Patent Application: US-7192905-A

Abstract:
a method for stimulating insulin secretion by anthocyanidins and anthocyanins is described . the secretion can be in vivo in mammals , including humans , or in vitro .

Description:
chemicals . fetal bovine serum ( fbs ) and rpmi - 1640 medium were obtained from invitrogen ( grand island , n . y .). all organic solvents used were acs reagent grade . hepes , penicillin - streptomycin , glutamine , sodium pyruvate , 2 - mercaptoethanol , trypsin - edta , bsa ( bovine , albumin ; ria grade ), folin - ciolatues reagent and chemicals used for the preparation of buffers were purchased from sigma - aldrich chemical co . ( st . louis , mo .). the anthocyanidins , cyanidin , delphinidin , pelargonidin , malvidin , and petunidin , used in the assay were purchased from chromadex ( laguna hills , calif .). anthocyanins . delphinidin - 3 - glucoside ( 2 ) was purified from c . officinalis fruits . cyanidin - 3 - galactoside ( 3 ) and pelargonidin - 3 - galactoside ( 4 ) were isolated from c . mas fruits . pure cyanidin - 3 - glucoside ( 1 ) used in this study was from our storage at − 20 ° c . isolation and purification of anthocyanins . the cornus fruits were blended with water ( ph = 3 ) and filtered . the filtrate was passed through xad - 16 amberlite resin in a column and the resin with the adsorbed anthocyanins was washed repeatedly with water ( 17 ). the xad - 16 resin was then eluted with acidic meoh ( ph = 3 ) and the resulting solution was concentrated under reduced pressure to yield a crude anthocyanin fraction . this fraction was purified by mplc column ( c 18 silica ) using meoh : h 2 o ( ph = 3 ) under gradient conditions . the anthocyanins were eluted with meoh : h 2 o ( 65 : 35 , v / v ) solvent system . the purity of the compounds was checked by hplc ( waters corp .) using capcell c 18 analytical column under gradient conditions . the solvents used were a : tfa : h 2 o ( 99 . 9 : 0 . 1 ; v / v ) and b : h 2 o : ch 3 cn : ch 3 cooh : tfa ( 50 . 4 : 48 . 5 : 1 . 0 : 0 . 1 ; v / v / v / v ). the gradient was 20 % b to 60 % b in 26 min and to 20 % b in 30 min at a flow rate of 0 . 8 ml / min . the peaks were detected at 520 nm using a pda . insulin secretion studies . ins - 1 832 / 13 cells ( kindly provided by dr christopher newgard , duke university , nc ) ( 18 ) were routinely cultured in 5 % co 2 / air at 37 ° c . in rpmi - 1640 medium containing 11 . 1 mm glucose and supplemented with 10 % fbs ( fetal bovine serum ), 10 mm hepes , 100 u / ml penicillin , 100 μg / ml streptomycin , 4 mm glutamine , 1 mm sodium pyruvate , and 50 μm 2 - mercaptoethanol . cells were passed weekly after trypsin - edta detachment . for static secretion studies , cells were plated on 24 well plates at a density of 0 . 64 × 10 6 cells per well and grown for 24 h . the cells were then cultured for an additional 24 h in rpmi - 1640 containing 4 mm glucose and the supplements described above . cells were then incubated twice for 30 min in krebs ringer bicarbonate buffer ( krbb ) containing 4 mm glucose and 0 . 1 % bsa . cells were rapidly washed with krbb and incubated for 60 min krbb containing 4 or 10 mm glucose with or without the indicated anthocyanins or anthocyanidins . the medium was then removed for determining insulin release . the cells were then washed twice with pbs and dissolved in 1 m naoh . cellular protein concentration was then determined by lowry assay . anthocyanins and anthocyanidins were dissolved in dmso to obtain desired concentrations . final concentration of dmso was 0 . 1 %. the insulin secreted into the medium by the cells was determined by radioimmuno assay and normalized to total cellular protein . radio immuno assay ( ria ). the kit for ria was purchased from linco research inc . ( st charles , mo . ), and the assay was conducted according to the manufacturer &# 39 ; s directions . briefly , 0 . 1 - 10 ng of insulin standards ( 100 μl ) were added to 12 × 75 mm test tubes . similarly , samples ( 25 μl ) from the insulin secretion studies were also added to the test tubes . to this , an aliquot ( 75 μl ) of assay buffer was added . the 125 i labeled insulin ( 100 μl ) was then added to each test tube . an aliquot of 100 μl anti rat insulin antibody was added to the tubes , mixed and incubated at 4 ° c . for 24 h and incubated further with 1 ml aliquot of the precipitating reagent for 20 min at 4 ° c . to precipitate the insulin bound to the antibody . the tubes were then centrifuged and the radioactivity was measured using a gamma counter . lowry protein assay . the amount of protein in the assay wells was determined by lowry method ( francis , j . a ., et al ., helv . chim . acta 87 317 - 326 ( 2004 )). the lowry assay solution was prepared by combining the lowry solution , cuso 4 . 