Patent Application: US-69183410-A

Abstract:
the present application describes a method of eliminating gallated catechins , especially epigallocatechin gallate , epicatechin gallate , as well as parathion , from green tea extraction by using at least one out of ethyl acetate , cetyl trimethyl ammonium bromide , cation exchange resin , anion exchange resin , c18 bead , peg bead and mpeg . and also , this method can be applied in the method of adding at least one of pre - mentioned components , when green tea is directly ingested in the form of an extract solution , leaf or powder . this is to be useful for people with metabolic disorders such as diabetes and obesity because of the resolved toxicity . this method is to maximize the beneficial luminal effect and to minimize the harmful circulatory effect of gallated catechins .

Description:
in the present application , “ a ” and “ an ” are used to refer to both single and a plurality of objects . egcg and ecg with gallate ester groups inhibited k atp channels via allosteric interactions with phospholipids of the lipid membrane . but the effective egcg concentration was more than 10 μm . further , egcg is the only green tea epicatechin that decreases the sensitivity of atp and pip to the k atp channel . by this mechanism , we concluded that egcg caused decrease in insulin secretion in response to glucose in the β - cell of pancreas . patients with diabetes or people with a risk factor for diabetes should be careful because the effective concentration of egcg for this mechanism could be 1 - 10 μm , which is an achievable concentration when a person drinks 2 - 8 cups of green tea while fasting . gallated catechins egcg and ecg inhibited normal glucose uptake mechanism after a meal into hepatocytes , adipocytes and muscle cells which play an important role in blood glucose clearance ( fig3 ). if the glucose clearance after a meal is not effective as stated above , long - term hyperglycemia will occur and will lead to emergence or worsening of diabetes and its complications such as vascular damage . these phenomena can occur after commonly drinking green tea because it can occur when the blood egcg level is lower than 10 μm . unlike the action of the k atp channel , the glucose intolerance caused by green tea can happen by various types of gallated catechins , not only egcg but also ecg ( fig1 ). therefore , to prevent this harmful effect of gallated catechins in the circulation , elimination of gallated catechins from green tea extracts before ingestion ( fig4 ) or ingestion together with one of the elimination methods ( fig5 ) provides diminution of gallated catechins absorbed into the circulation . the latter concepts are further recommended by the fact that gallated catechins , which are harmful in the circulation via delaying blood glucose removal , can inhibit glucose and lipid absorption into the circulation from the alimentary tract ( fig5 ), which is beneficial mechanism against diabetes and obesity . the method used in this invention to remove gallated catechins , which includes without limitation epigallocatechin gallate ( egcg ), epicatechin gallate ( ecg ), and parathion involves a step of using ethyl acetate , cetyl trimethyl ammonium bromide ( ctab ), cation exchange resin , anion exchange resin , bondesil - c18 bead , peg bead ( novasyn tg hydroxyl resin ) and / or mpeg . in detail , in one embodiment , egcg , ecg and parathion - removing methods include the step of mixing green tea extraction with ethyl acetate at a ratio of 100 to 50 - 200 ( volume to volume ) and the step of removing ethyl acetate from the extract . the mixing step should be carried out for about 5 to 20 minutes . the process of removing egcg , ecg and parathion may be carried out by directly adding ctab to the green tea extraction . in this situation , ctab is added to the green tea extraction at a ratio of green tea extract 100 to ctab 0 . 05 - 0 . 1 ( weight to weight ). in another embodiment , the toxin removal process may be carried out by passing the extraction through a filter paper or tube containing cation exchange resins . the cation exchange resins may be added at a ratio of green tea extraction 100 to cation exchange resins 0 . 05 - 100 ( weight to weight ). the cation exchange resins may be activated by a 4 to 6m hcl solution or a 4 to 6m nacl solution , 1 to 2 . 5m ca ( o 2 cch 3 ) solution or a 2 to 4m kcl solution . the egcg , ecg and parathion removing method may be carried out by directly adding the cation exchange resins to the green tea extraction . in this situation , green tea extraction may be mixed with cation exchange resins at a ratio of green tea extract 100 to cation exchange resin 0 . 