## 2 Phytochemicals Present in _Pulmonaria_
officinalis_ was used to prepare extracts in different concentrations of ethyl alcohol to optimally obtain the biologically active substances [21]. The total content of polyphenols in the _P. officinalis_ L. extract was determined using the Folin-Ciocalteu method. The concentration of flavonoids in the _P. officinalis_ extract was determined using the colorimetric method. The determination of proanthocyanidins was carried out using vanillin analysis in glacial acetic acid. The _P. officinalis_ extract showed a high yield of polyphenic compounds, i.e., 889.39 \(\pm\) 4.29 \(\mu\)g gallic acid/mL, flavonoids 728.90 \(\pm\) 6.98 \(\mu\)g rutin/mL, and proanthocyanidins 211.65 \(\pm\) 5.31 \(\mu\)g catechin/mL, respectively [21]. The content of these of types of phytochemicals was higher in the root culture extract than in the dried part of _P. officinalis_ obtained in the earlier study [21]. The fatty acid composition of seed oil was determined by the gas-liquid chromatography of fatty acid methyl esters. Heptadecanoic acid was used as an internal standard for the simultaneous quantification of the oil content. The oil content in the seeds was 25.5 and 24.3 (% seed weight) in _P. mollis_ and _P. obscura_, respectively. In _P. mollis_, the percentage composition of fatty acids such as palmitic acid, stearic acid, oleic acid, linoleic acid, alpha-linoleic acid, gamma-linoleic acid, and steardonic acid were found to be 10.2, 6.3, 17.6, 28.2, 10.5, 19.4, and 3.6, respectively. Similarly, in _P. obscura_, the percentage composition of palmitic acid, stearic acid, oleic acid, linoleic acid, \(\alpha\)-linoleic acid, \(\gamma\)-linolenic acid, and steardonic acid were found to be 14.1, 13.1, 16.9, 22.2, 9.4, 14.3, and 3.6, respectively. The total tocopherol content in the seeds of _P. mollis_ was observed to be 964 mg/Kg, which respectively comprises 6.2%, 51.9%, and 38.1% of \(\alpha\) (alpha), \(\gamma\) (gamma), and \(\delta\) (delta)tocopherol. The total tocopherol content in the seeds of _Pulmonaria obscura_ was observed to be 346 mg/Kg, which comprises 15.2%, 68.3%, and 16.5% of \(\alpha\), \(\gamma\), and \(\delta\)tocopherol, respectively [22]. The main components of the methanol extract of the aerial parts of _P. officinalis_ are derivatives of phenolic acid, primarily the conjugates of caffe acid with danshenus--rosemarinic, monadic, lithospheric, salvianolic, shimobashiric, and yunnanic acids. There are esters of caffe acid, quinic acid, threonine and glyceric acids, and coumaroylquinic acid. Several lignans (globoidnans A and B, pulmonariosides A and B) and common flavonoid glycosides (derivatives of quercetin and kaempfer) have been identified [20; 21; 22]. A recent study was conducted to understand the mechanism of color change in the _Pulmonaria_ flowers. Three important lungwort (_Pulmonaria_) cultivars, named [PERSON], [PERSON], and [PERSON] were cultivated and studied in the Tsukuba Botanical Garden, National Museum of Nature and Science, Ibaraki Pref., Japan. Three stages of corollas were used in the study and methanolic extract from the flowers was analyzed. Further, the preparative HPLC was used to separate the anthocyanins and phenolic acid. Finally, the identification of anthocyanins and phenolic acid was conducted through the LC-MS method [23]. Three anthocyanins (delphinidin 3-O-glucose, petunidin 3,5-di-O-glucose, and petunidin 3-O-glucose) and one phenolic acid (rosmarinic) were identified from the flower extract in the study (Table S1 and Figure 1) [23]. The Boragniaceae family is known for the presence of pyrrolidine alkaloids (PA), which can result in intoxication when present in herbal drugs. In the study, PA were analyzed in the leaves, roots, rhizomes, and infloresances of _P. obscun_ using gas chromatography-mass spectrometry.