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 . the starting point for a consideration of the claimed invention is the extract disclosed in wo2011 / 032502 . 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 : 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 ). 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 . 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 . 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 . 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 . 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 . the gc - ms chromatograms ( not shown ) of the ir120 fractions were all dominated by dnj , the un - retained material was mainly sugars and contained no dnj or other imino sugars after the second ir120 column . other imino sugars and imino sugar acids ( hydroxylated non - protein imino acids ) were also detected by their characteristic mass spectra . the dnj levels in the three retained fractions b ), c ) and d )— table 1 was determined and is shown in table 2 : 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 . 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 ) the inhibition of α - and β - glucosidases for the ir120 retained fractions was expected given the presence of significant amounts of dnj . 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 . elevated serum levels of glucuronidase and hexosaminidase activities are observed in many diseases including diabetes . 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 . 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 . some of these enzyme activities such as hexosaminidase and glucuronidase are known to be elevated in serum or urine in various diseases including diabetes . in the subsequent examples these fractions were further fractionated to more conclusively identify the more major compounds responsible for the activities . the extract of example 1 was fractionated into four fractions using cation exchange chromatography ( ir120 h + form ). the un - retained fraction contained sugars with imino - sugars displaced with pyridine and then ammonia . 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 . table 4 below shows the enzyme inhibitions noted for the fractions in example 1 . 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 . this example primarily involved further fractionation of the 10 g of morus alba leaf extract used in example 1 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 ). 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 . 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 . the sugar fractions obtained were colourless or slightly yellow . some were recombined for further separation later . the fractions are shown in table 5 below : 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 . 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 . 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 ). 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 . from the ir120 bound fractions obtained in example 1 several components were partially purified allowing further characterization . a compound with a retention time 10 . 37 min gave good matches by gc - ms with structures derived from dab . 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 . 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 . 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 . 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 ). the assay results for the fractions are given in table 6 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 . although dab is reported to give glycogen phosphorylase inhibition , the unfractionated extract clearly has several imino - sugar components which give this inhibition . 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 after removing the dnj , dab and fagomine , many components in the extract also inhibited the glucosidases . the analysis conducted on the fractions described in example 2 provided further information on the imino - sugars and sugars of the sample . the sugar fraction comprised almost half of the sample and showed potent activity against various glycosidases and glycogen phosphorylase . sugars such as sucrose , trehalose and glucose do not inhibit these enzymes significantly . in this example an attempt was made to characterise the active components of the sugar fraction and look further at its activity . in this regard it was fractionated further using a series of ion exchange columns . 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 . 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 . 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 . 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 . 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 . 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 . the un - retained fractions of sample 1 - 11 were white semi - crystalline material as before . 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 . the early 1 - 11 fractions had white solid semi - crystalline material that gave no gc peaks suggesting sugars that were larger than disaccharides . samples coded a - f in table 8 were submitted for nmr analysis . a , b , e and f were also submitted for glycosidase assays the results of which are tabulated in table 9 below . 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 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 . 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 . 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 ) 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 . 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 . 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 sought to confirm the activity in non - iminosugar fractions of the extracts . 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 . serum β - glucuronidase and hexosaminidases are elevated in several diseases including diabetes . 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 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 . the n - acids of dnj and dab are novel isolated natural products . an unusual trisaccharide , with a deoxy - sugar and an aryl - glycoside has also been tentatively identified . 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 . fractions were collected in water and freeze dried as described in example 3 retained material was removed using 1m acetic acid and these fractions were evaporated nearly to dryness by rotary evaporation and then freeze dried . 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 the samples iminonorm and phynoradiance ( diluted extracts as described in wo2011 / 032502 ) were additionally subjected to : the anion exchange retained material gave a more specific inhibition of rice α - glucosidase . the anion exchange resin un - retained material , of both samples , gave broader α - glucosidase inhibition . 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 ). 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 . sugars added to phynoradiance may have , therefore , contributed to the weight of anion exchange un - retained fractions of that sample . 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 . fig1 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 fig3 ( 9 . 5 minute ). fig4 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 . the iminonorm sample overloaded the cation exchange resin ( ir120 ) two times due to the high imino sugar content . bound fractions ( 2 and 3 ) are shown in fig7 and 8 with some preferential displacement of imino sugars evident . basic imino sugars tend to displace more neutral or acidic imino sugars . 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 ). 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 . fig6 a and b are the mass spectra of two iminosugars not yet identified from iminonorm . fig7 a is a gc - ms chromatogram of iminonorm ir120 retained ( iminosugar fraction 2 ) 23 g and fig7 b is a mass spec for fagomine . 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 . fig8 a , is a gc - ms chromatogram of iminonorm ir120 retained ( iminosugar fraction 3 ) 25 . 5 g . fig8 b and 8c are two mass spec of hydroxlylated pipecolic acids . 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 . fig9 is a mass spectra of n - ethanoic acid of dnj ( tms ) in phynoradiance . fig1 is a mass spectra of dab - n - ethanoic acid ( tms ) from phynoradiance . fig1 is a mass spectra of n - ethanoic acid of dnj ( tms ) in iminonorm . fig1 is a mass spectra of dab - n - ethanoic acid ( tms ) from iminonorm ; and fig1 is mass spectra of n - propanoic acid of dnj ( tms ) in iminonorm . the formula for these acids are illustrated in formula 8 to 10 below : 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 . the activity of these novel isolated acids is shown in table 14 below . 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 . dab - n - ethanoic acid has an ic 50 against β - glucuronidase of 523 um and 196 um on the iduronidase . dnj - ethanoic acid has an ic 50 on bovine β - glucuronidase of 208 um but only weakly inhibits the iduronidase at 0 . 8 mm . example 4 demonstrates that once dnj is removed there is great similarity in the glycosidase inhibition profiles of phynoradiance and iminonorm . 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 . selective fractions containing these compounds have clear application as pharmaceuticals and food ingredients . 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 . 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 . 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 . 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 . the extract of the invention was shown to significantly reduce blood glucose levels after a maltose challenge as illustrated in fig1 . 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 .
this invention relates to extracts rich in n - acids of imino sugars and / or pipecolic acids obtainable from the plant leaves of mulberry . 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 .