9  is a Mass Spectra of N-ethanoic acid of DNJ (tms) from PhynoRadiance 
           [0110]      FIG. 10  is a Mass Spectra of DAB-N-ethanoic acid (tms) from PhynoRadiance 
           [0111]      FIG. 11  is a Mass Spectra of N-ethanoic acid of DNJ (tms) from IminoNorm 
           [0112]      FIG. 12  is a Mass Spectra of DAB-N-ethanoic acid (tms) from IminoNorm; 
           [0113]      FIG. 13  is Mass Spectra of N-propanoic acid of DNJ (tms) from IminoNorm; and 
           [0114]      FIG. 14  is a graph showing the effect of an extract of the invention on blood glucose levels in mice. 
       
    
    
     DETAILED DESCRIPTION 
       [0115]    The Examples given below provide exemplification of how the claimed extracts and isolated compounds of the invention may be produced and used to treat the conditions claimed, and are provided by way of example only. 
         [0116]    The starting point for a consideration of the claimed invention is the extract disclosed in WO2011/032502. 
         [0117]    Contrary to conventional reasoning the Applicant, noting that the a glycosidase activity of the extract was greater than that of pure DNJ, decided to remove the DNJ and look at the activity of different sub-fractions. These experiments identified a fraction rich in the N-acids of imino sugars and pipecolic acids that exhibited good activity and details of the experiments giving rise to this finding are set out below: 
       Example 1 
     Starting Extract 
       [0118]    Dried extract of  Morus alba  leaves Batch ML091210 
       Method 
       [0119]    The sample was dissolved in water and fractionated by a strongly acidic cation exchange (IR120 H +  form resin in column of 2×30 cm) chromatography to give
       i. an un-retained sample displaced by water (expected to contain mainly sugars, flavonoids etc);   ii. a bound fraction displaced by 2M pyridine (usually removes remaining sugars, phenolics and neutral and acidic amino acids); and   iii. a bound fraction displaced by 2M ammonia solution (expected to contain any alkaloids [iminosugars] and basic amino acids).       
 
         [0123]    Some DNJ was detected in the un-retained material (perhaps due to overloading or pH of the sample) and so this was passed through a second IR120 column (same dimensions) to give a new un-retained sample and a second ammonia fraction. The second un-retained sample contained no DNJ. 
         [0124]    The fractions are set out in Table 1 below: 
         [0000]    
       
         
               
               
               
             
           
               
                   
                   
               
               
                   
                 Sample 
                 Weight in g 
               
               
                   
                   
               
             
             
               
                   
                 a) IR120 Un-retained fraction 
                 5.026 
               
               
                   
                 b) Pyridine displaced fraction 
                 1.743 
               
               
                   
                 c) 1 st  Ammonia displaced fraction 
                 1.592 
               
               
                   
                 d) 2 nd  ammonia displaced fraction 
                 0.469 
               
               
                   
                   
               
             
          
         
       
     
         [0125]    The fractions were freeze dried and weighed. Aliquots were analysed by GC-MS and by 500 MHz NMR spectroscopy (in D 2 O). DNJ quantity was determined both by GC-MS and NMR data. 
       GC-MS 
       [0126]    All samples were freeze dried before derivatisation. Trimethylsilyl (TMS) derivatives were prepared using a mixture of hexamethyldisilazane and trimethylchlorosilane in pyridine (Pierce Tri-Sir silylation reagent, HMDS:TMCS:pyridine in a ratio of 2:1:10). Samples were heated at 60° C. for 15 minutes and then left at room temperature for at least 60 min. Insoluble reaction products were sedimented by centrifugation, and the supernatant was transferred to fresh vials using a syringe. 
         [0127]    Analysis was carried out by GC-MS using a Perkin Elmer Autosystem XL gas chromatograph with a high polarity fused-silica column (Varian ‘Factor Four’ VF-5 ms column, 25 m×0.25 mm i.d., 0.25 μm phase thickness). The carrier gas (helium) flow rate was 1 ml min-1. Trimethylsilyl- (TMS) derivatives were separated using a temperature programme that started at 160° C. for 5 min, followed by a linear increase to 300° C. at a rate of 10° C. min-1. The temperature was held at 300° C. for an additional 10 min; the total analysis time was 29 min. Electron impact mass spectrometry of the column eluant was carried out using a Perkin Elmer TurboMass Gold mass spectrometer, with a quadrupole ion filter system, which was run at 250° C. constantly during analysis. The detector mass range was set to 100 to 650 amu. The temperature of the transfer line (GC to MS) was held at 250° C. Samples were injected onto the column via a split vent (split ratio 50:1) through a fused silica narrow bore injection liner packed with deactivated quartz wool; the injection port temperature was maintained at 200° C. The injection volume was 1 μl. System control, data collection and mass spectral analysis was carried out using Perkin Elmer TurboMass software (TurboMass v. 4.4). Quantification of DNJ was carried out by comparing peak areas in the samples (1 mg of each sample in a vial) with a calibration curve using pure DNJ (0.05-0.33 mg per vial). Quantification was confirmed by estimation of DNJ in the samples using NMR data. 
       Glycosidase Assays 
       [0128]    The assays used p-nitrophenyl-substrates and commercial enzymes purchased from Sigma-Aldrich. Enzymes were assayed at 27° C. in 0.1M citric acid/0.2M disodium hydrogen phosphate buffers at the optimum pH for the enzyme. The incubation mixture consisted of 10 μl enzyme solution, 10 μl of 10 mg/ml aqueous solution of extract and 50 μl of the appropriate 5 mM p-nitrophenyl substrate made up in buffer at the optimum pH for the enzyme. The reactions were stopped by addition of 70 μl 0.4M glycine (pH 10.4) during the exponential phase of the reaction, which had been determined at the beginning using uninhibited assays in which water replaced mulberry fractions. Final absorbances were read at 405 nm using a Versamax microplate reader (Molecular Devices). Assays were carried out in triplicate, and the values given are means of the three replicates per assay. Results are expressed as % inhibition. 
       Results 
       [0129]    The GC-MS chromatograms (not shown) of the IR120 fractions were all dominated by DNJ, 
         [0130]    The un-retained material was mainly sugars and contained no DNJ or other imino sugars after the second IR120 column. 
         [0131]    Other imino sugars and imino sugar acids (hydroxylated non-protein imino acids) were also detected by their characteristic mass spectra. 
         [0132]    The DNJ levels in the three retained fractions b), c) and d)—Table 1 was determined and is shown in Table 2: 
       Quantification of DNJ in Samples by GC-MS 
       [0133]      
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 2 
               
               
                   
               
               
                   
                 GC-MS  
                 DNJ  
                 Total wt  
                   
               
               
                   
                 Peak 
                 mg/mg of 
                 of sample 
                 DNJ in IR120 
               
               
                   
                 Area 
                 sample 
                 in mg 
                 fraction (mg) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 b) Pyridine 
                 92 
                 0.068 
                 1743 
                 118.5 
               
               
                 c) Ammonia 1 st   
                 313 
                 0.2 
                 1592 
                 318.4 
               
               
                 IR120 column 
                   
                   
                   
                   
               
               
                 d) Ammonia 2 nd   
                 61 
                 0.0461 
                 469 
                 21.6 
               
               
                 IR120 column 
                   
                   
                   
                   
               
               
                 Total DNJ 
                   
                   
                   
                 458.5 mg in 10 g 
               
               
                   
               
             
          
         
       
     
         [0134]    Assuming the same detector response for fagomine, DAB, pipecolic acids (combined) and the 13.35 minute imino sugar, the amount of these compounds in the 10 g are estimated as: 
         [0000]    
       
         
               
               
               
               
             
           
               
                   
                   
               
             
             
               
