## 2 Phytochemicals Present in _Pulmonaria_
In search of new sources for the treatment of neurodegenerative disorders, the content of different types of phytochemicals such as polyphenols, flavones, and prothocyani-dins was calculated from the ethanolic and aqueous extracts of _P. officinalis_. The content of polyphenols, flavones, and prothocyani-dins was calculated using Folin-Ciocalte (expressed in gallic acid equivalents), aluminum chloride colorimetric (expressed in rutin equivalents), and vanillin assay in glacial acetic acid (expressed in catechin equivalents) methods, respectively. The ethanolic extract was found to have a higher content of polyphenols (576.62 \(\pm\) 6.32 \(\upmu\)g GAE/mL in ethanolic extract and 486.60 \(\pm\) 2.12 \(\upmu\)g GAE/mL in aqueous extract), flavones (272.81 \(\pm\) 9.21 \(\upmu\)g RE/mL in ethanolic extract and 104.28 \(\pm\) 3.25 \(\upmu\)g RE/mL in aqueous extract), and proanthocyani-dins (61.5 \(\pm\) 6.23 \(\upmu\)g CE/mL in ethanolic extract and 41.83 \(\pm\) 3.2 \(\upmu\)g CE/mL in aqueous extract) compared to the aqueous extract [10]. Further, HPLC-MS was used to assess the polyphenol profiles of aqueous and ethanol extracts of _P. officinalis_. Using the selected ion monitoring (SIM) mode, the corresponding peaks of gallic acid, chlorogenic acid, vanillic acid, caffeic acid, p-coumaric acid, rutin, sinapic acid, hyperoside, ferulic acid, hesperidin, rosmarinic acid, myricetin, luteolin, quercetin, apigenin, kaempfer, and isorhamnetin fragment ions were found in ethanolic and/or aqueous extract (Table S1) [10]. To study the metabolites, the extracts from the aerial part _P. officinalis_ in different weather (spring and autumn) were analyzed [20]. Researchers isolated 45 compounds from the methanol extract of _P. officinalis_ through a multistep preparation procedure. These isolated compounds were further analyzed through high-resolution mass spectrometry, as well as one-dimensional 1H and 13C-NMR spectroscopy. In these analyses, nine new phenolic metabolites were identified along with 36 already known compounds. These new compounds were 3-O-(E)-caffeoyl-threonine acid, 3'-O-(E)-feruoyl-\(\alpha\)-sorbopyranosyl-(2'-1)-\(\alpha\)-glucopyranoside, 3-O-(E)-caffeoyl-glyceric acid, globoidnan B, yunnaneic acid E-1, pulmonarioside A, pulmonarioside B, pulmitric acid A, pulmitric acid B, and isosalvianic acid A-1 (Table S1). The difference in the concentration of phytochemicals in spring and autumn was observed, but rosmarinic acid was the most abundant compound in both seasons followed by Globoidnan B [20]. A similar analysis was also carried out including _P. obscura_. High-resolution mass spectrometric analysis (HR-MS) revealed the presence of numerous phenolic compounds from the aerial parts of _P. officinalis_ and _P. obscura_. The phytochemical profiles of the phenolic-rich fractions obtained from _P. officinalis_ and _P. obscura_ were very similar, both qualitatively and quantitatively [11]. These fractions contained different compounds belonging to multiple classes of phytochemicals. The phenolic acids were the main constituent of the extract, comprising more than 80% of the extract, with flavonoids constituting around 5%. The individual phytochemicals that were present in higher concentrations included rosemarinic, lithospheric, salvianolic, monardic, shimobashiric, and yunnanic acids. The esters of caffe acid with quinic, threonine, and glyceric acids were also found to be present in the extract. P-coumaroylquinic was found to be present in three isoforms, lignans such as globoidnans A and B, pulmonariosides A and B, and several commonly found flavonoid glycosides like quercetin and kaempfer derivatives were also found. A significant amount of menisdaurine, nitrile glucoside, was identified in the extract. A small amount of the megastigman glucoside actinidiotionoside and a tryptophan derivative, that is, lycoperodin-1, were also present in the extract [11; 20]. The root culture of _P.