Inhibitory Action on Aldose Reductase by Soybean Flavonoids

Flavonoids that are pres ent in higher plants, mainly le gu mi nous, have aroused in ter est be cause of their in hib i tory ac tion on aldose reductase1,2,3. Aldose reductase ca tal y ses the re duc tion of glu cose and galactose, pro duc ing sorbitol and dulcitol re spec tively, us ing NADH as a co-factor. This en zyme has been found in many an i mal tis sues, such as the ret ina, blood, the cor nea, the sci atic nerve, the liver, kid neys and sperm. The ex ces sive pres ence of sorbitol in di a betic rats and hu mans causes the for ma tion of cat a racts2,5,6. A sim i lar re sult was also ob served by the pres ence of dulcitol in the lenses of rats main tained on a diet rich in galactose. In the lenses of di a betic rats the con tent of sorbitol may be as high as 50 μmol/g, while in the lenses of galactosemic rats, the level of dulcitol can rise to ap prox i mately 80 μmol/g wet weight tis sue4. It has been dem on strated that cat a ract for ma tion ceases un der the sys temic ad min is tra tion of aldose reductase in hib i tors. In or der to solve this prob lem sev eral com pounds have been tested6,8 ,9 ,10, be sides flavonoids1,3. The pur pose of the pres ent study is to iso late, pu rify and iden tify flavonoidic com pounds of the UFV-5’ soy bean’s cultivar, as well as to test their in hib i tory ac tion in aldose reductase in vi tro.

Flavonoids that are pres ent in higher plants, mainly legu mi nous, have aroused in ter est be cause of their in hib i tory ac tion on aldose reductase 1,2,3 .Aldose reductase ca tal y ses the re duc tion of glu cose and galactose, pro duc ing sorbitol and dulcitol re spec tively, us ing NADH as a co-factor 4 .This en zyme has been found in many an i mal tis sues, such as the ret ina, blood, the cor nea, the sci atic nerve, the liver, kidneys and sperm.
The ex ces sive pres ence of sorbitol in di a betic rats and hu mans causes the for ma tion of cat a racts 2,5,6 .A sim i lar result was also ob served by the pres ence of dulcitol in the lenses of rats main tained on a diet rich in galactose 7 .In the lenses of di a betic rats the con tent of sorbitol may be as high as 50 µmol/g, while in the lenses of galactosemic rats, the level of dulcitol can rise to ap prox i mately 80 µmol/g wet weight tis sue 4 .It has been dem on strated that cat a ract for -ma tion ceases un der the sys temic ad min is tra tion of aldose reductase in hib i tors 7,8 .In or der to solve this prob lem several com pounds have been tested 6,8,9,10 , be sides flavonoids 1,3 .The pur pose of the pres ent study is to iso late, pu rify and iden tify flavonoidic com pounds of the UFV-5' soy bean's cultivar, as well as to test their in hib i tory ac tion in aldose reductase in vi tro .

Ma te rial and Methods
The flavonoids kaempherol (I), genistein (II), quercetin (III), naringenin (IV), morin (V), rutin (VI) and quercitrin (VIII) were ex tracted from the UFV-5'soybean's cultivar, fol low ing Hardin's tech nique 11 .The sam ple was heated in a bath-water with ace tic acid for 10 min, and then fil tered.The fil trate was treated with HCl 1 N for one hour, and then ex tracted with an hy drous ethyl ether which was evap o rated giv ing a res i due con tain ing flavonoidic com po nents isolated by chro ma tog ra phy.
Iden ti fi ca tion of the flavonoids was made by UV, NMR and IR spec tros copy tech niques and com par i son with authen tic sam ples avail able in our lab o ra tory.The ver i fi cation of the in hib i tory ac tion of aldose reductase in volved Shimizu's tech nique 1 at room tem per a ture, by us ing a phos phate buffer so lu tion (0.01 M; pH 6.2; 0.014 mM of NADPH, 10 mM of glu cose or galactose and 25 µL of aldose reductase with the ad di tion of H2O to a vol ume of 1.5 mL).The con trol test con tained all the com pounds, except ing the glu cose or galactose.
The re ac tion was ini ti ated by the ad di tion of aldose reductase, and the rate of ox i da tion from NADPH to NADP was ob served by the de crease in the absorbancy at 340 nm 1 .
The flavonoids were pre pared in con cen tra tions of 10 -4 M, 10 -5 M, 10 -6 M, and 10 -7 M and were dis solved in wa ter with the ad di tion of NaOH, the pH was ad justed to 7.0 and then added to the test mix ture.

Re sults and Dis cus sion
The in hib i tory ac tion of flavonoids on aldose reductase (D-mannitol-l-phos phate-dehydrogenase of E. coli) in differ ent con cen tra tions of flavonoids (Ta ble 1), dem on strates that a con cen tra tion of 10 -4 M is the best pro moter of the inhib i tory ac tion of the en zyme.On the other hand, it also con firms that the flavonoids morin and quercitrin presented higher in hib i tory ac tion rel a tive to the en zyme.Data on the Ta ble 4 shows that at the same con cen tra tion of 10 -5 M, the best re sults were ob tained from the flavonoids rutin, quercitrin, and naringenin.These re sults are in agree ment with those ob tained by Varma 12 for quercitrin, ac e tate of 2-quercitril and quercetin.
It is known that en zymes as aldose reductase re acts with sub strate as glu cose or galactose with the for ma tion of a complexe.Ac cord ing to these re sults it can be seen the most pref er ence of the en zyme for re act ing with flavonoids con tain ing sugar unit in its struc ture.The high est porcentage of in hi bi tion ob tained for quercitrin 97,66% is in agree ment with that pos si bil ity.
From the anal y sis of vari ance (Ta ble 2) a sig nif i cant inter ac tion be tween flavonoids and con cen tra tion can be seen.

Con clu sions
From the UFV-5'cultivar of Glycine max (soya) it was pos si ble to iso late and iden tify the flavonoids kaempherol, genistein, quercetin, naringenin, morin, rutin and quercitrin.The iden ti fi ca tion was achieved by UV, IV and NMR spec tro scopic stud ies.At a con cen tra tion of 10 -4 M, the flavonoids morin and quercitrin were those which demon strated higher in hib i tory ac tion.On the other hand, at a con cen tra tion of 10 -5 M, the best re sults were achieved by the flavonoids naringenin, rutin, and quercetrin.
These re sults showed the great pos si bil ity to use, in the fu ture, the soy bean as pre ven tion of cat a ract in foods or by us ing these com pounds as so ci ated to the med i cines, in order to avoid the cat a ract for ma tion.
Ta ble 1.In hib i tory ef fect of flavonoids at dif fer ent con cen tra tions on aldose reductase.

Com pounds
Con cen tra tions 13e val ues fol lowed by at least one equal let ter do not dif fer sta tis tically by Tukey's Test (5% prob a bil ity level).#-Average val ues of % in hi bi tion of flavonoids on aldose reductase.Ta ble 2. Anal y sis of Vari ance13.Sig nif i cant at level of 5% of prob a bil ity.