Biological activities of broad bean extracts cultivated in south Anatolia in favism sensitive subjects

Vural N., Sardas S.: Biological activities of broan bean (Vicia faba L.) extracts cultivated in south Anatolia in favism sensitive subjects. Toxicology, 31, 175-179, 1984.

Toxicology, 31 (1984) 175—179 Elsevier Scientific Publishers Ireland Ltd. Short Communication BIOLOGICAL ACTIVITIES OF BROAD BEAN (VICIA FABA L.) EXTRACTS CULTIVATED IN SOUTH ANATOLIA IN FAVISM SENSITIVE SUBJECTS* NEVIN VURAL? and SEMRA SARDAS? 4 Department of Toxicology, Ankara University, Faculty of Pharmacy, Ankara (Turkey) and b Department of Toxicology, Gazi University, Faculty of Pharmacy, Ankara (Turkey) (Received April 11th, 1983) (Accepted June 23rd, 1983) SUMMARY Aqueous extracts of a different variety of fresh broad bean seeds obtained from a favism endemic area in Turkey, were incubated with blood from sensitive and non-sensitive (control) subjects. Red blood cells were characterized by a whole blood glutathione (GSH) and a deficiency of Glucose- 6-phosphate dehydrogenase (G-6-PD) activity. As the decrease in GSH percent is taken as an index of haemolytic activity, the test results were as following: Sakiz, Milas-Region, French broad bean extracts reduced the blood GSH levels 48%, 70%, 46% and 53%, respectively, in favism sensitive subjects. Active principles which are responsible for the haemolysis (Vicine and Convicine) were isolated from broad beans and their effects on GSH levels of blood were 99% and 81%, respectively, in favism sensitive subjects and 33.3% and 19% in normal subjects. Key words: Favism; Glucose 6-phosphate dehydrogenase deficiency; Glutathione; Vicia faba; Vicine-Convicine; South Anatolia Favism, a remarkable type of acute haemolytic anemia developing as a result of ingestion of broad bean (Vicia faba) seeds and in some instances as a result of inhalation of Vicia faba pollen, is associated with glucose-6*This research was performed in partial fulfillment of the requirements for the degree of Doctor of Pharmacy by S. Sardas, Department of Toxicology, Ankara University of Pharmacy, Ankara, Turkey. Address correspondence to: Semra Sardas, Ph.D., Department of Toxicology, Gazi University-Faculty of Pharmacy, Etiler, Ankara, Turkey. 0300-483X/84/$03.00 © 1984 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

phosphate dehydrogenase (G-6-PD) deficiency and instability of glutathione. The incidence of this enzymatic defect is significantly higher in the Mediterranean people, particularly among Greeks, Jews, Sicilians, Sardinians and Arabs [1,2] Some investigators [3,4] suggested that pyrimidine derivatives iso-uramil (2,4,5-trihydroxy-6-amino pyrimidine) and divicine (2,6-diamino 4,5dihydroxy pyrimidine) occurring in the fava beans, as the aglycone moieties or their may corresponding ß-glycosides (convicine and vicine, respectively), be responsible for the induction of the haemolytic crisis known as favism [3,4]. The haemolytic activity was determined by the reducing effect of the extracts and toxic constituents of Vicia faba on G-6-PD deficient blood GSH levels [5,6]. Some investigations have been done on the incidence of G-6-PD deficiency and favism in the Turkish population, [7,8] but no research has been carried out to determine the relationship of broad bean to favism although consumption and cultivation is fairly high in Turkey. This present communication reports the investigation on the in vitro haemolytic activity of some varieties of Vicia faba extracts and pyrimidine glycosides isolated from broad beans cultivated in the South part of Turkey. MATERIALS AND METHODS Fresh broad bean varieties (Grex minor Milas 114, Grex major Sakiz 115, Grex equina Region 5108 and Grex equina French 116) cultivated in Turkey were obtained from the Mediterranean Institute of Agricultural Research, Antalya, Turkey. Blood specimens from 30 favism subjects and 20 normal (control) persons were collected in an acid-citrate-dextrose (ACD) solution and kept in a refrigerator. Favism subjects were traced by the help of Hematology Research Laboratories of the Childrens Hospital, Hacettepe University. GSH tests were performed, as described by Beutler, Duron and Kelly [9], and were based upon the development of a relatively stable colour when 5,5'-dithiobis (2-nitrobenzoic acid) was added to sulfhydryl compounds. G-6-PD was determined by specific procedures according to Beutler [10]. Before the quantitative determination, G-6-PD deficiency was screened by a rapid spot test. This method depends upon the fluorescence of reduced NADP (NADPH) as an indicator of G-6-PD activity. Extraction of broad bean samples A different variety of broad bean was extracted as described by Jamalian [11]. The aqueous extracts, obtained using 250 g of fresh seeds, were evaporated and the final residues were dissolved and taken up in 10.0 ml phosphate-buffered physiological saline (pH 7.4). They were stored frozen under N, in screw-cap sample bottles. Haemolytic activity of extracts Hematocrit (Hct) values of blood samples were measured by the micro-

