Archive of SIDMedical Laboratory Journal, Jan-Feb , 2019; Vol 13: No 1 Original Article E-ISSN: 2538-4449 Protective Effect of Ilex spinigera and Gleditsia caspica Extracts against Drug-Induced Hemolysis in Glucose-6-phosphate dehydrogenase-deficient Patients Maryam Mohadjerani (PhD) ABSTRACT Department of Molecular and Cell Background and Objectives: Glucose-6-phosphate dehydrogenase (G6PD) Biology, Faculty of Basic Sciences, University of Mazandaran, Babolsar, deficiency is the most common X-linked disorder of human erythrocytes in which cells are Iran susceptible towards hemolytic changes and could be destroyed by peroxides. Extracts of Ilex Mansoore Damanjany (MSc) spinigera and Gleditsia caspica leaves have excellent free radical scavenging activity. We Department of Molecular and Cell Biology, Faculty of Basic Sciences, investigated the protective effects of these extracts against hemolysis induced by some drugs University of Mazandaran, Babolsar, in G6PD-deficient erythrocytes. Iran Methods: Blood samples were collected from males with and without G6PD Corresponding author: Maryam Mohadjerani deficiency. Hemolysis induced by aspirin, phenylhydrazine hydrochloride and phenacetin Email: [email protected] was assessed in the presence and absence of the extracts. The amount of released Tel: +98-1135302455 hemoglobin was determined by reading absorbance at 540 nm. Address: Faculty of Basic Sciences, Results: The methanol extract of G. caspica had significant protective effects against University of Mazandaran, Babolsar, Iran phenacetin-induced hemolysis in G6PD-deficient human erythrocytes (P<0.05). However, the Received: 05 May 2018 I. spinigera extract had no significant anti-hemolytic effects on these cells. Revised: 05 Sep 2018 Conclusion: Our findings suggest that the extract of G. caspica could be a potential Accepted: 25 Sep 2018 drug with antioxidant and anti-hemolytic properties for patients with G6PD deficiency. Keywords: Antihemolytic activity, Medicinal plant, G6PD deficiency, Gleditsia caspica, Ilex spinigera. This work is licensed under a Creative Commons Attribution 4.0 License. Downloaded from goums.ac.ir at 10:40 +0330 on Monday February 4th 2019 [ DOI: 10.29252/mlj.13.1.21 ] This paper should be cited as: Mohadjerani M, Damanjany M [Protective Effect of Ilex spinigera and Gleditsia caspica Extracts against Drug-Induced Hemolysis in Glucose-6-phosphate dehydrogenase-deficient Patients]. mljgoums. 2019; 13(1):21-27 www.SID.ir Archive of SID 22/ Protective Effect of Ilex. INTRODUCTION Glucose-6-phosphate dehydrogenase against radical-mediated toxicity and free (G6PD, EC: 1.1.1.49) is an important and radical-induced damage (8). According to highly conserved enzyme in the pentose Flora Iranica, Ilex spinigera and Gleditsia phosphate metabolic pathway (1). It catalyzes caspica are plants native to the forests of the simultaneous oxidation of glucose-6- northern Iran, particularly in Guilan, Golestan, phosphate to 6-phosphogluconate and Mazandaran and Azerbaijan Provinces (9). reduction of NADP+ to NADPH. This process These two plants have numerous antioxidant, leads to the conversion of oxidized glutathione biological and therapeutic properties (10, 11). to reduced glutathione (GSH), which protects The present study investigates effects of I. human cells from oxidative damage (2). spinigera and G. caspica extracts on A free radical is any molecular species capable hemolysis induced by aspirin, phenacetin and of independent existence that contains one or phenylhydrazine hydrochloride in G6PD- more unpaired electrons. The oxygen radicals deficient human erythrocytes. and related species, singlet oxygen, superoxide radicals and hydrogen peroxide are known as MATERIALS AND METHODS reactive oxygen species (ROS). Imbalance Leaves of I. spinigera and G. caspica between ROS and antioxidants causes were collected from Mazandaran forests. The oxidative stress, which can lead to cell damage specimens of I. spinigera (voucher Nr.4034) (3). Oxidative stress could be due to increased and G. caspica (voucher Nr. 5502) were ROS production, and toxicity caused by drugs obtained from the Herbarium of University of (e.g. trimethoprim, sulfamethoxazole, dapsone, Mazandaran. The powdered plants samples (20 primaquine, etc.), acute infection (acute grams) were extracted using a method hepatitis A, hepatitis B, cytomegalovirus, described in our previous work (10). pneumonia, etc.), or some foods (fava beans) Total phenolic and flavonoid content of each can also cause G6PD deficiency-related extract were assessed using the Folin– hemolysis (4). Ciocalteu phenol and aluminum chloride Primaquine is the only drug currently licensed colorimetric methods (12, 13). All chemicals for the radical treatment of Plasmodium vivax used in the study were of analytical grade and malaria. However, it