05 - 100 ( wt / wt ). the used cation exchange resins are then removed from the green tea extraction . the mixing may be carried out for 5 to 30 minutes . in another embodiment , the toxin removal process may be carried out by passing the extraction through a filter paper or tube containing anion exchange resins . the anion exchange resins may be added at a ratio of green tea extraction 100 to anion exchange resins 1 - 50 ( weight to weight ). the anion exchange resin may be activated by a 2 to 6m naoh solution , 2 to 6 m koh solution , 2 to 6m nacl solution , or 2 to 4m kcl solution . the egcg , ecg and parathion removing method may be carried out by directly adding the anion exchange resins to the green tea extraction . in this situation , green tea extraction may be mixed with anion exchange resins at a ratio of green tea extract 100 to anion exchange resin 1 - 50 ( weight to weight ). the used anion exchange resins are then removed from the green tea extraction . the mixing may be carried out for 5 to 30 minutes . in another embodiment , the removal process may be carried out by passing the extraction through a filter paper or tube containing c18 beads . the c18 beads may be added at a ratio of green tea extraction 100 to c18 beads 1 - 50 ( weight to weight ). the egcg , ecg and parathion removing method may be carried out by directly adding the c18 beads to the green tea extraction . in this situation , green tea extraction may be mixed with the c18 beads at a ratio of green tea extract 100 to c18 beads 1 - 50 ( weight to weight ). the used c18 beads are then removed from the green tea extraction . the mixing may be carried out for 5 to 30 minutes . in another embodiment , the removal process may be carried out by passing the extraction through a filter paper or tube containing peg beads . the peg beads may be added at a ratio of green tea extraction 100 to peg beads 0 . 01 - 50 ( weight to weight ). the egcg , ecg and parathion removing method may be carried out by directly adding the peg beads to the green tea extraction . in this situation , green tea extraction may be mixed with the peg beads at a ratio of green tea extract 100 to peg beads 1 - 50 ( weight to weight ). the used peg beads are then removed from the green tea extraction . the egcg , ecg and parathion removing method may be carried out by directly adding mpeg ( methoxy peg ) to the green tea extraction . in this situation , green tea extraction may be mixed with the mpeg directly at a ratio of green tea extract 100 to mpeg 0 . 01 - 200 ( weight to weight ). the mixing may be carried out for 0 . 1 to 5 minutes . the production of the green tea beverage according to primary characteristics , includes the steps of preparing green tea extractions from dried leaves by water , removing ethyl acetate from the hydrophilic extraction , and eliminating the removed ethyl acetate . the inventive green tea beverage producing method in view of other aspects includes the steps of preparing the hydrophilic green tea extraction , passing the extraction through a filtering paper that includes or is layered with cation exchange resins , anion exchange resins , c18 beads or peg beads or a tube that is layered with them . in another aspect , the inventive green tea beverage producing method includes the steps of preparing green tea extractions by water from dried leaves , adding and mixing ctab , cation exchange resins , anion exchange resins , c18 beads or peg beads with the extraction , and eliminating the cation exchange resins , anion exchange resins , c18 beads and peg beads . in another aspect , the inventive green tea beverage producing method includes the steps of preparing extractions from dried green leaves by water and adding and mixing mpeg with the extraction . in another aspect , the inventive green tea powder producing method includes the steps of preparing extractions from dried green tea leaves by water , adding and mixing cation exchange resins , anion exchange resins , c18 bead or peg bead with the extraction , eliminating the cation exchange resins , anion exchange resins , c18 bead or the peg bead , and lyophilizing the extracts , and turning them into powder forms . the powderizing step may be carried out using well - known methods . in another aspect , the inventive green tea powder producing method includes the steps of adding mpeg to the powder ( 0 . 01 - 200 : 100 wt / wt ). this mixing procedure is carried out for about 0 . 1 to 5 minutes . selective removal of ecg and egcg from green tea leaves or green tea extracts may be accomplished by solid - liquid extraction , liquid - liquid extraction , liquid - liposome extraction or combination of those extractions . for extraction processes , some chemicals can be added to enhance the selectivity and extraction efficiency . in solid - liquid extraction , organic solvents such as methanol , ethanol , propanol , isopropanol , diethyl ether , hexane , acetone , ethyl acetate , acetonitrile , dichloromethane and toluene may be added to the extracting water . also , extracting temperature can be changed from 50 ° c . to 100 ° c . with variable extraction times . in liquid - liquid extraction , water insoluble organic solvents such as diethyl ether , hexane , ethyl acetate , dichloromethane , and toluene may be used . in liquid - liposome extraction , lipids which can form either micelle or liposome can be added to extracting water . selective removal of ecg and egcg