                   
                 a. Fagomine 
                 51  
                 mg 
               
               
                   
                 b. DAB 
                 7.5  
                 mg 
               
               
                   
                 c. Pipecolic acids (combined) 
                 167  
                 mg 
               
               
                   
                 d. 13.35 minute iminosugar 
                 7  
                 mg 
               
               
                   
                   
               
             
          
         
       
     
         [0135]    NMR analysis (not shown) showed the material not retained on IR120 to be mainly sugars; this fraction was approximately 50% of the 10 g sample by weight after cation exchange. 
         [0136]    The major nitrogen-containing components with DNJ were determined to be amino acids (precise identification not available but phenylalanine and proline were tentatively identified by GC-MS) 
       Glycosidase Assays 
       [0137]    In house glycosidase assay results are shown in Table 3. 
         [0138]    The inhibition of α- and β-glucosidases for the IR120 retained fractions was expected given the presence of significant amounts of DNJ. 
         [0139]    The inhibition of other glycosidases such as glucuronidase and hexosaminidases could not be attributed to DNJ, DAB or fagomine. It was probable that the major components identified as pipecolic acids were responsible for these other glycosidase inhibitions. 
         [0140]    Elevated serum levels of glucuronidase and hexosaminidase activities are observed in many diseases including diabetes. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 3 
               
               
                   
               
               
                 Glycosidase Results for Fractions 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                   
                   
                   
                   
                 α-D-gal 
               
               
                   
                 α-D-glu 
                 α-D-glu 
                 α-D-glu 
                 β-D-glu 
                 green coffee 
                 β-D-gal 
                 α-L-fuc 
                 α-D-man 
               
               
                 mulberry fraction 
                 Yeast 
                 
                   Bacillus 
                 
                 rice 
                 almond 
                 bean 
                 bovine 
                 bovine 
                 Jack bean 
               
               
                   
               
               
                 IR120 unb 
                 49 
                 68 
                 97 
                 0 
                 −17 
                 32 
                 −15 
                 −10 
               
               
                 ammonia 2 
                 85 
                 97 
                 100 
                 66 
                 60 
                 34 
                 0 
                 −12 
               
               
                 ammonia 1 
                 98 
                 100 
                 100 
                 89 
                 94 
                 67 
                 0 
                 25 
               
               
                 pyr 
                 85 
                 99 
                 100 
                 83 
                 77 
                 37 
                 0 
                 0 
               
               
                 DNJ 
                 weak 
                 potent 
                 potent 
                 moderate 
                 weak 
                 NI 
                 NI 
                 weak 
               
               
                   
               
             
          
           
               
                   
                   
                   
                 N- 
                 N- 
                   
                   
               
               
                   
                   
                   
                 acetyl- 
                 acetyl- 
               
               
                   
                   
                   
                 β- 
                 β- 
               
               
                   
                   
                   
                 D-gluc 
                 D-gluc 
                   
                 β-  
               
               
                   
                 β-D-man 
                 naringinase 
                 (Bovine 
                 (Jack 
                 amyloglucosidase 
                 glucuronidase 
               
               
                 mulberry fraction 
                 
                   Cellulomonas 
                 
                 
                   Penecillium 
                 
                 kidney 
                 bean) 
                 ( A. niger ) 
                 (bovine liver) 
               
               
                   
               
               
                 IR120 unb 
                 −6 
                 −7 
                 14 
                 −13 
                 8 
                 6 
               
               
                 ammonia 2 
                 0 
                 6 
                 22 
                 31 
                 71 
                 24 
               
               
                 ammonia 1 
                 8 
                 32 
                 39 
                 92 
                 96 
                 0 
               
               
                 pyr 
                 0 
                 16 
                 22 
                 84 
                 82 
                 6 
               
               
                 DNJ 
                 NI 
                 NI 
                 NI 
                 NI 
                 NI 
                 NI 
               
               
                   
               
               
                 Results are expressed as % inhibition at top concentration of 10 mg/ml (prior to dilution in the assay). 
               
               
                 NI = no inhibition. 
               
               
                 A negative value suggests promotion or stabilisation of the enzyme. 
               
               
                 The typical DNJ inhibitory activity is shown for reference. 
               
             
          
         
       
     
       DISCUSSION 
       [0141]    The IR120 retained fractions all contain DNJ but the analysis and glycosidase results show a wider range of glycosidase inhibitors to be present. 
         [0142]    The IR120 un-retained material also showed some glycosidase inhibitions that is surprising but this is likely attributed to the sugars which are dominant in this fraction and can interfere with glycosidase assays. 
         [0143]    DNJ, DAB and fagomine are all glucosidase inhibitors and it is interesting that the IR120 retained fractions give good inhibitions of glycosidases not affected by DNJ. 
         [0144]    Some of these enzyme activities such as hexosaminidase and glucuronidase are known to be elevated in serum or urine in various diseases including diabetes. 
         [0145]    In the subsequent Examples these fractions were further fractionated to more conclusively identify the more major compounds responsible for the activities. 
         [0146]    The pipecolic acids compounds are particularly interesting. 
       Example 2 
       [0147]    The Extract of Example 1 was fractionated into four fractions using cation exchange chromatography (IR120 H +  form). 
         [0148]    The un-retained fraction contained sugars with imino-sugars displaced with pyridine and then ammonia. 
         [0149]    Some DNJ was found in each retained fraction but most was in the first ammonia fraction. Other imino sugars were also detected including tentatively fagomine, DAB, some hydroxylated imino acids and other potentially novel imino-sugars. 
         [0150]    Table 4 below shows the enzyme inhibitions noted for the fractions in Example 1. 
         [0000]    
       
         
               
               
               
               
               
             
           
               
                   
               
               
                   
                   
                   
                   
                 Total  
               
               
                   
                   
                   
                 DNJ 
                 weight 
               
               
                   
                 Weight  
                 Glycosidases  
                 mg/mg 
                 of fraction  
               
               
                 Sample 
                 in g 
                 inhibited 
                 of sample 
                 in g 
               
               
                   
               
             
             
               
                 IR120  
                 5.026 
                 Gp 56% 
                 0    
                 5.026  
               
               
                 unretained 
                   
                 α-glucosidases 
                   
                   
               
               
                 fraction 
                   
                 β-glucuronidase (weak) 
                   
                   
               
               
                 Pyridine  
                 1.743 
                 Gp 84% 
                 0.068  
                 1.743 
               
               
                 fraction 
                   
                 α-glucosidases 
                   
                   
               
               
                   
                   
                 β-glucosidase 
                   
                   
               
               
                   
                   
                 α-galactosidase 
                   
                   
               
               
                   
                   
                 β-galactosidase 
                   
                   
               
               
                   
                   
                 Naringinase 
                   
                   
               
               
                   
                   
                 Hexosaminidase 
                   
                   
               
               
                   
                   
                 β-glucuronidase (weak) 
                   
                   
               
               
                 1 st   
                 1.592 
                 Gp 97% 
                 0.0461 
                 1.592  
               
               
                 ammonia  
                   
                 α-glucosidases 
                   
                   
               
               
                 fraction 
                   
                 β-glucosidase 
                   
                   
               
               
                   
                   
                 α-galactosidase 
                   
                   
               
               
                   
                   
                 β-galactosidase 
                   
                   
               
               
                   
                   
                 Naringinase 
                   
                   
               
               
                   
                   
                 Hexosaminidase 
                   
                   
               
               
                   
                   
                 α-mannosidase 
                   
                   
               
               
                 2 nd    
                 0.469 
                 Gp 84% 
                 0.0461 
                 0.469  
               
               
                 ammonia 
                   
                 α-glucosidases 
                   
                   
               
               
                 fraction 
                   
                 β-glucosidase 
                   
                   
               
               
                   
                   
                 α-galactosidase 
                   
                   
               
               
                   
                   
                 β-galactosidase 
                   
                   
               
               
                   
                   
                 Naringinase 
                   
                   
               
               
                   
                   
                 Hexosaminidase 
                   
                   
               
               
                   
                   
                 β-glucuronidase 
                   
                   
               
               
                   
               
             
          
         
       
     
         [0151]    In Example 2 the aim was to:
       a. Further resolve the components in all four fractions;   b. Analyse compounds by GC-MS and 500 MHz NMR to identify as many components as possible; and   c. Conduct glycosidase assays on purified compounds to further distinguish the inhibitions given by the different components.       
 
