Inhibitory Action on Aldose Reductase by Soybean Flavonoids
Tânia To ledo de Oliveiraa, Tanus Jorge Nagemb, Luiz Carlos Guedes de Mirandaa, Vanderlúcia Fonseca de Paulac, and Marco Antônio Teixeirad
a
Departamento de Bioquímica e Biologia Mo lec u lar, Universidade Fed eral de Viçosa,
Av PH Rolphs s/n 36571-000 Viçosa - MG, Brazil b
Departamento de Química, Universidade Fed eral de Ouro Preto, Cam pus Morro do
Cru zeiro, 35400-000 Ouro Preto - MG, Brazil c
Departamento de Química,Universidade Fed eral de Viçosa, Av. PH Rolphs s/n
36571-000 Viçosa - MG, Brazil d
Departamento de Química da Universidade Fed eral de Minas Gerais, Pampulha,
Cidade Universitária, 30 270-010 Belo Horizonte - MG, Brazil
Re ceived: April 28, 1995
Os flavonóides kaempherol, genisteína, naringenina, quercetina, morina, rutina, e quercitrina isolados do cultivar de soja UFV-5’ foram testados como inibidores de aldose redutase. Os melhores resultados foram obtidos usando morina e quercitrina.
The flavonoids kaempherol, genistein, naringenin, quercetin, morin, rutin and quercitrin iso lated from UFV-5’soybean’s cultivars were tested as in hib i tors of aldose reductase. The best re sults were ob tained by us ing morin and quercitrin.
Key words:flavonoids, soy bean, aldose reductase
In tro duc tion
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-factor4. 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 galactose7. 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 tors7,8. In or der to solve this prob lem sev -eral com pounds have been tested6 , 8 , 9 , 1 0, 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.
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 nique11. 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 iso -lated by chro ma tog ra phy.
Ar ti cle
J. Braz. Chem. Soc., Vol. 8, No. 3, 211-213, 1997. © 1997 Soc. Bras. Química
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 au then tic sam ples avail able in our lab o ra tory. The ver i fi ca -tion of the in hib i tory ac -tion of aldose reductase in volved Shimizu’s tech nique1 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, ex -cept 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 nm1. 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 dif -fer 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 in -hib i tory ac tion of the en zyme. On the other hand, it also con firms that the flavonoids morin and quercitrin pre -sented 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 Varma12 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 in -ter 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 dem -on strated higher in hib i tory ac ti-on. 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 or -der to avoid the cat a ract for ma tion.
Ref er ences
1. Shimizu, M.; Terashima, I.S.; Hayashi, T.; Arisawa, M.; Morita, N.; Kurokawa, S.K.K.; Ito; Hashimoto, Y. Phytochemistry 1984, 9,1885.
2. Varma, S.D.; Kinoschita, J.H. Biochem. Pharmacol. 1976, 25, 2505.
3. Chaudry, P.S.; Cabrera, J.; Juliani, H.R.; Varma, S.D. Biochem. Pharmacol . 1983, 32, 1995.
4. Kinoschita, J.H. In vest. Ophtalmol & Vis Sci. 1965,
4, 786.
5. Heyningen, R.Van. Na ture 1959, 184, 194. 6. Lerner, B.C.; Varma, S.D.; Rich ards, R.D. Arch.
Ophthalmol. 1984, 102, 917.
7. Varma, S.D.; Kinoschita, J.H. Documenta Ophtalmol. 1976, 8, 305.
8. Dvornick, D.; Simard-Duquesne, N.; Krami, M.; Sestanj, K.; Gabbay, K.H.; Kinoschita, J.H.; Varma, S.D.; Merola, L.O. Sci ence1973, 182, 1146. 9. Varma, S.D.; Shocket, S.S.; Rich ards, R.D. In vest.
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212 Oliveira et al. J. Braz. Chem. Soc.
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
10-4 M 10-5M 10-6 M 10-7M
Kaempherol 80.33 D *# 60.33 D 40.33 E 7.00 C
Genistein 48.66 F 28.66E 18.66 F 0.00 D
Quercetin 83.66 C 63.66 C 44.33 D 30.66 A
Naringenin 75.66 E 74.66 A 54.66 A 21.66 B
Morin 96.66 A 67.66 B 47.66 C 0.00 D
Rutin 91.66 A 71.66 A 51.66 AB 0.00 D
Quercitrin 97.66 A 71.66 A 51.66A 20.66 AB
* The val ues fol lowed by at least one equal let ter do not dif fer sta tis ti -cally by Tukey’s Test (5% prob a bil ity level).
#-Av er age val ues of % in hi bi tion of flavonoids on aldose reductase.
Ta ble 2. Anal y sis of Vari ance13.
free grade mean square
Flavonoid 6 1777.43*
Con cen tra tion 3 18966.14*
Flav x Con cen tra tion 18 251.87*
Res i due 56 1.6093
10. Varma, S.D.; El-Aguizy, H.K.; Rich ards, R.D. Acta Ophthalmol. 1980, 58, 748.
11. Hardin, J.M.; Stutte, C.A. Anal. Biochem. 1980, 102, 171.
12.Varma, S.D. Pharmacol. Struct. Activ. Relat.1986, 1,
343.
13. Gomes, F.P. In Curso de Estatística Ex per i men tal . 6a Ed. Piracicaba, Livraria No bel, 1976, p 430.