from green tea leaves or green tea extracts can be accomplished by hydrolysis of gallate ester . hydrolysis can be carried out by either thermal decomposition or chemical decomposition . for the chemical hydrolysis processes , inorganic acids or bases may be used . selective removal of ecg , egcg and parathion from green tea leaves or green tea extracts can be accomplished by the separation techniques such as absorption , excursion or partition chromatography . in one aspect , the invention comprises formulations of green tea polyphenols , in particular , catechins , for the production of specialized tea products for hyperglycemia patients . the green tea utilized was seijak from boseongnokchamyoungga ( chunnam , south korea ). hplc grade water , acetonitrile , ethyl acetate , methanol , and ethanol were obtained from samchun chemical , south korea . analytical grade trifluoroacetic acid was obtained from acros . ion exchange resins were obtained from aldrich ( usa ). epicatechin , epicatechin gallate , epigallocatechin , and epigallocatechin gallate were obtained from sigma ( usa ). the inventive purification methods to remove gallated catechins , including without limitation epigallocatechin gallate ( egcg ), epicatechin gallate ( ecg ) and parathion from the green tea extraction include without limitation settling the extraction with ethyl acetate , with cetyl trimethyl ammonium bromide ( ctab ), with cation exchange resins , with anion exchange resins , with c18 bead , with peg bead and finally with mpeg . the relative amounts of catechins were determined by high pressure liquid chromatography ( hplc ) using aqueous acetonitrile containing 0 . 1 % trifluoroacetic acid as eluent . quantification of catechins was performed using calibration curves obtained using pure catechins . eliminating toxic substances by treating green tea extraction with ethyl acetate ( ea ) ethyl acetate is liquid in form and to use it for egcg , ecg and parathion elimination it needs to be mixed with the green tea extraction for a certain amount of time . the ethyl acetate is included with a ratio of the tea extraction 100 volume versus 50 - 200 volume of ea . there are no problems in the case of the quantity of ea exceeding 200 , but the efficiency rate of removal does not increase as much . preferably , ethyl acetate is mixed with the amount of 80 - 120 volume relative to the extraction 100 volume . the mixing time for the extraction and ethyl acetate may be about 2 to 20 minutes , during which time egcg , ecg and parathion are selectively dissolved . ethyl acetate may be separated from the extraction such as by centrifugation or layer separation methods . as a result , egcg , ecg and parathion in the extraction can be selectively removed . other catechin substances are also dissolved but egcg is the most dominant . this method of removing egcg , ecg and parathion can be included in the process of green tea production thus allowing the emergence of a better health benefiting green tea beverage . therefore , the method above can be accomplished by primarily preparing the extraction from dried leaves by using hot water and successively mixing ethyl acetate to the extraction and then removing ethyl acetate after the appropriate amount of time has passed . eliminating toxic substances by treating green tea extraction with cetyl trimethyl ammonium bromide ( ctab ) another method to remove egcg , ecg and parathion from green tea is to treat the green tea extraction with the solid substance cetyl trimethyl ammonium bromide ( ctab ) by directly adding it to the green tea extraction and then centrifuging the extraction after an adequate amount of time has passed . ctab is included at a ratio of tea extraction 100 versus 0 . 05 - 0 . 1 ctab ( wt / wt ). eliminating toxic substances by treating green tea extraction with cation exchange resins another way to remove egcg , ecg and parathion is to use a filter paper or a tube containing cation exchange resins and passing the extraction through thus allowing selective egcg , ecg and parathion clearance . alternatively , the cation exchange resin may be added directly to the extraction and passed through the filter thus clearing the toxic substances from the extraction . the cation exchange resin amount used in this method may include the tea extraction 100 versus cation exchange resin 0 . 05 - 100 ( wt / wt ). the layering includes simply putting the cation resins on the filtering paper in which the extraction is vertically moved and filtered . another method of using the cation resins includes first adding the resins directly to the extraction , then after the right amount of time has passed , clearing the resins by filtering . this method , compared to the method using layers , relatively has more contact surface and time with the extraction and therefore can save the amount of resins used . this clearing method may be used in massive production by small scaled beverage stores or by consumers . for example , in small scaled production , a functional green tea beverage may be made by primarily producing the extraction by filtering and then additionally using the inventive method . for the cation exchange resins used in this method 4 to 6m of hcl solution is used for activation . 