## 3 _Nelumbo nucifera_
### Antioxidant Health Promoting Effect of Nelumbo uncifera Bioactives and Extracts
#### 3.2.1 In Vitro Antioxidant Effects of Nelumbo uncifera's Bioactives and Extracts
The generation of the ABTS\({}^{+}\) radical cation forms the basis of one of the spectrophotometric methods used to measure the total antioxidant activity of solutions of pure substances, aqueous mixtures and drinks [31; 32]. A decolorization technique, in which the radical is directly generated in a stable form prior to reaction with putative antioxidants, is a more suitable format for the assay. The improved technique for generating ABTS described here involves the direct generation of the blue/green ABTS\({}^{-+}\) chromophore by reacting ABTS with potassium persulphate. Moreover, superoxide anion radicals are produced by several enzyme systems in autoxidation reactions and by non-enzymatic electron transfer reducing molecular oxygen. These systems are also capable of reducing certain iron complexes, such as cytochrome c [33]. The Ferric Reducing Antioxidant Power (FRAP) assay evaluates the reducing power of antioxidants by measuring their ability to reduce ferric ions (Fe\({}^{3+}\)) to ferrous ions (Fe\({}^{2+}\)). This assay provides information on the electron-donating capacity of antioxidants. While the total flavonoid content in the combined fractions I and II of lotus seed embryos was lower than that in lotus leaves, the \(C\)-glycosides in the seed embryos showed higher antioxidant properties compared to the \(O\)-glycosides in lotus leaves. This suggests that lotus seed embryos could be valuable sources of natural flavonoids with antioxidant properties, potentially suitable for incorporation into dietary supplements to promote health [21]. Other studies also employed the DPPH assay and yielded similar outcomes. [PERSON] et al. analyzed the methanol extract of lotus seed in six different lotus varieties, identifying 27 phytochemical constituents, with methyl-alpha-D-galactopyranoside being the predominant compound across all varieties. The study demonstrated notable antioxidant activity of the methanolic extracts of lotus seed varieties, with DPPH radical scavenging ranging from 9.86% to 77.97% and IC\({}_{50}\) values lower than those of the control, ascorbic acid [25]. [PERSON] et al. fractionated a methanol extract from lotus stames using organic solvents (dichloromethane, ethyl acetate, n-butanol), leading to the isolation of seven flavonoids and one sterol glucopyranoside from ethyl acetate fraction, which demonstrated strong antioxidant activity in all model systems [26]. These compounds were evaluated for their antioxidant properties in various model systems (DPPH, reactive oxygen species [ROS], and ONOO-) to assess their ability to scavenge free radicals and inhibit ROS generation. ROS test involves measuring ROS levels in biological samples to assess oxidative stress and potential damage to cells. The ROS test typically involves using specific probes or dyes that react with different types of ROS present in the biological sample. These probes undergo a chemical reaction with ROS, leading to a measurable change in fluorescence, absorbance or color. ONOO\({}^{-}\) test evaluates levels of peroxynitrite, a reactive nitrogen species (peroxynitrite is formed by the rapid reaction between nitric oxide NO and superoxide anion O\({}_{2}\)\({}^{-}\)), to understand its role in oxidative and nitrosative stress-related conditions. These tests help assess the impact of these reactive species on cellular function and the effectiveness of antioxidants in combating oxidative damage. The isolated bioactive compounds assessed exhibited antioxidant properties. Kaempfer demonstrated strong antioxidant activity in all three tests, while 13-sitosterol glucopyranoside did not exhibit any activity in the model systems. Specific flavonoids like Kaempfer 3-\(O\)-\(\beta\)-D-glucopyranoside and Kaempfer 3-\(O\)-\(\beta\)-D-galactopyranoside showed activity in the ONOO test [26]. Utilizing the DPPH free radical scavenging assay as a standard method for assessing antioxidant capacity, [PERSON] et al. demonstrated a correlation between the concentration of bioactive compounds in a plant extract and its antioxidant potential.