       Analysis 
       [0155]    This Example primarily involved further fractionation of the 10 g of  Morus alba  leaf extract used in Example 1 
       GC-MS 
       [0156]    This was conducted as per Example 1. 
       NMR 
       [0157]    This was conducted as per Example 1. 
       Glycosidase Assays 
       [0158]    This was conducted as per Example 1. 
       Methods of Purification 
       [0159]    a) Sugar Components (IR120 Un-Retained) 
         [0160]    The IR120 un-retained fraction from Example 1 was shown by GC-MS to contain primarily sugars but gave rather surprising activity in the glycosidase assays (including glycogen phosphorylase). This sample was therefore further fractionated to separate the common sugars from more unusual components (probably giving the activity) and to remove some brown colour (probably phenolics). 
         [0161]    The 5 g was dissolved in a minimum volume of distilled water and applied to a column (2×10 cm) of Amberlite CG400 anion exchange resin in the OH− form. 
         [0162]    Eleven 2 ml water fractions were collected before removing retained material with 1M HOAc. Samples were dried and analysed by GC-MS, NMR and on the basis of the analysis some were provided for glycosidase and glycogen phosphorylase assays. Some brown material was bound permanently to the resin. 
         [0163]    The sugar fractions obtained were colourless or slightly yellow. Some were recombined for further separation later. The fractions are shown in Table 5 below: 
         [0000]    
       
         
               
               
               
             
               
               
               
             
           
               
                   
                   
               
               
                   
                 Fraction from CG400 
                 Weight in mg 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 1-10 
                 765 
               
               
                   
                 11 
                 364 
               
               
                   
                 HOAc 1 
                 100 
               
               
                   
                 HOAc 2 
                 1,2697 
               
               
                   
                   
               
             
          
         
       
     
         [0164]    The total weight of sugars, therefore, appears to be in the region of 3.728 g in the 10 g sample although it is possible that some inorganic material is not being detected by GC or NMR. 
         [0165]    b) Pyridine and Ammonia Fractions 
         [0166]    The IR120 pyridine and ammonia fractions 1 and 2 were further fractionated to reduce the DNJ present with other components to allow their structural elucidation by NMR. 
         [0167]    To achieve this a series of columns were run using:
       a. Anion exchange chromatography (Amberlite CG400 in the OH −  and Ac −  forms) and   b. Cation exchange chromatography using a weakly acidic resin (Amberlite CG50 in the ammonium form).       
 
       Results 
       [0170]    a) Sugar Fractionation 
         [0171]    The CG400 fractionation of sugars (IR120 un-retained material from Example 1) showed sugar alcohols in earlier fractions. Some common monosaccharides (furanose and pyranose) occurred throughout the fractions. Disaccharides increased and then further monosaccharides showed in the main acetic acid fraction (some perhaps due to acid hydrolysis). Interestingly all fractions tested gave both glycosidase and glycogen phosphorylase inhibition. Several unusual components were detected. 
         [0172]    b) Iminosugar Fractionation (from IR120 Pyridine and Ammonia Fractions) 
         [0173]    From the IR120 bound fractions obtained in Example 1 several components were partially purified allowing further characterization. 
         [0174]    A compound with a retention time 10.37 min gave good matches by GC-MS with structures derived from DAB. 
         [0175]    The compounds with retention times of 12.91 and 13.31 minutes gave good matches to piperidine N-acid type compounds which might be derived from DNJ. 
         [0176]    A component with GC retention time of 19.36 minutes was purified—3.3 mg. This component gave mass spectral data consistent with a glycoside although it is unusual to have glycosides bound to IR120 resin. 
         [0177]    Attempts were made to further purify the fagomine and DAB type compounds. Some of these were eventually obtained in a single fraction of 2.0 mg. 
         [0178]    Pipecolic acids were identified in fraction JH0806/101/23. 
         [0179]    All five fractions were submitted for enzyme assays in addition to sugar fractions and trigonelline (observed in NMR spectra Formula 6) and 6-hydroxytrigonelline (Formula 7) (possibly observed in NMR spectra but very minor component). 
         [0000]    
       
                 
         
             
             
         
       
     
       Glycosidase Assays on Compounds and Fractions 
       [0180]    The assay results for the fractions are given in Table 6 
         [0000]    
       
         
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 6 
               
               
                   
               
             
             
               
                   
                   
                 α-D- 
                 α-D- 
                 α-D- 
                 β-D- 
                 α-D- 
                 β-D- 
                 α-L- 
               
               
                   
                   
                 glucosidase 
                 glucosidase 
                 glucosidase 
                 glucosidase 
                 galactosidase 
                 galactosidase 
                 fucosidase 
               
               
                 compound 
                 PQ code 
                 Yeast 
                 
                   Bacillus 
                 
                 rice 
                 almond 
                 coffee bean 
                 bovine 
                 bovine 
               
               
                   
               
               
                 19.4 min disacch. 
                 JH806/78/45 
                 8 
                 53 
                 90 
                 97 
                 9 
                 50 
                 −6 
               
               
                 comp 
               
               
                   Calliandra  pipecolic 
                 JH806/87/4 
                 0 
                 34 
                 54 
                 0 
                 −7 
                 0 
                 0 
               
               
                 acid 
               
               
                 DAB N-acid tentative 
                 JH806/93/4 
                 23 
                 58 
                 97 
                 8 
                 0 
                 0 
                 0 
               
               
                 pipecalic acids 
                 JH806/101/23 
                 0 
                 33 
                 59 
                 0 
                 −6 
                 0 
                 0 
               
               
                 DAB and fagomine 
                 JH806/106/8 
                 98 
                 96 
                 56 
                 25 
                 −11 
                 21 
                 ND 
               
               
                   
               
             
          
           
               
                   
                   
                 β-D- 
                   
                   
                   
                   
               
               
                   
                 α-D- 
                 mannosidase 
                   
                 N-acetyl-β-D- 
                 β-N-acetyl-β-D- 
                 β- 
               
               
                   
                 mannosidase 
                 
                   Cellulomonas 
                 
                 naringinase 
                 gluc 
                 gluc 
                 glucuronidase 
               
               
                 compound 
                 Jack bean 
                 
                   fimi 
                 
                 
                   P. decumbens 
                 
                 Bovine kidney 
                 
                   A. oryzae 
                 
                 bovine liver 
               
               
                   
               
               
                 19.4 min disacch. comp 
                 −18 
                 −12 
                 0 
                 15 
                 0 
                 0 
               
               
                   Calliandra  pipecolic acid 
                 0 
                 −6 
                 0 
                 0 
                 0 
                 −6 
               
               
                 DAB N-acid tentative 
                 −6 
                 0 
                 0 
                 8 
                 0 
                 0 
               
               
                 pipecalic acids 
                 0 
                 0 
                 0 
                 0 
                 −6 
                 −6 
               
               
                 DAB and fagomine 
                 0 
                 15 
                 0 
                 −7 
                 −6 
                 0 
               
               
                   
               
             
          
           
               
                   
                   
                   
                   
                 α-D- 
                 β-D- 
                 α-D- 
                 β-D- 
                 α-D- 
               
               
                   
                   
                 α-D-glucosidase 
                 α-D-glucosidase 
                 glucosidase 
                 glucosidase 
                 galactosidase 
                 galactosidase 
                 mannosidase 
               
               
                 type 
                 number 
                 Yeast 
                 
                   Bacillus 
                 
                 rice 
                 almond 
                 coffee beans 
                 bovine 
                 jack bean 
               