4 ˜ 6m nacl solution , 1 ˜ 2 . 5m ca ( o 2 cch 3 ) solution or 2 ˜ 4m kcl solutions also can be used for activation . eliminating toxic substances by treating green tea extraction with anion exchange resins an additional method to eliminate egcg , ecg and parathion is to use solid anion exchange resin layered filtering paper or anion exchange resin layered tube and passing the green tea extraction through it resulting in egcg , ecg and parathion clearance from the green tea extract . the layering includes simply putting the anion exchange resins on the filtering paper and moving the extraction vertically and filtering . 2 ˜ 6m naoh can be used to activate the anion exchange resins or one or more of 2 ˜ 6m koh , 2 ˜ 6m nacl or 2 ˜ 4 kcl solution can be used to activate the resins . the anion exchange resin amount used may be in a ratio of resin 1 - 50 versus tea extraction 100 by weight . directly adding the anion exchange resin to the extraction and then filtering and thus clearing the tea extraction of the anion exchange resin is another method that can be used to clear the toxic substances . this method , compared to the method using layers , relatively has more contact surface and time with the extraction and therefore can save the amount of resins used . the anion exchange resin amount used may be in a ratio of resin 1 - 50 versus tea extraction 100 by weight . this clearing method may be used in massive production by small scaled beverage stores or by consumers . for example , in small scaled production , a functional green tea beverage may be made by primarily producing the extraction by filtering and then additionally using the inventive method . eliminating toxic substances by treating green tea extraction with c18 bead another method to selectively remove egcg , ecg and parathion is to use a c18 bead layered filter paper or tube to pass the extraction . the layering includes simply putting the c18 beads on the filtering paper and moving the extraction vertically and filtering . another way to clear the toxic substance is to directly add the c18 bead to the extraction and clearing the c18 bead by filtering . the amount of the c18 bead should be used at a ratio of c18 bead 1 - 50 versus the tea extraction of 100 ( wt / wt ). another method of using the c18 beads can be performed by first adding the beads directly to the extraction , then after the right amount of time has passed , clearing the beads by filtering . this method , compared to the method using layers , relatively has more contact surface and time with the extraction and therefore can save the amount of beads used . this clearing method may be used in massive production by small scaled beverage stores or by consumers . for example , in small scaled production , a functional green tea beverage may be made by primarily producing the extraction by filtering and then additionally using the inventive method . eliminating toxic substances by treating green tea extraction with polyethylene glycol ( peg ) bead another method to selectively remove egcg , ecg and parathion is to use a solid peg bead layered filter paper or tube to pass the extraction . the right amount of the peg bead may be 0 . 01 - 50 weight versus the tea extraction of 100 . the layering includes simply putting the peg bead on the filtering paper and moving the extraction vertically and filtering . another method of using the peg beads can be performed by first adding the beads directly to the extraction , then after the right amount of time has passed , clearing the beads by filtering . this method , compared to the method of using layers , relatively has more contact surface and time with the extraction and therefore can save the amount of beads used . this clearing method may be used in massive production by small scaled beverage stores or by consumers . for example , in small scaled production , a functional green tea beverage may be made by primarily producing the extraction by filtering and then additionally using the inventive method . eliminating toxic substances by treating green tea or green tea extraction with methoxy polyethylene glycol ( mpeg ) another way to clear egcg , ecg and parathion is to add and agitate the mpeg to the extraction and selectively clear the toxic materials . the right amount of mpeg should be 0 . 01 - 200 weight versus the tea extraction of 100 . in this application , use of only cation exchange resins , anion exchange resins , c18 beads , peg beads and mpeg are exemplified but also other resins , beads and other types of peg macromolecules may be used by a person of skill in the art . the present invention is not to be limited in scope by the specific embodiments described herein . indeed , various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures . such modifications are intended to fall within the scope of the appended claims . the following examples are offered by way of illustration of the present invention , and not by way of limitation . removal of egcg and ecg from green tea extraction by using ethyl acetate we confirmed that egcg and ecg were removable by separating ethyl acetate by centrifuging after mixing ethyl acetate with the green tea extract . quantitative analysis of the catechin content in the green tea extraction such as egcg , ecg was done by hplc ( high pressure liquid chromatography ) using acetonitrile containing 0 . 