haematocrit procedure, described by Dacie and Lewis [12], and adjusted to have a Hct value of 30% ACD solution before incubation. One millilitre of blood sample was incubated (at 37°C for 3 h) with 0.5 ml extract solution in buffered physiological saline (20% total extract) in 5-ml rubber stoppered glass tubes. One millilitre of blood incubated with buffered physiological saline was used as an internal control for each subject. Blanks were prepared by mixing 0.5 ml of each extract solution with 1 ml of buffered physiological saline and were incubated simultaneously. At the end of 3 h GSH was determined in duplicate [9]. Isolation of toxic principles Vicine was isolated from dry seeds [13] and convicine was isolated from fresh seeds of the Milas variety [14]. Physical and chemical properties, UV, IR and NMR spectras and elementary analysis of isolated substances were determined and compared with standards according to Bien [14]. Solutions of vicine and convicine crystals were prepared in buffered physiological saline (100 mg/10 ml) and 0.5 ml of the solution was used for the incubation. RESULTS AND DISCUSSION 1. The effects of different varieties of fresh broad beans on the blood GSH levels is shown in Table I. All the favism subjects tested have shown G-6-PD deficiency (0 units/g Hb). It was noted that each of the extracts decreased the level of GSH in favism subjects to a greater extent than in the normal control. Using mean values of Table I, the following can be calculated; Sakiz and Milas broad bean extracts decreased the blood GSH levels in normal subjects by 15% and 18.5% and in favism subjects by 48% and 70%, respectively. It was observed that the Milas variety has the greatest effect (Table 1). TABLE I EFFECTS OF VICIA FABA EXTRACTS ON BLOOD GSH LEVELS OF SENSITIVE AND NON-SENSITIVE SUBJECTS GSH in mg/100 m! red blood cells + standard deviation. Groups No. of Treatment Mean GSH subjects Before increase After increase Non-sensitive 20 Milas extract 108.535 + 19.404 88.315 + 19.675 Non-sensitive 20 Sakiz extract 108.535 + 19.404 92.275 + 20.645 Sensitive 80 Milas extract 58.913 + 17.792 17.757 + 12.602 Sensitive Sensitive Sensitive 30 12 12 Sakiz extract Bölge extract Fransiz extract 58.913 + 17.792 53.583 + 16.232 53.583 + 16.232 30.620 + 13.566 24.817 + 7.391 28.533 + 8.824

FABLE Il EFFECTS OF VICINE AND CONVICINE PYRIMIDINE GLUCOSIDES ON GSH LEVELS OF SENSITIVE AND NON-SENSITIVE SUBJECTS GSH in mg/100 ml red blood cells + standard deviation. Groups No. of Treatment Mean GSH subjects Before increase After increase Non-sensitive 10 Vicine 81.250 + 24.848 54.310 + 23.248 Non-sensitive 10 Convicine 81.250 + 24.848 65.930 + 25.481 Sensitive 10 Vicine 46.970 + 21.858 2.090 + 3497 Sensitive 10 Convicine 46.970 + 21.858 8.730 + 4.819 The extracts other than the Milas variety had no statistically significant difference on the fall of the GSH level (Table III). Some investigators such as Walker and Bowman [5], Bottini et al. [6] and Jamalian [14] reported that aqueous extracts of broad bean reduced the blood GSH levels between 27% and 91% (average 60%) in favism sensitive subjects. 2. The effect of isolated pyrimidine glycosides vicine and convicine on blood GSH levels of sensitive and non-sensitive subjects is shown in Table Il. Vicine and convicine reduced the GSH levels in normal subjects by 33% and 19% and in favism subjects by 99% and 81%, respectively. The yield was 9.6 vicine from 1 kg dry seed Milas variety and 0.6 g convicine from 1 kg Milas variety fresh Faba beans. Statistical results are shown in Table Ill. The haemolytic. effect of vicine has been found more potent than convicine (P < 0.01). Vicine content of some varieties of broad bean was reported earlier by TABLE IN EFFECTS OF VARIOUS BROAD BEAN EXTRACTS, VICINE AND CONVICINE GLYCOSIDES ON BLOOD GLUTATHIONE LEVELS (Chi-square test, No, 12) No statistically significant difference (P > 0.05) was observed, P < 0.001 was found to be significant. Comparison of various X t P extract types Bolge-Milas Bölge-Sakiz Bölge-Fransiz Milas-Sakiz Milas-Fransiz Sakiz-Fransiz 24.817 24.817 24.817 14.750 14.750 26.375 14.750 26.375 28.535 26.375 28.533 28.533 5.508 0.590 1.119 7.752 6.527 0.955 <0.001 >0.05 >0.05 <0.001 <0.001 > 0.05 Convicine-Vicine 8.730 2.090