               
                   
               
               
                 compound 
                 900439 
                 0 
                 0 
                 0 
                 0 
                 −6 
                 0 
                 0 
               
               
                 commercial 
                 trigonellin 
                 0 
                 −12 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                   
                 HCl 
               
               
                 6OH trig 
                 Jh808/6/13 
                 0 
                 −36 
                 0 
                 0 
                 −16 
                 8 
                 0 
               
               
                 sugar 
                 JH808/4/35 
                 0 
                 −9 
                 0 
                 0 
                 0 
                 0 
                 5.7 
               
               
                 RN sugars 
                  5* 
                 7 
                 41 
                 92 
                 0 
                 −9 
                 0 
                 ND 
               
               
                 RN sugars 
                 10* 
                 75 
                 81 
                 97 
                 6 
                 0 
                 6 
                 ND 
               
               
                 RN sugars 
                 11* 
                 25 
                 26 
                 82 
                 0 
                 −8 
                 0 
                 ND 
               
               
                 RN sugars 
                 HOAc* 
                 0 
                 −14 
                 57 
                 0 
                 −15 
                 16 
                 ND 
               
               
                   
               
             
          
           
               
                   
                   
                   
                 β-D- 
                   
                 N-acetyl-β-D- 
                 N-acetyl-β-D- 
                   
                   
               
               
                   
                   
                   
                 mannosidase 
                 naringinase 
                 gluc 
                 gluc 
                 amyloglucosidase 
                 β-glucuronidase 
               
               
                   
                 type 
                 number 
                 
                   C. fimi 
                 
                 
                   Penecillium 
                 
                 Bovine kidney 
                 Jack bean 
                 
                   A. niger 
                 
                 bovine liver 
               
               
                   
                   
               
               
                   
                 compound 
                 900439 
                 0 
                 −6 
                 9 
                 8 
                 0 
                 0 
               
               
                   
                 commercial 
                 trigonellin HCl 
                 0 
                 0 
                 −8 
                 0 
                 0 
                 0 
               
               
                   
                 6OH trig 
                 Jh808/6/13 
                 0 
                 0 
                 12 
                 −11 
                 0 
                 0 
               
               
                   
                 sugar 
                 JH808/4/35 
                 0 
                 0 
                 14 
                 11 
                 8 
                 0 
               
               
                   
                 RN sugars 
                  5* 
                 ND 
                 −7 
                 0 
                 0 
                 0 
                 −7 
               
               
                   
                 RN sugars 
                 10* 
                 ND 
                 0 
                 9 
                 7 
                 24 
                 0 
               
               
                   
                 RN sugars 
                 11* 
                 ND 
                 0 
                 0 
                 0 
                 6 
                 0 
               
               
                   
                 RN sugars 
                 HOAc* 
                 ND 
                 −6 
                 0 
                 0 
                 0 
                 0 
               
               
                   
                   
               
               
                   
                 *run at 2 mg/ml. 
               
               
                   
                 ND = not determined 
               
             
          
         
       
     
       Glycogen Phosphorylase Assay Results 
       [0181]    These assay results are given in Table 7 below: 
         [0000]    
       
         
               
               
               
             
               
               
               
               
             
           
               
                   
               
               
                   
                 Compound 
                 GP Inhibition (%) 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 Trigonelline 
                 6.9%  
                 at 400 μM 
               
               
                   
                 JH0808/6/13 6-hydroxytrigonelline 
                 11.9%  
                 at 400 μM 
               
               
                   
                 JH0808/4/35 disaccharide 
                 1.7%  
                 at 400 μg/mL 
               
               
                   
               
               
                   
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 0%  
                 at 400 μM 
               
               
                   
               
               
                   
                 Sugars 5 
                 63.5%  
                 at 1 mg/m 
               
               
                   
                 Sugars 11 
                 55.3%  
                 at 1 mg/ml 
               
               
                   
                 Sugars HOAc 
                 48.9%  
                 at 1 mg/ml 
               
               
                   
               
             
          
         
       
     
       Discussion and Conclusion 
       [0182]    It has been possible to identify imino-sugars including some novel natural compounds tentatively identified as N-acids of DAB and DNJ. 
         [0183]    The sugar fraction of the sample also shows both glycosidase and glycogen phosphorylase inhibition. 
         [0184]    Sugars such as glucose and sucrose do not inhibit glycogen phosphorylase or glycosidases. 
         [0185]    Some unusual components were identified as present in the sugar fractions by GC-MS and it is presumed that these are responsible for the inhibition of the glycosidases and glycogen phosphorylase. The sugar fractions are a high proportion of the unfractionated extract and so may contribute significantly to the activity of the sample. 
         [0186]    Although DAB is reported to give glycogen phosphorylase inhibition, the unfractionated extract clearly has several imino-sugar components which give this inhibition. 
         [0187]    The pipecolic acids and trigonelline identified in Extracts were not greatly inhibitory to the glycogen phosphorylase. DNJ and fagomine do not inhibit this enzyme. It may well be that the N-acids of DAB and DNJ are the active components 
         [0188]    After removing the DNJ, DAB and fagomine, many components in the extract also inhibited the glucosidases. 
       Example 3 
       [0189]    The analysis conducted on the fractions described in Example 2 provided further information on the imino-sugars and sugars of the sample. 
         [0190]    The sugar fraction comprised almost half of the sample and showed potent activity against various glycosidases and glycogen phosphorylase. 
         [0191]    Sugars such as sucrose, trehalose and glucose do not inhibit these enzymes significantly. 
         [0192]    In this Example an attempt was made to characterise the active components of the sugar fraction and look further at its activity. 
         [0193]    In this regard it was fractionated further using a series of ion exchange columns. 
       Method 
       [0194]    The IR120 un-retained fraction from Example 1 was submitted to chromatography on an anion exchange resin CG400 (OH −  form) to yield water fractions (1-11) and two acetic acid fractions (Table 5—Example 2). These were still further fractionated in this Example and the activity of the fractions was further investigated. 
       Analysis 
     GC-MS 
       [0195]    This was as per Example 1. 
       NMR 
       [0196]    This was as per Example 1. 
       Glycosidase Assays 
       [0197]    This was as per Example 1. 
       Methods of Purification 
       [0198]    The further fractionation of the sugar samples involved use of a strongly basic anion exchange resin CG400 used in the OH− form and also in the acetate form. 
         [0199]    Fractions were collected in water and freeze dried. Retained material was removed using 1M acetic acid and these fractions were evaporated nearly to dryness by rotary evaporation and then freeze dried. 
       Results 
       [0200]    To further purify the sugar samples and to remove any possible inorganic material all samples were re-chromatographed through CG400 OH− form. Sugar fractions 1-11 were combined into sample 1-11 and the two acetic acid samples were also recombined. 
         [0201]    Both samples were loaded on a CG400 OH− 2×20 cm column and water fractions collected before a final wash with 1M HOAc to remove retained material. 
         [0202]    The fraction weights are shown in Table 8 below. 
         [0000]    
       
         
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                   
               
               
                   
                 Weights of 
                   
                   
                   
                   
               
               
                 Fraction 
                 fractions  
                   
                 Weights of 
                   
                   
               
               
                 number 
                 from 
                   
                 fractions  
                   
                   
               
               
                 CG400 
                 combined 
                   
                 from 
                   
                   
               
               
                 OH- 
                 sample 1-11 
                   
                 acid fraction 
                   
                 Notes 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 1 
                 0 
                   
                   
                 0.7  
                 mg 
                 Yellowish 
                   
               
               
                 2 
                 55.3  
                 mg 
                 White 
                 1.8  
                 mg 
                 White 
                   
               
             
          
           
               
                   
                   
                 semi-crys 
                   
                 amorphous 
                   
               
             
          
           
