IL35986A - Amylase inhibitor,its production and use - Google Patents

Description

its production and use BAYER A It is known that can be inhibited by various lo substances such as salicylic acid and abiscisine Larsson 861 1665 is known there also exist substances higher molecular weight which are capable of unspecifically inhibiting the activity of some amylases by physical adsorption and 28 296 or by denaturing and precipitation of the enzyme Acta it has been observed that it is possible to elute with distilled from wheat a substance which reduces the dextrifying activity of saliva but has little influence on the activity of pancreas amylase 235 It is a disadvantage of the substances described above that the inhibition of the amylase is either unspecific as our own investigations have inhibiting activity Of the described substances is particularly in relation to pancreas that is to say that almost complete inhibition of the amylases to 0 and are onl achieved with a very high ratio of inhibitor to enzyme this shows the inhibition course of the amylese inhibitor according to the invention in comparison with an inhibitor prepared according to the instructions given by Kneen in relation to amylase units of pancreas am It has now been found that it is possible to extract a highly active inhibitor for pancreas amylase which wheat flour or with the aid of aqueous electrolyte preferably dilute acids alcohol preferably at acid pH values The so obtained inhibits pancreas amylase at a very low ratio to the extent of than 1 The same substance also inhibits saliva amylase to a high Other hydrolases so far as they have been chymotrypsin and ribonuclease are not inhibited by this The inter as starting material is the protein component of wheat and is obtained as a byproduct in the production of wheat starch Handbuch der pharmazeutischen Julius von 1925 page 332 Blish in Advances in Protein New 1 page The new substance is colourless and dissolves readily in dilute acids and alkaline solutions as well as in aqueous The absorption spectrum of a aqueous solution of the inhibitor is shown in The absorption maximum lies at The is insoluble in acetone and absolute The new inhibitor cannot be The substance can with 2 trichloroacetic the precipitate is soluble in H2P and still exhibits 60 to of its initial The substance is slowly inactivated by proteases Solutions of the substance are comparatively stable to acid 1 to 2 but labile to incubation at in pH 1 leads to an loss f Neutral aqueous From inaetivation the amylase inhibitor oecors rapidly increases as the duration of incubation is increased in m g CaCl2 pH By the addition of urea up to a concentration of 2 the inhibitor is not up to it is inactivated to 50 According to the amylase inhibitor is a The inhibiting capacity of the best inhibitor preparations in relation to pig pancreas amylase amounts to 700 to 800 pancreas amylase units per in relation to saliva amylase to 900 to 1000 saliva amylase units per The high activity of this inhibitor also against pancreas amylase curve distinguishes inhibitor from that described by curve which saliva amylase with about half the capacity of 500 saliva but has only a poor activit against pancreas amylases 10 pancreas and can inhibit hardly more than of the pancreas amylase activity even if added in large amounts curve One amylase inhibitor unit is defined as the amount of inhibitor which inhibits one amylase unit to 90 90 inhibition was chosen because of easier tion when determining the inhibition Complete inhibition is achieved asymptotically 1 and One amylase unit is the amount of enzyme which splits 1 μ equivalent of glucosidic bonds in starch within one minute under the stated test The μ gramme equivalent of split bonds are colorimetrically determined with dinitroealicylic acid as μ gramme equivalent of reducing sugars formed and are To carry out the ml amylase solution to 22 are mixed with 0 to inhibitor in 04 of m sodium glycerophosphate buff m pH and equilibrated on a water bath of for about 10 to 20 The mixture is then incubated at for minutes with ml of a starch solutio facturers which been heated to and subsequently mixed with 1 ml of salicylic acid reagent to Bernfeld in page To develop the the mixture is heated on a boiling water bath for 5 is then cooled and 10 ml of distilled water are The extinction at is measured against a blank value prepared the amylase activity which is still effective after addition of the inhibitor is read from a previously plotted amylase curve and the percentage inhibition of the amylase used is calculated The percentage inhibition is plotted as a function of the quotient inhibitor referred to dry substance in the mixture the seriee the 0 inhibition point is read from the curve and recalculated for mg The extraction of the inhibitor according to the invention from wheat shredded wheat