Spinach (Spinacia oleracea) is an annual plant belonging to the Amaranthaceae family (Amir et al. 2024). It is widely cultivated as a green leafy versatile vegetable for various cuisines, including fresh ones, soups, sauces, etc (Zhou et al. 2023). Spinach is widely recognized for its nutritional value, containing sugars, organic acids, amino acids, vitamins, minerals, and phytochemicals (Salehi et al. 2019; Waseem et al. 2021; Sharma et al. 2023). Recent studies have highlighted the association between phytochemicals found in spinach and several health benefits (Roberts and Moreau 2016). Spinach has been found to have positive impacts on eye health, as well as anti-inflammatory and antioxidant effects (Metha and Belemkar 2014; Khalid et al. 2017; Thandapani et al. 2023). Moreover, it has also been linked to the prevention and management of chronic diseases such as depression, obesity, and hypolipidemia (Son et al. 2018). With its impressive benefits, spinach has valuable potential for development as a nutraceutical.
The healthy benefits of spinach have been noted to involve its specific phytochemicals, such as carotenoids, flavonoids, terpenoids, polyketides, lignans, saponins, and peptides (Riso et al. 2004; Park et al. 2011; Karasawa et al. 2021). One of the key components responsible for these benefits is flavonoids, a type of phytochemical in spinach (Bergqust et al. 2005; Tao et al. 2023). The types of spinach flavonoids have been known as patuletin derivatives, spinacetin derivatives, spinach flavonoids glucuronide derivatives, and other basic skeleton flavonoids (Mi et al. 2008; Singh et al. 2018; Yuk et al. 2019). One of the patuletin derivatives, patuletin-3-O-(2''-coumaroylglucosyl)-(1→6)-[apiosyl-(1→2)]-β-D-glucopyranoside, has been reported to inhibit the formation of advanced glycation end products (AGEs) and aldose reductase activity (RLAR) (Perez Gutierrez and Velazquez 2020). 3',4',5-Trihydroxy-3-methoxy-6,7-methylenedioxyflavone 4'-glucuronide (TMG) is a specific flavonoid glucuronide derivative found in spinach leaves (Aritomi and Kawasaki 1984; Ferreres et al. 1997; Koh et al. 2012). TMG has inhibitory activity on granulation in basophilic leukemia RBL-2G3 cells (Yuta Morishita 2015). The results indicate that spinach flavonoids have therapeutic potential and specific skeletons for standard compounds of spinach.
Establishing and verifying standard compounds in bioresources play a crucial role in the development of nutraceuticals (Dillard and Bruce German). These compounds serve as indicators or reference points for quality control and standardization of nutraceutical products (Morris; 2001). The selection of standard compounds is typically based on their presence in the botanical source or their known bioactivity (Moreno et al. 2006; Devi et al. 2024). For the validation, it is imperative to ensure their safety, efficacy, and quality according to Association of Official Analytical Collaboration (AOAC) international guidelines (2023).
In this study, 3',4',5-trihydroxy-3-methoxy-6,7-methylenedioxyflavone 4'-glucuronide (TMG) was identified as a standard compound to develop nutraceuticals of spinach extract using UPLC-Q-TOF/MS analysis. In addition, the effectiveness of the analytical method validation was verified by confirming specificity, precision, repeatability, accuracy, LOD, LOQ, and recovery rate according to the AOAC international guideline using HPLC.