               
                 3 
                 82.3  
                 mg 
                 White 
                 0.9  
                 mg 
                 White 
                 3-7 
               
             
          
           
               
                   
                   
                 semi-crys 
                   
                 amorphous 
                 combined  
               
               
                   
                   
                 A 
                   
                   
                 as 
               
               
                   
                   
                   
                   
                   
                 E 2.1 mg 
               
             
          
           
               
                 4 
                 86  
                 mg 
                 White 
                 0.9  
                 mg 
                 White 
                   
               
             
          
           
               
                   
                   
                 semi-crys 
                   
                 amorphous 
                   
               
             
          
           
               
                 5 
                 63.8  
                 mg 
                 White 
                 l  
                 mg 
                 White 
                   
               
             
          
           
               
                   
                   
                 semi-crys 
                   
                 amorphous 
                   
               
             
          
           
               
                 6 
                 53.5  
                 mg 
                 White 
                 0.8  
                 mg 
                 White 
                   
               
             
          
           
               
                   
                   
                 semi-crys 
                   
                 amorphous 
                   
               
             
          
           
               
                 7 
                 81.1  
                 mg 
                 White 
                 0.9  
                 mg 
                 White 
                   
               
             
          
           
               
                   
                   
                 semi-crys 
                   
                 amorphous 
                   
               
               
                   
                   
                 B 
                   
                   
                   
               
             
          
           
               
                 8 
                 53.3  
                 mg 
                 White 
                 0.9  
                 mg 
                 White 
                 8-14 
               
             
          
           
               
                   
                   
                 semi-crys 
                   
                 amorphous 
                 combined  
               
               
                   
                   
                   
                   
                   
                 as 
               
               
                   
                   
                   
                   
                   
                 F 11.7 mg 
               
             
          
           
               
                 9 
                 34  
                 mg 
                 White 
                 1.1  
                 mg 
                 White 
                   
               
             
          
           
               
                   
                   
                 semi-crys 
                   
                 amorphous 
                   
               
             
          
           
               
                 10 
                 7.1  
                 mg 
                 Clear 
                 2.1  
                 mg 
                 White 
                   
               
             
          
           
               
                   
                   
                 syrupy 
                   
                 amorphous 
                   
               
             
          
           
               
                 11 
                 1.4  
                 mg 
                 Clear 
                 3.8  
                 mg 
                 White 
                   
               
             
          
           
               
                   
                   
                 syrup 
                   
                 amorphous 
                   
               
             
          
           
               
                 12 
                 1  
                 mg 
                 Clear 
                 3  
                 mg 
                 White 
                   
               
             
          
           
               
                   
                   
                 syrup 
                   
                 amorphous 
                   
               
             
          
           
               
                 13 
                 0.1  
                 mg 
                 Clear 
                 2.8  
                 mg 
                 White 
                   
               
             
          
           
               
                   
                   
                 syrup 
                   
                 amorphous 
                   
               
             
          
           
               
                 14 
                 1.4  
                 mg 
                 Clear 
                 1  
                 mg 
                 White 
                   
               
             
          
           
               
                   
                   
                 syrup 
                   
                 amorphous 
                   
               
             
          
           
               
                 15 
                 1.2  
                 mg 
                 Clear 
                 — 
                 — 
                   
               
             
          
           
               
                   
                   
                 syrup 
                   
                   
                   
               
             
          
           
               
                 16 
                 1.2  
                 mg 
                 Clear 
                 — 
                 — 
                   
               
             
          
           
               
                   
                   
                 syrup 
                   
                   
                   
               
             
          
           
               
                 17 
                 0.8  
                 mg 
                 Clear 
                 — 
                 — 
                   
               
             
          
           
               
                   
                   
                 syrup 
                   
                   
                   
               
             
          
           
               
                 HOAc 
                 545  
                 mg C 
                 clear light 
                 989 
                 mg D 
                 clear light 
                 The  
               
             
          
           
               
                 wash 
                 HS119 
                 yellow 
                 HS120 
                 yellow  
                 retained 
               
               
                   
                   
                 syrup 
                   
                 syrup 
                 material  
               
               
                   
                   
                   
                   
                   
                 was 
               
               
                   
                   
                   
                   
                   
                 further 
               
               
                   
                   
                   
                   
                   
                 fraction- 
               
               
                   
                   
                   
                   
                   
                 ated 
               
               
                   
                   
                   
                   
                   
                 on CG400 
               
               
                   
                   
                   
                   
                   
                 acetate  
               
               
                   
                   
                   
                   
                   
                 form 
               
               
                   
                   
                   
                   
                   
                 resin 
               
               
                   
               
             
          
         
       
     
         [0203]    Surprisingly the sample 1-11 that was un-retained on the first CG400 OH-column now gave a considerable weight that was retained. This suggests a pH effect or possible overloading of the first column. 
         [0204]    The un-retained fractions of sample 1-11 were white semi-crystalline material as before. 
         [0205]    The acid sample gave most weight in the retained fraction as expected but small amounts of material were un-retained and sample 3-7 had most glycosidase inhibition activity. 
         [0206]    The early 1-11 fractions had white solid semi-crystalline material that gave no GC peaks suggesting sugars that were larger than disaccharides. 
         [0207]    Later fractions showed some sugar alcohols but low amounts. 
         [0208]    Samples coded A-F in Table 8 were submitted for NMR analysis. 
         [0209]    A, B, E and F were also submitted for glycosidase assays the results of which are tabulated in Table 9 below. 
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 9 
               
               
                   
               
               
                 Activity (% inhibition) of Sugar Fractions on Glycosidases 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                   
                   
                   
                   
                   
                 α-D- 
                   
               
               
                   
                   
                   
                   
                   
                 galactosidase 
               
               
                   
                 α-D- 
                 α-D- 
                 α-D- 
                 β-D- 
                 (green 
                 β-D- 
               
               
                   
                 glucosidase 
                 glucosidase 
                 glucosidase 
                 glucosidase 
                 coffe 
                 galactosidase 
               
               
                 sample 
                 (Yeast) 
                 ( Bacillus ) 
                 (rice) 
                 (almond) 
                 bean) 
                 (bovine) 
               
               
                   
               
               
                 orig frac 5* 
                 7 
                 41 
                 92 
                 0 
                 −9 
                 0 
               
               
                 orig 10* 
                 75 
                 81 
                 97 
                 6 
                 0 
                 6 
               
               
                 orig 11* 
                 25 
                 26 
                 82 
                 0 
                 −8 
                 0 
               
               
                 orig HOAc* 
                 0 
                 −14 
                 57 
                 0 
                 −15 
                 16 
               
               
                 1-11 (frac3) 
                 37 
                 85 
                 99 
                 0 
                 10 
                 12 
               
               
                 1-11 (frac7) 
                 81 
                 89 
                 99 
                 9 
                 12 
                 11 
               
               
                 1-11(frac14- 
                 69 
                 68 
                 87 
               
               
                 17) 
               
               
                 1-11 (frac10) 
                 96 
                 97 
                 100 
               
               
                 acids 3-7 E 
                 89 
                 81 
                 98 
               
               
                 acids 8-14 F 
                 7 
                 −15 
                 7 
                 0 
               
               
                 HS804/119/1 
                 0 
                 −21 
                 26 
                 −18 
               
               
                 HS804/120/1 
                 −10 
                 0 
                 26 
                 −11 
               
               
                 HS804/120/2 
                 −13 
                 0 
                 81 
                 0 
                 0 
                 10 
               
               
                 HS804/120/3 
                 26 
                 30 
                 54 
                 13 
                 −12 
                 66 
               
               
                 HS804/120/5 
                 −21 
                 −61 
                 35 
                 13 
               
               
                 HS804/122/5 
                 0 
                 6 
                 30 
                 −8 
                   
                   
               
               
                 HS804/122/8 
                 0 
                 −8 
                 92 
                 0 
                 −9 
                 29 
               