5h 2 o ( 1 %), and sodium tartarate ( 1 %). briefly , the protein sample ( 100 μl ) and lowry mixture ( 1 ml ) were mixed in a test tube ( 12 × 75 ). the folin - ciolatues reagent ( 100 μl ) was added to these tubes , mixed , and incubated for 30 min at room temperature . the optical density of resulting solutions was read at 700 nm using a uv spectrophotometer . the cornus fruits are used in antidiabetic traditional chinese prescription medicines such as “ hachimi - gan ” ( yamahara , j ., et al ., yakugaku zasshi , 101 86 - 90 ( 1981 )). it was recently reported the quantification of anthocyanins in cornus spp . fruits ( seeram , n . p ., et al ., j . agri . food chem . 50 2519 - 2523 ( 2002 )). the investigation of cornus fruits indicated that the primary bioactive components in them were cyanidin , delphinidin and pelargonidin glycosides . therefore , we have focused our attention on the insulin secreting ability of these anthocyanins and their aglycones using pancreatic beta cells in order to substantiate the anecdotal use of cornus fruits in antidiabetic preparations . petunidin , malvidin and peonidin aglycones were also included the assay since they are abundant in other fruits . anthocyanins are water - soluble compounds . the aqueous extracts of c . mas fruits contained sugars , bioflavonoids and anthocyanins and hence was fractionated by xad - 16 resin . the resulting anthocyanin fraction eluted from the resin was purified by mplc to afford pure anthocyanins . the glucose - induced insulin production by ins - 1 832 / 13 cells was determined at 4 , 10 and 16 mm glucose concentrations and found that the insulin secretion reached a lag phase at 10 mm glucose concentration ( data not presented ). the glucose concentration at 4 mm level is representative of the normal glucose level in human ( christison , g . b ., et al ., med . boil . eng . comp 31 284 - 290 ( 1993 )). the insulin secretion per mg of protein by cells at 10 mm glucose was three fold higher when compared to the insulin secretion at 4 mm glucose concentration . anthocyanins and anthocyanidins were also tested at 4 and 10 mm glucose loads in the cell growth medium . anthocyanins and anthocyanidins were assayed initially at 50 μg / ml concentration . the anthocyanin , cyanidin 3 - glucoside ( 1 ) showed an increase in insulin secretion at 4 mm glucose by 9 ng / mg of protein ( 1 . 3 fold ) whereas it enhanced the insulin secretion by 1 . 43 fold ( 119 ng / mg protein ) at 10 mm glucose concentration ( fig2 a ). delphinidin - 3 - glucoside ( 2 ) was the most active anthocyanin tested and showed a 1 . 8 - fold increase ( 49 ng / mg of protein ) in insulin secretion at 4 mm glucose concentration . however , at 10 mm glucose it exhibited only a 1 . 4 - fold ( 113 ng ) increase ( fig2 a ) in insulin production . the insulin secreted by cells at 4 and 10 mm glucose concentrations in this assay were 27 and 83 ng of insulin per mg protein , respectively . the anthocyanins , cyanidin - 3 - galactoside ( 3 ) and pelargonidin - 3 - galactoside ( 4 ), did not increase the insulin secretion at 4 mm glucose concentration . however , cyanidin - 3 - galactoside showed an increase of 17 ng / mg of protein of insulin ( 1 . 2 fold ) at 10 mm glucose concentration ( fig3 ). the pelargonidin - 3 - galactoside ( 4 ) was tested only once due to the limitation of sample . the anthocyanin cyanidin - 3 - glucoside ( 1 ) was evaluated for dose dependent insulin secretion at 5 , 10 , 50 , 100 and 250 μg / ml concentrations . the glucose concentration used in this assay was 4 mm level which is representative of the normal glucose level in human ( christison , g . b ., et al ., med . boil . eng . comp . 