or gluten is carried out according to the invention either with 10 to preferabl 30 to 70 mixtures soluble lower alcohols such as propanol preferably dilute aqueous at pH values of to preferably at pH values of 2 to or with alcohol mixtures of the composition stated under but at acid pH values of 1 to 6 preferably 2 to the presence of mineral acids or organic For this part by weight of wheat shredded wheat or gluten is homogenized with 1 to parts by preferably 2 to parts by of the appropriate extraction medium for 1 to 2 minutes and the mixture is subsequently stirred for to hours 1 to 2 at room temperature to The mixture is then filtered or centrifuged at 3000 to 10000 g for 10 to 20 If the residue is reextracted 1 to 2 parts by weight of the extraction as described centrifuged or The supernatant solutions or filtrates are in the case of acidic they are preferably with a concentrated and freed from the ballast proteins precipitated in the course of neutralisation by filtration or In contrast to aqueous wheat flour extracts which are capable of very effectively inhibiting amylases but not pancreas amylases 3 curve the extracts prepared according to the invention by the methods to show an high inhibiting activity also against pancreas amylases curves 3 illustrates the inhibition of pig pancreas amylase by a an acidic aqueous a aqueous ethanolic 3 and an aqueous neutral extract from wheat In each 1 part by weight of wheat flour was extracted with parts b of extraction a ent at for one s amylase inhibitor according to the invention is the extraction of wheat flour or gluten with acidic aqueous alcohols to methanol or pH 2 to This method gives higher yields of inhibitor than the extraction with neutral aqueous alcohols compared with the acidic aqueous extraction mixtures it has the advantage that the mixture is less viscous and therefore easier to for centrifuging the coextraction observed in processes and of an amylase which is present in wheat flour and cannot be inhibited is obviated in this process and inactivation or separation of the vegetable amylase is therefore When gluten is used as starting the yields are 5 to 8 higher with the use of wheat Another advantage consists in that the extraction mixtures are easier to filter and centrifuge and that the amylase inhibitor is obtained with a higher specific activit when gluten is used as starting Isolation of the inhibitor from the extraction solutions are previously neutralised in processes B and can be carried out in various Concentration of the extracts under reduced pressure to 20 mm at temperatures up to to about to of the starting The concentrated extract is filtered or centrifuged and the clear filtrate the clear natant is lyophilized after Precipitation of inhibitors from the extracts with the aid of This process is mainly suitable extracts still having a high content of alcohol concentration above since relatively complete not too high can only be attained from such extracts The precipitation is achieved by pouring 1 part by volume of the solut ion to be precipitated into 2 to parts by volume acetone upon the alcohol content of the extraction solution to be precipitated Drying of the inhibitor preparation in a vacuum at room temperature after washing with absolute alcohol Precipitation of the inhibitors from the extracts or the extracts concentrated according to by the addition of lower alcohols up to a per cent by volume content of 95 for ethanol 90 for or 80 for isopropanol The amylase inhibitor can also be precipitated methanol but in lower yields than are obtained with the use of the aforesaid alcohols Since low alcohol concentrations lead to the precipitation of inactive accompanying substances proteins the precipitation process is also for fractional precipitation out of the amylase inhibitor from the extracts from the extracts concentrated according to e with sodium chloride ammonium sulphate etc Precipitation of the amylase inhibitor from extracts with a certain alcohol content at low temperatures For this purpose the extracts are firet ad justed to alcohol concentrations of 10 to 50 preferably 20 to subsequently kept for several hours to hours at below preferably at to The settle s well and the clear supernatant liquid can be drawn off The remaining precipitate is centrifuged absolute ethanol or about 5 parts volume of absolute preferably in the cold and subsequently dried at room preferably in a This method is also suitable for fractional precipitation different It is known that after the intake of containing food potato alimentary occur in animals and humans this is caused by a rapid decomposition of the amylase and pancreas amylase and according to the following scheme maltase starch maltose glucose or glycogen These hyperglycaemiae are particularly marked and persistent in In adipose persons the alimentary caemia frequently