               
                 HS804/122/10 
                 0 
                 0 
                 90 
                 0 
               
               
                 HS804/122/18 
                 0 
                 −11 
                 98 
                 19 
                 0 
                 62 
               
               
                 sucrose 
                 0 
                 −7 
                 0 
                 0 
                   
                 0 
               
               
                 10 mg/ml 
               
               
                   
               
             
          
           
               
                   
                   
                   
                 N- 
                 N- 
                   
                   
               
               
                   
                   
                   
                 acetyl- 
                 acetyl- 
               
               
                   
                 α-D- 
                   
                 β-D- 
                 β-D- 
                   
                 β- 
               
               
                   
                 mannosidase 
                   
                 gluc 
                 gluc 
                   
                 glucuronidase 
               
               
                   
                 (Jack 
                 naringinase 
                 (Bovine 
                 (Jack 
                 amyloglucosidase 
                 (bovine 
               
               
                 sample 
                 bean) 
                 ( P. decumbens ) 
                 kidney 
                 bean) 
                 ( A. niger ) 
                 liver) 
               
               
                   
               
               
                 orig frac 5* 
                   
                 −7 
                 0 
                 0 
                 0 
                 −7 
               
               
                 orig 10* 
                   
                 0 
                 9 
                 7 
                 24 
                 0 
               
               
                 orig 11* 
                   
                 0 
                 0 
                 0 
                 6 
                 0 
               
               
                 orig HOAc* 
                   
                 −6 
                 0 
                 0 
                 0 
                 0 
               
               
                 1-11 (frac3) 
                 0 
                   
                   
                 0 
               
               
                 1-11 (frac7) 
                 0 
                   
                   
                 11 
               
               
                 1-11 (frac14- 
               
               
                 17) 
               
               
                 1-11 (frac10) 
               
               
                 acids 3-7 E 
               
               
                 acids 8-14 F 
               
               
                 HS804/119/1 
               
               
                 HS804/120/1 
               
               
                 HS804/120/2 
                 0 
                   
                   
                 −12 
               
               
                 HS804/120/3 
                 0 
                   
                   
                 −16 
               
               
                 HS804/120/5 
               
               
                 HS804/122/5 
                   
                   
                   
                 0 
               
               
                 HS804/122/8 
                 0 
                   
                   
                 0 
               
               
                 HS804/122/10 
               
               
                 HS804/122/18 
                 8 
                   
                   
                 −37 
               
               
                 sucrose 
                   
                   
                 0 
                 0 
               
               
                 10 mg/ml 
               
               
                   
               
               
                 Substrates are 10 mM p-nitrophenyl-glycopyranosides. 
               
               
                 Enzymes are purchased from Sigma and run at the pH optima 
               
             
          
         
       
     
         [0210]    The CG400 OH− retained material samples (C and D) were further fractionated using CG400 in the acetate form. 
         [0211]    Sample C (HS0804/119) gave a good separation as did sample D (HS0804/120) 
         [0212]    The compounds in C resembled mono and disaccharide sugars but the common sugars would not be retained on CG400. 
         [0213]    Sample D showed some similar compounds but also some larger molecules around 24-25 minute retention times. 
         [0214]    The amount and activity of fractions C and D are set out in the tables below: 
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 10 
               
             
             
               
                   
               
               
                 Weights of fractions from acid sample C  
               
               
                 fractionated on CG400 acetate form 
               
             
          
           
               
                   
                 Fraction 
                 Weight mg 
                 Notes 
               
               
                   
                   
               
             
          
           
               
                   
                 HS0804/119/1 
                 437 
                 Assays 
               
               
                   
                 119/2 
                 31 
                   
               
               
                   
                 119/3 
                 4.4 
                   
               
               
                   
                 119/4 
                 2 
                   
               
               
                   
                 119/5 
                 9.9 
               
               
                   
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 11 
               
             
             
               
                   
               
               
                 Weights of fractions from acid sample 
               
               
                 D fractionated on CG400 acetate form 
               
             
          
           
               
                   
                 Fraction 
                 Weight mg 
                 Notes 
               
               
                   
                   
               
             
          
           
               
                   
                 HS0804/120/1 
                 125 
                 Assays 
               
               
                   
                 120/2 
                 643 
                 Assays 
               
               
                   
                 120/3 
                 11.4 
                 Assays 
               
               
                   
                 120/4 
                 6.5 
                   
               
               
                   
                 120/5 
                 76.2 
                 Assays 
               
               
                   
                   
               
             
          
         
       
     
         [0215]    The samples C and D fractionated well on the acetate columns. 
         [0216]    Sample HS120/2 was further fractionated on a 2×10 cm column of CG400 acetate form resin using 5 ml water fractions followed by 1M HOAc to give samples HS0804/122 
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 12 
               
             
             
               
                   
               
               
                 Weights of Fractions from HS0804/120/2 
               
             
          
           
               
                   
                 Fraction 
                 Weight mg 
                 notes 
               
               
                   
                   
               
             
          
           
               
                   
                 HS0804/122/1-3  
                 0 
                   
               
               
                   
                 122/4 
                 204 
                   
               
               
                   
                 122/5 
                 189 
                 assays 
               
               
                   
                 122/6 
                 141 
                   
               
               
                   
                 122/7 
                 55 
                   
               
               
                   
                 122/8 
                 22 
                 assays 
               
               
                   
                 122/9 
                 15 
                   
               
               
                   
                 122/10 
                 15 
                 assays 
               
               
                   
                 122/11 
                 8 
                   
               
               
                   
                 122/12 
                 3.4 
                   
               
               
                   
                 122/12-17  
                 19 
                   
               
               
                   
                 122/18 HOAc 
                 11.9 
                 assays 
               
               
                   
                   
               
             
          
         
       
