## 3 Results and Discussion
### Evaluation of Cytotoxicityoxicity
#### 3.2.1 In Vitro_ ROS Induced by _A. agallocha_ and _A. malacensis_ on Cancer Cells  
Reactive oxygen species (ROS) are essential for producing cytotoxicity in cancer cells higher levels of ROS buildup have been found to trigger apoptosis, cell cycle arrest, and autophagy, in a number of human cancer cell lines [62, 63, 64]. Reactive oxygen species (ROS) contribute to cellular signaling and are produced naturally as byproducts of regular cell activity [65]. Because of a mismatch among oxidizing substances and antioxidants, cancer cells usually have baseline ROS levels that are higher than those of normal cells. ROS at low levels serve as second messengers inside cells. Because they can enhance cancer metabolism and growth signals and suppress antioxidants, which promote oncogenesis, moderate levels of ROS are advantageous to cancer cells. In contrast, high ROS concentrations may lead to DNA damage-related cell death [66, 67, 68]. Due to the production of reactive oxygen species (ROS), _A. agallocha_ and _A. malacensis_ are essential reputable materials for anti-cancer action [69]. The DCFH-DA reagent was added in this instance to determine the intracellular ROS level in both samples on A549 cells. In comparison to the control, a dose-dependent increase in internal ROS production was seen in the malignant A549 cells after treatment with _A. agallocha_ and _A. malacensis_ at higher concentrations (1000 \(\mathrm{\SIUnitSymbolMicro g}\)/mL) (Figure 6). Research findings have revealed that an elevate in ROS formation and apoptosis are correlated with a decrease in mitochondrial membrane potential. Reactive oxygen species (ROS) are created by mitochondria during apoptosis [70, 71]. The buildup of ROS may also trigger the development of p53, which has a substantial impact on the beginning of apoptosis by activating pro-apoptotic proteins (_Bax_) or inhibiting anti-apoptotic mitochondrial proteins (_BCL-2_) [31, 72]. _A. agallocha_ and _A. malacensis_ extracts inhibited cell growth and produced ROS, which suggests that ROS generation promotes apoptosis via the mitochondrial pathway in A549 lung cancer cells. Figure 5: An assessment of the cytotoxicity of _A. agallocha_ and _A. malacensis_ to cells (**A**) on (RAW 264.7), (**B**) on lung carcinoma (A549) cells, (**C**) HaCaT cell lines compared to non-treated cells. The graph displays the means and standard deviations for the four replicates. Significant differences from control sets are mentioned by **_p_ < 0.01; and **_p_ < 0.001. 2.2 A. agallocha and _A. malacensis_ Extract Increased NO Production and Inhibited ROS Generation Induced by LPS  
Appropriate NO production in mammalian cells is important for many basic physiological processes [73; 74]. However, excessive NO production can result in a number of chronic and acute illnesses, including inflammation, cancer, and sepsis. One of the most significant pro-inflammatory mediators is nitric oxide (NO). Inhibiting the overproduction of nitric oxide levels is thus a desirable target in the search for new anti-inflammatory drugs. We examined the anti-inflammatory properties of the chosen _A. agallocha_ and _A. malacensis_ on the reduction of NO generation in LPS-stimulated RAW 264.7 cells at concentrations of (15.625 to 1000 \(\upmu\)g/mL). At 1000 \(\upmu\)g/mL concentration, both samples showed strong significant reductions in LPS-induced NO production (44.73% and 46.91%, respectively), in a dose-dependent way, whereas L-NMMA was employed in this investigation as a positive control for inhibiting NO production. On the other hand, as a result of the immune response's increased ROS production during inflammation and the body's altered oxidant/antioxidant balance, DNA, proteins, and lipids are damaged [75]. RAW 264.7 cells were subjected to various meditations of _A. Agallocha_ and _A. malacensis_ in the presence or absence of LPS for 24 h to examine the inhibitory effects of both species on LPS-induced ROS generation.