gives rise to a particularly strong of insulin which in turn leads to increased formation of fat and decomposition of It has been found that the amylase according to the isolated by the above significantly reduces alimentary hyperglycaemia six healthy test persons after with wheat starch Experiment Example is therefore suitable as therapeutical agent for diabetes and Doeage to once or several times daily per during Forms of granules kg mouse per os were tolerated without Combinations with the oral sulphonyl derivatives blood depressing biguanides are also of Example 1 1 kg of wheat flour is homogenized 2 litres of pH 3 to to pH to with and stirred at room temperature for 30 The mixture is then centrifuged at 6000 for 10 minutes the supernatant liquid is stored and the residue is reextracted as described above with further 2 litres ethanol at pH 3 to The combined supernatant liquids are with concentrated aiimonia and the flocculated ballast proteins are removed by centrifuging 10 The clear slightly yellowish supernatant liquid litres is trated of its volume at about 20 mm Hg and a bath temperature of to again freed from precipitated proteins by centrifuging and after dialysis distilled water room it is frozen and 10 g with 000 pancreas lyophilisate g inhibitor dry substance with pancreas kg of wheat flour is homogenized wit litres of ethanol in a adjusted to pH 3 to with hydrochloric acid and stirred at room temperature for 60 The is centrifuged at 6000 for 10 the supernatant liquid is adjusted with to pH and centrifuged from the ballast The clear supernatant liquid is stored overnight the precipitated sediment is collected in centrifuging glass vessels at 20 cooling 10 6000 It is mixed with cold absolute ethanol temperature from now with absolute alcohol and dried in a vacuum 10 g with pancreas 8g inhibitor substance with pancreas Example 200 g of flour are homogenized with 800 ml of adjusted with HCl to pH and stirred at room temperature for 30 The mixture is subsequently centrifuged for 30 minutes at the turbid supernatant liquid is neutralised with and again centrifuged at 30 The 600 ml of turbid supernatant liquid are mixed with 1100 ml of absolute filtered and the inhibitor is precipitated from the filtrate with a further 5 litres of absolute It is washed with absolute ethanol and dried in a 1 g with pancreas if 500 g of wheat flour are homogenized with 2 litres of 7 methanol for 2 stirred at room temperature for 1 and subsequently filtered through two large fluted The clear filtrate is added dropwise with stirring to parts by volume The inhibitor settles at the bottom in the form of thick white The clear supernatant liquid is decanted and the white sediment is taken Up in absolute washed and finally dried at room Example 5 The antihyperglycaemic effect of the amylase hibitor was tested the following test arrangement 100 g of cereal starch were administered in the of an aqueous suspension os to 6 healthy test The blood sugar was determined immediately before the start of the test and at short intervals until 3 hours tion of the starch by means of the to 51 In two further the amylase inhibitor taken up in slime was applied in the same test arrangement to the same six test persons per os immediately before application the Example 6 kg gluten manufacturers Crespel and were introduced in portions with intense stirring into litres of The mixture was then with hydrochloric acid to the viscosity of solution increased The mixture was stirred fqr minutes and subsequently neutralised with Flocculation The mixture was centrifuged for 10 minutes at about 2000 and the clear centrifugate was cooled to The precipitate flocculating in the cold settled well over The supernatant clear solution was the sediment collected in a cooling centrifuge at after washing with alcohol and dried in a vacuum at room 180 g pancreas Table re Example 5 Change of blood glucose as of initia of six healthy test persons value 1 at different times after oral administration of wheat starch after previous application of the amylase as against control test as against control test as against control test insufficientOCRQuality

Claims (1)

1. CLAIMS Amylase inhibitor derived from being a colorless readily soluble in dilute alkaline solutions and aqueous alcohol and insoluble in absolute having high activity against pancreas and saliva while being inactive against pepsin and ribonuclease and having the absorption spectrum of a aqueous solution as shown in Figure 2 of the accompanying Amylase inhibitor according Claim being obtained from k process for the production of an inhibitor according to 1 or characterised in that wheat shredded wheat or gluten is extracted with alcohol or with aqueous 3 electrolyte solutions at pH values of 1 to and the amylase inhibitor is isolated from the For the Applicants AND PARTNERS insufficientOCRQuality