Inhibitory Action on Aldose Reductase by Soybean Flavonoids

Arti cl e

Tânia Toledo de Oliveira a, Tanus Jorge Nagem b, Luiz Carlos Guedes de Miranda a, Vanderlúcia Fonseca de Paula c, and Marco Antônio Teixeira d

aDepartamento de Bioquímica e Biologia Molec u lar, Universidade Federal de Viçosa, Av PH Rolphs s/n 36571-000 Viçosa - MG, Brazil bDepartamento de Química, Universidade Federal de Ouro Preto, Campus Morro do Cruzeiro, 35400-000 Ouro Preto - MG, Brazil cDepartamento de Química,Universidade Federal de Viçosa, Av. PH Rolphs s/n 36571-000 Viçosa - MG, Brazil dDepartamento de Química da Universidade Federal de Minas Gerais, Pampulha, Cidade Universitária, 30 270-010 Belo Horizonte - MG, Brazil

Received: 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

mation ceases under the systemic admin is tra tion of aldose In tro duc tion reductase inhib i tor s 7,8 . In order to solve this problem sev - Flavonoids that are present in higher plants, mainly le - eral com pounds have been tested 6,8,9,10 , be sides gumi nous, have aroused inter est because of their in hib i tory flavonoids 1,3 . The purpose of the present study is to isolate , action on aldose reductase 1,2,3 . Aldose reductase catal y se s purify and identify flavonoidic compounds of the UFV-5’ the reduc tion of glucose and galactose, produc ing sorbitol soybean’s cultivar, as well as to test their inhib i tory actio n and dulcitol respec tively, using NADH as a co-factor 4. This in aldose reductase in vitr o . enzyme has been found in many ani mal tissues, such as the retina, blood, the cornea, the sciatic nerve, the liver, kid - Ma te rial and Methods neys and sperm. The flavonoids kaempherol (I), genistein (II), quercetin The exces sive presence of sorbitol in dia betic rats and (III), naringenin (IV), morin (V), rutin (VI) and quercitrin 2,5,6 humans causes the forma tion of cata ract s . A simi lar re - (VIII) were extracted from the UFV-5’soybean’s cultivar, sult was also observed by the presence of dulcitol in the follow ing Hardin’s techniqu e 11 . The sample was heated in 7 lenses of rats maintained on a diet rich in galactose . In the a bath-water with ace tic acid for 10 min, and then filtered . lenses of dia betic rats the con tent of sorbitol may be as high The filtrate was treated with HCl 1 N for one hour, and then as 50 mmol/g, while in the lenses of galactosemic rats, the extracted with anhy drous ethyl ether which was evap o rated level of dulcitol can rise to approx i mately 80 mmol/g wet giving a resi due contain ing flavonoidic compo nents iso - weight tissu e 4. It has been demon strated that cata ract for - lated by chroma tog ra phy . 212 Oliveira et al. J. Braz. Chem. Soc.

