Annato extract and β-carotene modulate the production of reactive oxygen species/nitric oxide in neutrophils from diabetic rats

Abstract

Annatto has been identified as carotenoids that have antioxidative effects. It is well known that one of the key elements in the development of diabetic complications is oxidative stress. The immune system is especially vulnerable to oxidative damage because many immune cells, such as neutrophils, produce reactive oxygen species and reactive nitrogen species as part of the body's defense mechanisms to destroy invading pathogens. Reactive oxygen species/reactive nitrogen species are excessively produced by active peripheral neutrophils, and may damage essential cellular components, which in turn can cause vascular complications in diabetes. The present study was undertaken to evaluate the possible protective effects of annatto on the reactive oxygen species and nitric oxide (NO) inhibition in neutrophils from alloxan-induced diabetic rats. Adult female rats were divided into six groups based on receiving either a standard diet with or without supplementation of annatto extract or beta carotene. All animals were sacrificed 30 days after treatment and the neutrophils were isolated using two gradients of different densities. The reactive oxygen species and NO were quantified by a chemiluminescence and spectrophotometric assays, respectively. Our results show that neutrophils from diabetic animals produce significantly more reactive oxygen species and NO than their respective controls and that supplementation with beta carotene and annatto is able to modulate the production of these species. Annatto extract may have therapeutic potential for modulation of the balance reactive oxygen species/NO induced by diabetes.

Keywords: annatto; diabetes; neutrophils; nitric oxide; reactive oxygen species.

Fig. 1

Annatto extract and β-carotene treatment decreased reactive oxygen species (ROS) production in neutrophils from non-diabetic and diabetic rats. The data are presented as the means ± SD. C: Control (n = 8), CAn: Control + 0.09% annatto extract treatment (n = 7), Cβcar: Control + 0.1% β-carotene treatment (n = 8), D: Diabetic (n = 6), DAn: Diabetic + 0.09% annatto extract treatment (n = 10), Dβcar: Diabetic + 0.1% β-carotene treatment (n = 9). *p<0.0001 compared to C, **p<0.0001 compared to D, ^†† p<0.01 compared to ROS in C and CAn, ^† p<0.05 compared to ROS in Cβcar, ^##p = 0.0008 compared to ROS D, ^#p<0.03 compared to ROS in DAn and Dβcar.

Fig. 2

Annatto extract and β-carotene decreased reactive oxygen species (ROS) production in neutrophils from diabetic rats after incubation with ZC3b particles. The data are presented as the means ± SD. C: Control (n = 8), D: Diabetic (n = 6), DAn: Diabetic + 0.09% annatto extract treatment (n = 10), Dβcar: Diabetic + 0.1% β-carotene treatment (n = 9). ***p<0.0005 compared to C, *p<0.05 compared to D; **p<0.001 compared to D.

Fig. 3

Annatto extract and β-carotene decreased nitric oxide (NO) production in neutrophils from diabetic rats. The data are presented as the means ± SD. C: Control (n = 4), CAn: Control + 0.09% annatto extract treatment (n = 4), Cβcar: Control + 0.1% β-carotene treatment (n = 6), D: Diabetic (n = 4), DAn: Diabetic + 0.09% annatto extract treatment (n = 6), Dβcar: Diabetic + 0.1% β-carotene treatment (n = 6). *p<0.0001 compared to C, ^#p<0.0001 as compared to D.

Fig. 4

Evaluation of ROS/NO balance in neutrophils from treated or untreated diabetic rats. The data are presented as the means ± SD. D: Diabetic (ROS/NO), DAn: Diabetic + 0.09% annatto extract treatment (ROS/NO), Dβcar: Diabetic + 0.1% β-carotene treatment (ROS/NO).