Lin and Ling [3] and Jamalian [13]. Jamalian reported that vicine concentration of Vicia faba can change due to the variety, culture area, different parts of pod and pod development [15]. There are some investigations on the isolation methods, characterisation and effect of reduction on GSH levels [4,14,16] dealing with convicine but we could not find any research on its comparative haemolytic activity. The toxic constituents vicine and convicine of Vicia faba seem mainly to be the responsible active principles in the haemolytic activity although some other factors such as L-DOPA may also be effective [16]. Our results, until now, confirm and amplify previous reports and serve to indicate the degree of variability in toxicity that occur in broad beans. REFERENCES 1 E. Sartori, On the pathogenesis of favism, J. Med. Genet., 8 (1971) 462. 2 C.A Kattamis, M. Kyriazakou, S. Chaidas, Favism clinical and biochemical data. J. Med. Genet. 6 (1969) 34. 3 J.Y. Lin and K. Ling, Isolation of an active principle from Faba beans (Vicia faba). J. Formoson Med. Assoc., 61 (1962) 484. 4 J. Mager, G Glaser, A. Razin, G. Izak, S. Bién and M. Noam, Metabolic effects of pyrimidines derived from Fava bean glycosides on human erythrocytes deficient in glucose-6-phosphate dehydrogenase. Biochem. Biophys. Res. Commun., 20 (1965) 235. 5 D.G. Walker and J.E. Bowman, In vitro effect of Vicia faba extracts upon reduced glutathione of erythrocytes. Proc. Soc. Exp. Biol. Med., 103 (1960) 476. 6 E. Bottini, P Lucarelli, G.F. Spennati, L. Besinco and R. Palmarino, Presence in Vicia faba, of different substances with activity in vitro on Gd(—)Med red blood cell glutathione. Clin. Chim. Acta, 30 (1970) 831. 7 B Say, P. Ozand, J. Berkel and N. Cevik, Erythrocyte glucose-6-phosphate dehydrogenase deficiency in Turkey. Acta Pediatr, Scand., 54 (1965) 319. 8 H. Sipahioglu, 8 favizm vakasi münasebeti ile G-6-PD enzimi noksan hayvanlarda 9 E. Beutler, O. Duron and M.K. Kelly, Improved method for the determination of tecrübi favizm arastirilmasi. Turk Tip Cem, Mec., 35 (1969) 376. blood glutathione. J. Lab. Clin. Med., 61 (1963) 882. Beutler, A series of new screening procedures for pyruvate kinase deficiency, glucose-6-phosphate dehydrogenase deficiency and glutathione reductase deficiency. 10 E. Blood, 28 (1966) 553. 11 J. Jamalian, (Vicia faba). F. Alyward, B.J.F. Hudson, Favism-inducing toxins in broad beans Examination of bean extracts for pyrimidine glycosides. Anal. Plan.- PI.Fd s Hum. Nutr. XXVI, 4 (1976) 331. 12 J.V. Dacie, The Haemolytic Anaemias, J. and A. Churchill Ltd. London, 2nd Edition, Part IV, 1967, p. 1060. 13 J. Jamalian, (Vicia faba), F. Alyward and B.J. Hudson, Favism-inducing toxins in broad beans Estimation of the vicine contents of broad bean and other legume samples. Qual. Plant. Pl. Fds. Hum. Nutr. XXVII, 2 (1977) 207. 14 S. Bien, G. Salemnik, L. Zamir and M. Rosenbaum, The structure of convicine. J. Chem. Soc. (C) (1968) 496. 15 J. Jamalian, A. Bassiri, Variation in vicine concentration during pod development in broad beans (Vicia faba L.). J. Agric. Food. Chem., 26 (1978) 1454. 16 A.B. Engel, Detection of the toxic factors in broad beans (Vicia faba), Progress Report I, Rapport Nr (R 3195) (Central Institute for Nutrition and Food Research, Utrechtseweg 48, Zeist) World Health Organization of U.N. Geneva, Switzerland,