Antioxidant activity of Ocimum Genus Against H2O2 Induced Oxidative Stress and Apoptosis in Human Lung Fibroblasts

Cell apoptosis, caused by oxidative stress, is an important step leading to Chronic Obstructive Pulmonary Disease (COPD). Chronic bronchitis and pulmonary emphysema are forms of COPD. This project explores natural antioxidants that can help inhibit apoptosis, preventing oxidative lung damage that leads to COPD. The purpose of this project is to find out which of the Ocimum genus species Ocimum basilicum, Ocimum gratissimum, and Ocimum tenuiflorum exhibits the most antioxidant properties by inhibiting apoptosis, due to oxidative stress induced by H2O2 in MRC-5 cells. Copper reducing equivalents (CRE) of the Ocimum genus species were determined. CRE is directly proportional to the total antioxidant capacity. The average CRE of O.tenuiflorum (16,229 μM) was the highest, the second highest was that of O.basilicum (15,207μM) and the least was that of O.gratissimum (14,876 μM). MRC-5 cell lines treated with various concentrations of H2O2, ranging from 450 μM to 0 μM, were incubated for 45 hours. Trypan Blue staining and microscopic counting of dead cells led to the median lethal dose of H2O2 as 100 μM. MRC5 cells were pre-treated with the individual Ocimum genus infusions, incubated for 60 hours, then treated with 100 μM H2O2 concentration, and incubated for 22 hours. The ANOVA test showed that the average percentage of viable cells was significantly different among the herbal groups: O.tenuiflorum (90.25%), O.gratissimum (75.49%), and O.basilicum (74.28%). The two tailed p-value tests showed that O.tenuiflorum inhibited apoptosis significantly more than the other two species. Introduction This study investigated the inhibitory effects of the Ocimum genus against apoptosis caused by oxidative stress in lung fibroblasts, and the possible use of the Ocimum genus as a natural medicine for lung diseases caused by oxidative stress. Chronic Obstructive Pulmonary Disease (COPD) is one such progressive disease that restricts breathing and is a major worldwide health problem being the cause of increasing mortality. In normal lungs, the air that is breathed in goes down the windpipe into airways called the bronchial tubes. Within the lungs, the bronchial tubes branch into smaller, thinner channels called bronchioles and these channels end in tiny round air sacs called alveoli. In normal lungs, the airways and alveoli are highly elastic. In COPD, breathing is restrictedless air flows in and out of the lungs because of several reasons: the airways and air sacs lose elasticity, walls between alveoli are destroyed, walls of airways are thick Antioxidant activity of Ocimum Genus Against H2O2 Induced Oxidative Stress and Apoptosis in Human Lung Fibroblasts Sangamithra Vardhan1*, Mary Anderson2, and William Safranek3 Student1, Teacher2: West Shore Jr./Sr. High School, 250 Wildcat Alley, Melbourne, FL 32935 Mentor3: University of Central Florida, 4000 Central Florida Boulevard, Orlando, FL 32816 *Corresponding author: [email protected] INTERNSHIP ARTICLE and inflamed, and the airways produce mucus that tends to clog the channels. Further, COPD consists of two main pathological conditionsemphysema and chronic bronchitis. Emphysema is the condition in which the walls between alveoli are damaged, causing the alveoli to lose shape. This major damage can cause the destruction of the walls of the alveoli, leading to fewer and larger alveoli and reduced amount of gas exchange. Chronic bronchitis is a medical condition where the lining of the airways is inflamed and thus thickens. The increased, heavy mucus production causes difficult breathing1. Recently, research of patients with COPD has provided considerable evidence that shows cigarette smoke increases oxidative burden in the lungs. Oxidants are either produced endogenously (intracellular oxidants generated by the mitochondrial electron transport chain) or exogenously (oxidants originating from cigarette smoke, a major external causative agent of COPD). Cigarette smoke contains 5000 different chemicals of which many are reactive oxygen species including H2O2, O2-, OH-, and NO. There are numerous sources of reactive oxygen species and free radicals in the body as well that can cause the pathogenesis of COPD. The lung is the only organ in the body that has the highest exposure to oxidants due to its large surface area and important interactions with atmospheric oxygen in the breathing function of the body. The reactive oxygen species are generated by the initial reduction of oxygen in the air breathed into the lung. The addition of one electron to oxygen results in the production of the superoxide anion (O2-). Further, the addition of a second electron to oxygen results in hydrogen peroxide (H2O2), and a third addition of an electron will result in the formation of a hydroxyl radical (OH-). These three reduction reactions generate a major amount of reactive oxygen species leading to the onset of oxidative stress. Oxidants inactivate antiproteases, such as alpha-1-antitrypsin and elastase, thus causing a deficiency in antiproteases. This creates a proteaseantiprotease imbalance, which in turn sets the foundation of the pathogenesis of emphysema. Oxidant generating systems, prominently the xanthine/xanthine oxidase, cause the release of mucus from the airway epithelium. Also, oxidants are involved in signaling pathways for the epidermal growth factor receptor, which plays a critical role in the release of mucus. Oxidants, like H2O2 and hypochlorous acid, in low concentrations (100 μM) have been shown to cause impairment of ciliary beating and even complete ciliary stasis. These oxidant-mediated mucus hyper secretion and impaired mucociliary clearance result in the accumulation of mucus in airways contributing to airflow limitation characterized in COPD2. Oxidative stress plays an important role in the pathogenesis of COPD by being the major