     
         [0217]    GC-MS analysis of these samples revealed a good separation and several components of interest. 
         [0218]    Fractions 5, 8, 10 and 18 were submitted for assays. 
         [0219]    These samples showed an interesting selectivity for the rice glucosidase which was also evident in the first HOAc samples produced in Example 2 
         [0220]    The fractions from samples 1-11 tended to give good inhibition of not only the rice glucosidase but also the yeast and bacillus enzyme. 
         [0221]    GC-MS analysis of the HS0804/122 fractions showed several potentially novel components. One component in the 122/18 gave a mass spectrum fairly typical of an imino-sugar and some other compounds had some similarities in fragmentation to imino-sugars. 
       DISCUSSION AND CONCLUSIONS 
       [0222]    From the 10 g PhynoRadiance sample was obtained 5.026 g of a sugar fraction. After anion exchange chromatography this reduced to 3.728 g by possible removal of inorganic or aromatic material. This fraction comprising 37% of the PhynoRadiance sample had significant glycogen phosphorylase inhibition activity and also potently inhibited three alpha-glucosidases. This Example has confirmed the activity of the sugars of PhynoRadiance and determined that there are some larger sugars with activity and some possible sugar acids (imino-sugar acids) giving inhibition more selectively to a rice alpha-glucosidase. 
         [0223]    Selectivity of activity amongst glucosidases has been shown to be important for treating some disorders because there are many glucosidases active in mammalian systems and blanket inhibition of all glucosidases can cause some off-target effects. It should also be noted that inhibitors of glucosidases can also have beneficial effects on cells producing deficient glycoproteins by chaperoning activity; such effects could be surmised to be related to anti-aging properties (Best et al., 2010) 
         [0224]    The glycosidase assays of the original sugar fractions from Example 2 were extended and show that the PhynoRadiance sugar fraction shows particularly good inhibition of a range of alpha-glucosidases but an extensive set of other glycosidases has not been run. This inhibition profile is somewhat similar to that of DNJ except that DNJ does not also inhibit glycogen phosphorylase. The sugar fractionation and assays tends to show that the retained components inhibit particularly the rice glucosidase whereas the un-retained components inhibit all three alpha-glucosidases tested. Glycogen phosphorylase inhibition was found in both CG400 OH− retained and un-retained fractions. 
         [0225]    GC-MS analysis of the CG400 OH− unretained fractions from sample 1-11 was not very informative but NMR revealed quite pure looking sugars presumed to be larger than disaccharides. The CG400 retained components gave good GC-MS data and appear quite complex; some disaccharides are present (not sucrose) but also unusual sugars or imino-sugars (presumably with carboxyl-groups or aromatic units to have retained on CG400 initially). It was concluded that the sugar fraction contains some novel sugars with potent glycosidase and glycogen phosphorylase activity. The likelihood is that the larger sugars seen in the un-retained CG400 OH− fractions are novel and have imino-sugars conjugated to them giving activity. The retained compounds on CG400 OH− may be smaller units that are being released from the larger sugars or are novel sugar acids. 
         [0226]    The NMR data collected for sample A (a highly active un-retained CG4000H-fraction from sample 1-11) showed good purity but very little structural information is obtained on such larger sugars. 
       Example 4 
       [0227]    Example 4 sought to confirm the activity in non-iminosugar fractions of the extracts. 
       Summary 
       [0228]    This Example confirmed that N-acids of DNJ and DAB present in a mulberry extract give more selective inhibition of alpha-glucosidases than DAB and DNJ and these compounds also give good inhibition of mammalian β-glucuronidase, hexosaminidase and α-L-iduronidase. 
         [0229]    Serum β-glucuronidase and hexosaminidases are elevated in several diseases including diabetes. 
         [0230]    Elevation of β-glucuronidase could result in poor excretion of toxins as glucuronides and hence inhibition of this enzyme could be beneficial to health 
         [0231]    It is reported that imino sugars can act as both inhibitors and chaperoning molecules for glycosidases and hence may also increase the activity of glycosidases they inhibit at sub-inhibitory concentrations. 
         [0232]    The N-acids of DNJ and DAB are novel isolated natural products. 
         [0233]    An unusual trisaccharide, with a deoxy-sugar and an aryl-glycoside has also been tentatively identified. 
       Material Studied 
       [0234]    IminoNorm 200 ml crude extract; and 
         [0235]    PhynoRadiance 50 g batch as set out in Table 13 
         [0000]    
       
         
               
               
               
               
             
               
               
               
               
             
           
               
                   
                 TABLE 13 
               
               
                   
                   
               
               
                   
                   
                 IR120 retained  
                 IR120 unretained 
               
               
                   
                 Sample 
                 Weight in g 
                 weight in g 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 IminoNorm 
                 54.64  
                 28 
               
               
                   
                   
                 (too viscous to dry fully) 
                   
               
               
                   
                 PhynoRadiance 
                 1.38 
                 32 
               
               
                   
                   
               
             
          
         
       
     
       Analysis 
     GC-MS 
       [0236]    This was as per Example 1. 
       NMR 
       [0237]    This was as per Example 1. 
       Glycosidase Assays 
       [0238]    This was as per Example 1. 
       Methods of Purification 
       [0239]    The further fractionation of the sugar samples involved use of the strongly basic anion exchange resin CG400 in the OH− form and the acetate form. 
         [0240]    Fractions were collected in water and freeze dried as described in Example 3 
         [0241]    Retained material was removed using 1M acetic acid and these fractions were evaporated nearly to dryness by rotary evaporation and then freeze dried. 
       Results 
       [0242]    Table 14 below provides α-Glycosidase results (% inhibition at 2 mg/ml) for IminoNorm and PhynoRadiance Anion Exchange Resin (CG400) un-retained and retained Fractions 
         [0000]    
       
         
               
               
               
               
               
             
               
               
               
               
               
             
           
               
                   
                   
               
               
                   
                   
                 (Yeast) 
                 ( Bacillus ) 
                 (rice) 
               
               
                   
                   
               
             
             
               
                   
               
             
          
           
               
                   
                 IminoNorm unretained 
                 85 
                 99 
                 100 
               
               
                   
                 PhynoRadiance unretained 
                 33 
                 88 
                 99 
               
               
                   
                 PhynoRadiance retained 
                 0 
                 12 
                 96 
               
               
                   
                 IminoNorm retained HOAc 
                 6 
                 24 
                 97 
               
               
                   
                   
               
             
          
         
       
     
         [0243]    The samples IminoNorm and PhynoRadiance (diluted extracts as described in WO2011/032502) were additionally subjected to: 
         [0244]    1) Cation exchange chromatography; and 
         [0245]    2) Anion exchange chromatography, as described in the previous examples. 
         [0246]    The anion exchange retained material gave a more specific inhibition of rice α-glucosidase. 
         [0247]    The anion exchange resin un-retained material, of both samples, gave broader α-glucosidase inhibition. 
         [0248]    The N-acids of the imino sugars DAB and DNJ appear to be responsible for the specific rice α-glucosidase inhibition (See glycosidase Table 16). 
         [0249]    There was proportionally more weight of material retained by the anion exchange resin (Table 15) from IminoNorm (71%) probably due to its greater dilution with sugars. 
         [0250]    Sugars added to PhynoRadiance may have, therefore, contributed to the weight of anion exchange un-retained fractions of that sample. 
         [0251]    Both samples had inhibitory activity, however, and so the added sugars of PhynoRadiance do not necessarily have the activity which seems to come from mulberry itself. 
         [0000]    
       
         
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 15 
               
               
                   
               
               
                   
                 % retained on  
                 % not  
                   
               
               
                   
                 anion exchange  
                 retained on 
                 % weight not 
               
               
                   
                 resin but 
                 anion  
                 retrieved from 
               
               
                   
                 displaced  
                 exchange 
                 anion exchange 
               
               
                 Sample 
                 by HOAc 
                 resin 
                 resin 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 IminoNorm  
                 71 
                 29 
                 38 
               
               
                 (diluted to 2% DNJ) 
                   
                   
                   
               
               
                 PhynoRadiance 
                 56 
                 44 
                 20.5 
               
               
                 (diluted to 5% DNJ) 
                   
                   
                   
               
               
                   
               
             
          
         
       
     
         [0252]    DNJ was the major component of both pre-fractionated extracts. 
         [0253]    GC-MS Chromatograms (and mass spectrum) are provided in  FIGS. 1-13   
         [0254]    Referring to these Figs the following is noted: 
         [0255]      FIGS. 1 and 2  are GC-MS Chromatograms of PhynoRadiance and IminoNorm Crude Extracts. It will be noted that in the IminoNorm Chromatogram some imino sugars appear in relatively higher concentrations. One is the compound having a mass spectrum as shown in  FIG. 3  (9.5 minute). 
         [0256]      FIGS. 4 and 5  are GC-MS Chromatogram of PhynoRadiance IR120 Retained (iminosugar fraction) 1.38 g and IminoNorm IR120 Retained (iminosugar fraction 1) 6.14 g respectively. 
         [0257]    The iminoNorm sample overloaded the cation exchange resin (IR120) two times due to the high imino sugar content. 
         [0258]    Bound fractions (2 and 3) are shown in  FIGS. 7 and 8  with some preferential displacement of imino sugars evident. Basic imino sugars tend to displace more neutral or acidic imino sugars. 
         [0259]    Two related imino sugars only just visible in PhynoRadiance are quite major in the IminoNorm IR120 bound fraction 1 and this could be used to purify and identify those iminosugars (8.72 and 9.49 minutes). 
         [0260]    The selective displacement also allowed the further characterisation of three N-acids of DAB and DNJ which are new natural products. These N-acids also occur in PhynoRadiance. 
         [0261]      FIG. 6   a  and  b  are the Mass spectra of two iminosugars not yet identified from IminoNorm. 
         [0262]      FIG. 7   a  is a GC-MS Chromatogram of IminoNorm IR120 Retained (iminosugar fraction 2) 23 g and  FIG. 7   b  is a Mass spec for Fagomine. 
         [0263]    DNJ is by far the major imino sugar in the 2 nd  IminoNorm retained fraction on IR120. The iminosugar at 7.4 minutes is fagomine. 
         [0264]      FIG. 8   a , is a GC-MS Chromatogram of IminoNorm IR120 Retained (iminosugar fraction 3) 25.5 g.  FIGS. 8B and 8C  are two Mass spec of hydroxlylated pipecolic acids. 
         [0265]    Hydroxylated pipecolic acids are more major than DNJ in the 3 rd  IR120 retained fraction of IminoNorm. Their mass spectra and one identified in Example 2 are shown below. The major component at 3.59 minutes could be phenylalanine. 
         [0266]      FIG. 9  is a Mass Spectra of N-ethanoic acid of DNJ (tms) in PhynoRadiance. 
         [0267]      FIG. 10  is a Mass Spectra of DAB-N-ethanoic acid (tms) from PhynoRadiance. 
         [0268]      FIG. 11  is a Mass Spectra of N-ethanoic acid of DNJ (tms) in IminoNorm. 
         [0269]      FIG. 12  is a Mass Spectra of DAB-N-ethanoic acid (tms) from IminoNorm; and 
         [0270]      FIG. 13  is Mass Spectra of N-propanoic acid of DNJ (tms) in IminoNorm. 
         [0271]    The formula for these acids are illustrated in Formula 8 to 10 below: 
         [0000]    
       