can cause severe purchased from Fluka, Sigma and Merck Co. hemolysis in individuals with G6PD All kits were obtained from Kimia Pajouhan deficiency, a common genetic disorder that is Co., Iran. We randomly collected 315 blood positively associated with P. vivax incidence. samples from men in Babolsar. G6PD G6PD deficiency is largely asymptomatic until deficiency was screened in the samples using individuals are exposed to external sources of the fluorescent spot test (Kimia Pajouhan kits) oxidative stress, including certain nutrients and (14). To determine the concentration of the drugs, but most notably fava beans (5). GSH is drugs that could produce approximately 50% a major antioxidant that protects cells against hemolysis in the G6PD-deficient erythrocytes, oxidative damage (6). suspension aliquots were incubated with A proposed mechanism for the lack of varying amounts of the drugs (0 to 4 mM) resistance of G6PD-deficient RBCs to (15). The percentage of hemolysis was Downloaded from goums.ac.ir at 10:40 +0330 on Monday February 4th 2019 [ DOI: 10.29252/mlj.13.1.21 ] oxidative stress is shown in figure 1. The calculated by dividing the absorbance reading interaction of drugs with RBCs leads to (at 540 nm) from the experimental tubes by the hydrogen peroxide production, either directly mean value of complete hemolysis induced by or through ROS. GSH can remove the distilled water multiplied by 100. Data were produced hydrogen peroxide in the presence of expressed as means ± standard deviation. All glutathione peroxidase. The produced oxidized measurements were replicated three times. glutathione will be reduced to regenerate GSH Half-maximal inhibitory concentration (IC50) through the activity of glutathione reductase. values were calculated via linear regression Then, continuous reduction of NADP to analysis. One-way analysis of variance NADPH relies on the G6PD activity (7). (ANOVA) and Tukey’s post-test were carried Antioxidants, flavonoids and phenolic out for comparison of data. P-values less than compounds act as a major defense system 0.05 were considered statistically significant. www.SID.ir Medical Laboratory Journal Jan-Feb, 2019; Vol 13: No 1 Archive of SID 23/ Mohadjerani and Damanjany Figure 1. A plausible series of reactions that could explain the lack of resistance of G6PD-deficient red blood cells (RBCs) to oxidative stress (7) RESULTS (0.459±0.007 mg quercetin equivalents per The extraction yield and total phenolic gram of dried leaves). and flavonoid content of the extracts are Our results revealed that the frequency of reported in table 1. G6PD deficiency is 15% among males in The total phenolic content was higher in the Babolsar, Iran. Figures 2-4 show that G6PD- methanol extract of G. caspica (0.123 ± 0.003 deficient and normal human erythrocytes mg gallic acid equivalents per gram of dried undergo hemolysis when exposed to aspirin, leaves). Similarly, the total flavonoid phenylhydrazine hydrochloride and content was higher in the G. caspica extract phenacetin. Table 1- Total phenolic and flavonoid content of G. caspica and I. spinigera extracts Extract Total phenolic content* Total flavonoid content** Extraction yield (w/w %) G. caspica 0.123 ± 0.003 0.459±0.007 10.35 0.042±0.004 0.030±0.005 19.54 I. spinigera * mg gallic acid equivalents per gram of dried leaves ** mg mg quercetin equivalents per gram of dried leaves Figure 2- Hemolysis of G6PD-deficient and -sufficient erythrocytes after 2 hours of incubation at 37°C with different concentrations of aspirin. Water-induced hemolysis was considered 100%. Downloaded from goums.ac.ir at 10:40 +0330 on Monday February 4th 2019 [ DOI: 10.29252/mlj.13.1.21 ] Figure 3- Hemolysis of G6PD-deficient and -sufficient erythrocytes after 2 hours of incubation at 37°C with different concentrations of phenylhydrazine hydrochloride. Water-induced hemolysis was considered as 100%. Medical Laboratory Journal Jan-Feb, 2019; Vol 13: No 1 www.SID.ir Archive of SID 24/ Protective Effect of Ilex. The degree of hemolysis is dependent on extract at a high dose (600 µg/ml) did not have concentration of the hemolytic agent present in any side effects on the RBCs when used alone. the medium. The I. spinigera extract had no The inhibitory activity of the extract was significant effect on the hemolysis induced by compared with that of the positive control, aspirin, phenacetin and phenylhydrazine glutathione (0.5 mM). hydrochloride in G6PD-deficient erythrocytes In all cases except for phenylhydrazine (P>0.05). However, the plant extract showed hydrochloride, glutathione had significant higher anti-hemolytic activity at higher inhibitory activity against the tested drugs concentrations (Figure 5). The I.
Details
-
File Typepdf
-
Upload Time-
-
Content LanguagesEnglish
-
Upload UserAnonymous/Not logged-in
-
File Pages9 Page
-
File Size-