31 284 - 290 ( 1993 )). at this concentration , untreated cells secreted 33 ng of insulin / mg of protein . the insulin secreted by cyanidin - 3 - glucoside ( 1 ) treated cells was 46 ng of insulin per mg protein at 5 μg / ml . however , there was no significant difference in insulin secretion at 10 , 50 , 100 and 250 μg / ml concentrations of compound 1 . we did not have adequate supply of delphinidin - 3 - glucoside to conduct dose dependent assays . the anthocyanidins were assayed at 50 μg / ml concentration . the aglycone of cyanidin - 3 - glucoside , cyanidin ( 5 ), enhanced insulin secretion by 1 . 5 fold ( 29 ng / mg of protein ) at 4 mm glucose whereas at 10 mm glucose it secreted 88 ng / mg of protein ( fig2 b ). the untreated cells at 4 and 10 mm glucose secreted 19 and 83 ng insulin / mg of protein , respectively , in this set of assay . the aglycone delphinidin ( 6 ) showed an increase in insulin secretion by 6 ng / mg of protein at 4 mm glucose concentration and was not significant . delphinidin did not show glucose - induced insulin secretion at 10 mm glucose ( fig2 b ). pelargonidin was the most active anthocyanidin and it secreted 49 ( 1 . 4 fold ) and 91 ( 1 . 2 fold ) ng of insulin / mg of protein at 4 and 10 mm glucose , respectively ( fig3 ). the aglycone petunidin ( 9 ) increased insulin secretion by 4 ng of insulin / mg protein at 4 mm glucose concentration . however , malvidin ( 8 ) did not show an increase in insulin secretion with respect to the untreated cells . reports indicate that consumption of fruits and vegetables , especially rich in polyphenols , decreased the incidence of type - 2 diabetes ( anderson , r . a ., et al ., j . agric . food chem . 50 7182 - 7186 ( 2002 ); anderson , r . a ., et al ., j . agric . food chem . 52 65 - 70 ( 2004 ); and landrault , n ., et al ., j . agric . food chem . 51 311 - 3188 ( 2003 )). also , it is known that dietary antioxidants protect pancreatic β - cells from glucose - induced oxidative stress . anthocyanins are abundant in fruits , vegetables and processed food products such as wine , cider and tea ; however , little is known of its ability to reduce or prevent diabetes . our results suggested that both anthocyanins and anthocyanidins are insulin secretagogues . the most potent among them was delphinidin - 3 - glucoside and it significantly induced the insulin secretion at 4 and 10 mm glucose concentrations compared to the untreated cells . although cyanidin - 3 - glucoside was less active than delphinidin - 3 - glucoside at lower glucose concentration , it was more active at higher glucose concentration . among the galactosides , pelargonidin - 3 - galactoside did not induce insulin secretion at 4 and 10 mm glucose concentrations studied where as cyanidin - 3 - galactoside showed significant increase in insulin secretion . the ability of anthocyanins studied to secrete insulin was in the increasing order of delphinidin - 3 - glucoside & gt ; cyanidin - 3 - glucoside & gt ; pelargonidin - 3 - galactoside . this indicated that the number of hydroxyl groups in ring - b of anthocyanins played an important role in their ability to secrete insulin . among the anthocyanidins tested , pelargonidin was the most active at 4 mm glucose . other aglycones did not potentiate significant insulin secretion at 4 or 10 mm glucose concentrations studied . this is the first report of insulin secretion by anthocyanins and anthocyanidins when exposed to pancreatic beta cells . our results suggest that cornus fruits , cherries and berries containing these anthocyanins are useful for the prevention of type - 2 diabetes . also , isolated and purified anthocyanins and anthocyanidins from fruits and vegetables may be useful to treat type - 2 diabetes . 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( 23 ) landrault , n . ; poucheret , p . ; azay , j . ; krosniak , m . ; gasc , f . ; lenin , c . ; cros , g . ; teissedre , p . effect of a polyphenols - enriched chardonnay white wine in diabetic rats . j . agric . food chem . 2003 , 51 , 311 - 318 . the methods for the separation of and production of the anthocyanins and anthocyanidins are described in u . s . pat . nos . 6 , 194 , 469 ; 6 , 423 , 365 ; 6 , 623 , 743 ; 6 , 676 , 978 and 6 , 656 , 914 ; and u . s . patent application ser . no . 10 / 084 , 575 , filed feb . 27 , 2002 which are incorporated by reference herein in their entireties . it is intended that the foregoing description be only illustrative of the present invention and that the present invention be limited only by the hereinafter appended claims .