                 
         
             
             
         
       
     
         [0272]    The N-acids of DAB and DNJ are visible in the PhynoRadiance extract without concentration but are less obvious in IminoNorm. They are, however, present in both samples. 
         [0273]    The activity of these novel isolated acids is shown in Table 14 below. 
       Glycosidase Results for Iminosugar N-Acids (% Inhibition at 0.8 mM) 
       [0274]      
         [0000]    
       
         
               
               
               
               
               
               
             
               
               
               
               
               
               
             
           
               
                   
               
               
                   
                   
                   
                 DAB-N- 
                 DNJ-N- 
                 DNJ-N- 
               
               
                 Assay 
                 DNJ 
                 DAB 
                 ethanoic 
                 propanoic 
                 ethanoic 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 α-D-glucosidase  
                 36 
                 100 
                 10.7 
                 0.0 
                 2.5 
               
               
                 (Yeast) 
                   
                   
                   
                   
                   
               
               
                 α-D-glucosidase 
                 100 
                 93 
                 24.2 
                 45.0 
                 9.1 
               
               
                 ( Bacillus ) 
                   
                   
                   
                   
                   
               
               
                 α-D-glucosidase 
                 100 
                 100 
                 42 
                 100 
                 100 
               
               
                 (Rice) 
                   
                   
                   
                   
                   
               
               
                 β-D-glucosidase 
                 64 
                 100 
                 7 
                 32 
                 14 
               
               
                 (yeast) 
                   
                   
                   
                   
                   
               
               
                 α-D-galactosidase 
                 −3 
                 7 
                 −5 
                 15 
                 −8 
               
               
                 (Green coffee beans) 
                   
                   
                   
                   
                   
               
               
                 β-D-galactosidase 
                 0 
                 100 
                 0 
                 12 
                 2 
               
               
                 (Bovine) 
                   
                   
                   
                   
                   
               
               
                 α-L-fucosidase 
                 6 
                 5 
                 3 
                 11 
                 5 
               
               
                 (Bovine) 
                   
                   
                   
                   
                   
               
               
                 α-D-mannosidase 
                 27 
                 3 
                 28 
                 40 
                 38 
               
               
                 (Jack bean) 
                   
                   
                   
                   
                   
               
               
                 β-D-mannosidase 
                 −19 
                 95 
                 −5 
                 −5 
                 −10 
               
               
                 ( C.   fimi ) 
                   
                   
                   
                   
                   
               
               
                 Naringinase 
                 21 
                 3 
                 0 
                 4 
                 0 
               
               
                 ( P.   decumbens ) 
                   
                   
                   
                   
                   
               
               
                 N-acetyl-β-D-gluc 
                 0 
                 −8 
                 82 
                 86 
                 84 
               
               
                 (Bovine kidney) 
                   
                   
                   
                   
                   
               
               
                 Amyloglucosidase 
                 32 
                 47 
                 −2 
                 53 
                 0 
               
               
                   
               
             
          
         
       
     
         [0275]    DNJ-N-propanoic acid has an IC 50  against bovine liver β-glucuronidase of 340 uM and 383 uM on human recombinant α-L-iduronidase. 
         [0276]    DAB-N-ethanoic acid has an IC 50  against β-glucuronidase of 523 uM and 196 uM on the iduronidase. 
         [0277]    DNJ-ethanoic acid has an IC 50  on bovine β-glucuronidase of 208 uM but only weakly inhibits the iduronidase at 0.8 mM. 
       DISCUSSION AND CONCLUSIONS 
       [0278]    Example 4 demonstrates that once DNJ is removed there is great similarity in the glycosidase inhibition profiles of PhynoRadiance and IminoNorm. 
         [0279]    There are N-acids of DNJ and DAB in both samples which give more selective inhibition of α-glucosidases than DAB and DNJ but these compounds also give good inhibition of mammalian β-glucuronidase, hexosaminidase and α-L-iduronidase. 
         [0280]    Selective fractions containing these compounds have clear application as pharmaceuticals and food ingredients. 
         [0281]    Serum β-glucuronidase and hexosaminidases are elevated in several diseases including diabetes and Alzheimer&#39;s. Elevation of β-glucuronidase could result in poor excretion of toxins as glucuronides and hence inhibition of this enzyme could be beneficial to health. It is reported that iminosugars can act as both inhibitors and chaperoning molecules for glycosidases and hence may also increase the activity of glycosidases they inhibit at sub-inhibitory concentrations. 
         [0282]    The N-acids of DNJ and DAB are novel natural products. 
         [0283]    One can conclude from the Examples that there are several compounds contributing to glycosidase inhibitions in both PhynoRadiance and IminoNorm. These include DNJ, DAB, fagomine, hydroxylated pipecolic acids, N-acids of DAB and DNJ and other, as yet, not characterised iminosugars. An aryl-glycoside and sugars including a trisaccharide may also be giving inhibition of glycosidases and glycogen phosphorylase. 
         [0284]    Thus selective water soluble  Morus  extracts rich in hydroxylated pipecolic acids, N-acids of DAB and DNJ and which contain significantly reduced levels of the imino acids DNJ, DAB and Fagomine or substantially pure hydroxylated pipecolic acids, N-acids of DAB and DNJ can be used in medicine. The acids include N-ethanoic DAB and N-ethanoic DNJ. 
       Example 5 
     Methodology 
       [0285]    The efficacy of an extract of the invention (PYN 8) to reduce post-prandial blood glucose spikes was evaluated in an acute sugar challenge ddy mouse model. Ten mice were divided into two groups, the treatment groups and placebo group. Animals orally received 2.5 g/kg body weight maltose, with either 500 mg/kg PYN8 (active arm) or saline (control). Blood glucose concentration was measured at time points 0, 15 min, 30 min, 60 min and 120 min to evaluate any effects on reduction of blood glucose following the ingestion of maltose. 
       Results: 
       [0286]    The extract of the invention was shown to significantly reduce blood glucose levels after a maltose challenge as illustrated in  FIG. 14 . 
       CONCLUSIONS 
       [0287]    The in vivo study confirmed the in vitro findings that the extract of the invention could significantly reduce post-prandial blood glucose levels. The extract inhibits multiple glucosidase enzymes that are involved in glucose metabolism. The in vivo study shows that the extract is bioavailable and is not inactivated by stomach acid, stomach enzymes or by an active metabolism.