Identi fi ca tion of the flavonoids was made by UV, NMR Ta ble 2. Anal y sis of Vari ance 13 . and IR spectros copy techniques and compar i son with au - thentic samples available in our labo ra tory. The ver i fi ca- free grade mean square tion of the inhib i tory action of aldose reductase involve d Flavonoid 6 1777.43* 1 Shimizu’s techniqu e at room temper a ture, by using a Concen tra tio n 3 18966.14* phosphate buffer solu tion (0.01 M; pH 6.2; 0.014 mM of Flav x Concen tra tio n 18 251.87* NADPH, 10 mM of glucose or galactose and 25 mL of Resi du e 56 1.6093 aldose reductase with the addi tion of H 2O to a volume of 1.5 mL). The control test contained all the compounds, ex - *Sig nif i cant at level of 5% of prob a bil ity. cepting the glucose or galactose. The reac tion was initi ated by the addi tion of aldose con tain ing sugar unit in its struc ture. The high est reductase, and the rate of oxi da tion from NADPH to NADP porcentage of inhi bi tion obtained for quercitrin 97,66% is was observed by the decrease in the absorbancy at 340 nm 1. in agreement with that possi bil ity . The flavonoids were prepared in concen tra tions of 10 -4 From the analy sis of variance (Table 2) a signif i cant in - M, 10 -5 M, 10 -6 M, and 10 -7 M and were dissolved in wate r terac tion between flavonoids and concen tra tion can be with the addi tion of NaOH, the pH was adjusted to 7.0 and seen. then added to the test mixture . Re sults and Dis cus sion Con clu sions The inhib i tory action of flavonoids on aldose reductase From the UFV-5’cultivar of Glycine max (soya) it was (D-mannitol-l- phosphate-dehydrogenase of E. coli ) in dif - possi ble to isolate and identify the flavonoids kaempherol, ferent concen tra tions of flavonoids (Table 1), dem on strates genistein, quercetin, naringenin, morin, rutin and -4 quercitrin. The identi fi ca tion was achieved by UV, IV and that a concen tra tion of 10 M is the best pro moter of the in - -4 hibi tory action of the enzyme. On the other hand, it also NMR spectro scopic studies. At a concen tra tion of 10 M, confirms that the flavonoids morin and quercitrin pre - the flavonoids morin and quercitrin were those which dem - onstrated higher inhib i tory action. On the other hand, at a sented higher inhib i tory action rela tive to the enzyme. Data -5 on the Table 4 shows that at the same concen tra tion of 10 -5 concen tra tion of 10 M, the best results were achieved by M, the best results were obtained from the flavonoids rutin, the flavonoids naringenin, rutin, and quercetrin. quercitrin, and naringenin. These results are in agreemen t These results showed the great possi bil ity to use, in the with those obtained by Varma 12 for quercitrin, ace tate of future, the soybean as preven tion of cata ract in foods or by 2-quercitril and quercetin. using these compounds asso ci ated to the medi cines, in or - It is known that enzymes as aldose reductase reacts with der to avoid the cata ract forma tion . substrate as glucose or galactose with the forma tion of a Ref er ences complexe. Accord ing to these results it can be seen the most prefer ence of the enzyme for react ing with flavonoids 1. Shimizu, M.; Terashima, I.S.; Hayashi, T.; Arisawa, M.; Morita, N.; Kurokawa, S.K.K.; Ito; Hashimoto, Y. Phytochemistry 1984 , 9, 1885. Ta ble 1. In hib i tory ef fect of flavonoids at dif fer ent con cen tra tions on 2. Varma, S.D.; Kinoschita, J.H. Biochem. Pharmacol. aldose reductase. 1976 , 25 , 2505. Compound s Concen tra tion s 3. Chaudry, P.S.; Cabrera, J.; Juliani, H.R.; Varma, S.D. Biochem. Pharmacol . 1983 , 32 , 1995. 10 -4 M 10 -5 M 10 -6 M 10 -7 M 4. Invest. Ophtalmol & Vis Sci. 1965 D D E C Kinoschita, J.H. , Kaempherol 80.33 *# 60.33 40.33 7.00 4, 786. Genistein 48.66 F 28.66 E 18.66 F 0.00 D 5. Heyningen, R.Van. Na ture 1959 , 184 , 194. Quercetin 83.66 C 63.66 C 44.33 D 30.66 A 6. Lerner, B.C.; Varma, S.D.; Richards, R.D. Arch. Naringenin 75.66 E 74.66 A 54.66 A 21.66 B Ophthalmol . 1984 , 102 , 917. 7. Varma, S.D.; Kinoschita, J.H. Documenta Morin 96.66 A 67.66 B 47.66 C 0.00 D Ophtalmol 1976 8 A A AB D . , , 305. Rutin 91.66 71.66 51.66 0.00 8. Dvornick, D.; Simard-Duquesne, N.; Krami, M.; Quercitrin 97.66 A 71.66 A 51.66 A 20.66 AB Sestanj, K.; Gabbay, K.H.; Kinoschita, J.H.; Varma, * The val ues fol lowed by at least one equal letter do not dif fer sta tis ti- S.D.; Merola, L.O. Scienc e 1973 , 182 , 1146. cally by Tukey’s Test (5% prob a bil ity level). 9. Varma, S.D.; Shocket, S.S.; Richards, R.D. Invest . #-Av er age val ues of % in hi bi tion of flavonoids on aldose reductase. Ophtalmol. & Vis. Sci. 1979 , 18 , 237. Vol. 8, No. 3, 1997 Inhib i tory Action in Aldose-Reductase by Soybean Flavonoids 213

10. Varma, S.D.; El-Aguizy, H.K.; Richards, R.D. Acta 12.Varma, S.D. Pharmacol. Struct. Activ. Relat.1986, 1, Ophthalmol. 1980 , 58 , 748. 343. 11. Hardin, J.M.; Stutte, C.A. Anal. Biochem . 1980 , 102 , 13. Gomes, F.P. In Curso de Estatística Exper i men ta l . 6 a 171. Ed. Piracicaba, Livraria Nobel, 1976, p 430.