1 % trifluoroacetic acid as an expansion liquid . catechin quantity was obtained from the quantity curve with pure catechin at a known concentration . extraction was carried out in a 60 ° c . water for 5 minutes from dry green tea leaf ( product name : sejac , manufacturer : bosung nok cha myoung ka , jeonnam , korea ). after removing solid leaf material with a 0 . 45 μm filter , 5 minutes of agitation was carried out in 60 ° c . water after adding 10 ml of ethyl acetate . separation of the ethyl acetate layers was done by centrifugation and by gathering the water layer . catechin content in the water layer was analyzed and the results are shown in table 1 . extraction was carried out in 10 ml of 60 ° c . water for 5 minutes from the 0 . 2 g dry green tea leaf , and then without removing solid leaf material agitation was done at 10 ml 60 ° c . water after mixing it with 10 ml of ethyl acetate . we analyzed the catechin content and the results are shown on table 2 . as we can see in table 2 with the presence of solid material there was almost no change in the egcg content even after the ethyl acetate treatment . without being bound by any theory to explain the results , one explanation for this is that the green tea leaf persistently provides egcg . therefore , to remove egcg effectively green tea extraction should be prepared primarily after the elimination of solid material and then secondary process may proceed . extraction was done in 10 ml of 60 ° c . water for 5 minutes from the 0 . 2 g dry green tea leaf . and then filtering the leaf at a 0 . 45 fall filter agitation was done at a 60 ° c . for 5 minutes after adding ctab . centrifugation was done and precipitated ctab . catechin content was analyzed by high pressure liquid chromatography ( hplc ) using aqueous acetonitrile containing 0 . 1 % trifluoroacetic acid as eluent . quantifications of catechins were performed using calibration curves obtained from authentic catechins . the amount of addition was 5 mg and 10 mg and the catechin content was analyzed in each case . the results are shown in table 3 . extraction was done in 10 ml of 60 ° c . water for 5 minutes from the 0 . 2 g dry green tea leaf . next , agitation at 60 ° c . for 5 minutes after adding cation exchange resin ( dowex 50wx2 - 100 ) ( activated in a 6m hcl solution ) into the remaining extraction ( filtered in a 0 . 45 fall filter ) was carried out . the relative amounts of catechins were determined by high pressure liquid chromatography ( hplc ) using aqueous acetonitrile containing 0 . 1 % trifluoroacetic acid as eluent . quantifications of catechins were performed using calibration curves obtained using pure catechins . cation exchange resin was used after it was activated in a pipe in which 6m hcl solution ( 20 times larger in volume than the resin &# 39 ; s ) was slowly flowing and cation exchange was washed with deionized water before its use . after removing resin , catechin content in water soluble state was analyzed . the results are shown in table 4 . extraction was carried out in 10 ml of 60 ° c . water for 5 minutes from 0 . 2 g dry green tea leaf . next , agitation was carried out at 60 ° c . for 5 minutes after adding 1 . 5 g cation exchange resin ( activated in a 6m nacl , 2 . 5m ca ( o 2 cch 3 ), 3 . 5m kcl solution ) into the remaining extraction ( filtered in a 0 . 45 fall filter ). the cation exchange resin was used after it was activated in a pipe in which 6m nacl , 2 . 5 m ca ( o 2 cch 3 ) or 3 . 5 m kcl solution ( 20 times larger in volume than the resin &# 39 ; s ) was slowly flowing and cation exchange was washed with deionized water before its use . after removing the resin , catechin content in a water soluble state was analyzed . the results are shown in table 5 . extraction was carried out from 0 . 2 g dried green tea leaf in 10 ml of 60 ° c . water for 5 minutes . it was then filtered by a 0 . 45 fall filter . and then 0 . 1 g of peg bead was added . next , it was agitated at 60 ° c . for 5 minutes . after the agitation resin was removed by filter the catechin content in the water soluble fraction was measured using hplc . the results are shown in table 6 . extraction from 30 g dried green tea leaf was done in 500 ml of 80 ° c . water for 3 minutes . after the green tea leaf was filtered in a 0 . 45 μm filter 7 . 6 g of mpeg was added . and then it was mixed for 2 minutes in ambient temperature . we presumed the absorption of egcg indirectly by measuring the amount of glucose in blood during oral glucose challenge after a person drank this solution . the fact that the increase in the amount of glucose in the blood by green tea is by egcg and ecg was indicated by experimentation ( fig1 ). accordingly , it is plausible that the reason of lower blood glucose in the group treated with green tea plus mpeg is due to the blocking effect of mpeg in absorption of egcg and ecg from the alimentary tract ( fig2 ). after 10 minutes of stirring of 0 . 2 g of dried green tea leaf in 10 ml of 100 ° c . water it was filtered and the green tea leaf was removed . next the quantity of parathion was analyzed with hplc standard solution was made by diluting the 17 % parathion ( commercially available ) 100 times with methanol ( manufactured by sam - jeon chemistry , republic of korea ). solution diluted with methanol 100 times was analyzed with hplc . also the solution diluted by 100 times by adding 100 μl of standard solution into 9 . 9 ml of water was analyzed with hplc . 20 minutes of agitation was done after adding 10 ml of ethyl acetate in the diluted solution . after separating it into ethyl acetate and water layer the parathion content was each measured with hplc . 100 times diluted 17 % parathion was used as a standard solution . solution made by adding 10 μl of standard solution in 9 . 9 ml of water was analyzed with hplc . after adding 1 g of activated cation exchange resin into the previous solution , minutes of agitation was done . and then cation exchange resin was removed by filter . next the amount of parathion remaining in the solution was measured . after adding 2 g of activated cation exchange resin into the previous solution , 5 minutes of agitation was done . and then cation exchange resin was removed by filter . next the amount of parathion remaining in the solution was measured by hplc . after adding 0 . 4 g of activated c18 bead into the previous solution , 5 minutes of agitation was done . and then c18 bead was removed by filtration . next , the amount of parathion remaining in the solution was measured by hplc . status about extracted and detected parathion from 0 . 2 g / 10 ml green tea is shown in fig6 . fig6 is a hplc graph showing the amount of parathion remaining in 0 . 2 g / 10 ml of green tea . a line indicated as “ p ” is for parathion 0 . 17 mg / l , and the line indicated as “ g ” is for green tea extracts . from the extractions from green tea a substance with the same retention time with parathion was detected . other analysis tools are needed to examine the exact structure of this material but still when we presumed it as a parathion the amount is lesser than 0 . 17 mg / l . but again , considering 1 cup of green tea is 2 g green tea / 100 ml water , the amount of parathion from a cup of green tea is almost the same as the daily intake limit ( 0 . 02 mg / kg / day ) that fda approved . the result of eliminating parathion dissolved in the water is shown in fig7 . fig7 is a hplc graph showing the quantity change of parathion by the method using ethyl acetate . in fig7 , the line indicated as “ w ” refers to the water solution before it was treated with ethyl acetate , and the line indicate as “ ea ” refers to the ethyl acetate layer after the solution was treated with ethyl acetate . the line indicated as “ wea ” refers to the water solution after it was treated with ethyl acetate . the result of removal of parathion dissolved in water with absorption methods are shown in fig8 and 9 . fig8 is an hplc graph showing the quantity change in parathion by treating with cation exchange resin . the parathion amount in the solution decreased in proportion to the amount of ion exchange resin used . ( parathion solution : line indicated as “ p ”, solution treated with 1 g of ion exchange resin : line indicated as “ c1g ”, solution treated with 2 g of ion exchange resin : line indicated as “ c2g ”). fig9 is an hplc graph showing the quantity change of parathion according to the use of c18 bead . the amount of parathion in the solution was decreased by adding c18 bead . ( parathion solution : line indicated as “ p ”, solution treated with 0 . 4 g of c18 bead : line indicated as “ c18 ”). according to this invention we can effectively and selectively remove egcg , ecg and parathion from the green tea extraction liquid . so it is possible to develop an adequate green tea drink and green tea powder product to those who are vulnerable to diabetes with a help of this invention . and also by removing parathion which is a harmful agricultural chemical it is possible to make a safer green tea drink from a green tea extract . finally applying this invention to the industry may provide healthful drink or food to the people . all of the references cited herein are incorporated by reference in their entirety . those skilled in the art will recognize , or be able to ascertain using no more than routine experimentation , many equivalents to the specific embodiments of the invention specifically described herein . such equivalents are intended to be encompassed in the scope of the claims . 1 . qanungo , s ., das , m ., haldar , s ., and basu , a . 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