Histochemical and Phytochemical Analysis of Lamium album subsp. album L. Corolla: Essential Oil, Triterpenes, and Iridoids

The aim of this study was to conduct a histochemical analysis to localize lipids, terpenes, essential oil, and iridoids in the trichomes of the L. album subsp. album corolla. Morphometric examinations of individual trichome types were performed. Light and scanning electron microscopy techniques were used to show the micromorphology and localization of lipophilic compounds and iridoids in secretory trichomes with the use of histochemical tests. Additionally, the content of essential oil and its components were determined using gas chromatography-mass spectrometry (GC-MS). Qualitative analyses of triterpenes carried out using high-performance thin-layer chromatography (HPTLC) coupled with densitometric detection, and the iridoid content expressed as aucubin was examined with spectrophotometric techniques. We showed the presence of iridoids and different lipophilic compounds in papillae and glandular and non-glandular trichomes. On average, the flowers of L. album subsp. album yielded 0.04 mL/kg of essential oil, which was dominated by aldehydes, sesquiterpenes, and alkanes. The extract of the L. album subsp. album corolla contained 1.5 × 10−3 ± 4.3 × 10−4 mg/mL of iridoid aucubin and three triterpenes: oleanolic acid, β-amyrin, and β-amyrin acetate. Aucubin and β-amyrin acetate were detected for the first time. We suggest the use of L. album subsp. album flowers as supplements in human nutrition.


Introduction
Volatile substances are produced by plants mainly in glandular trichomes, which are spread over aerial vegetative organs [1]. In flowers, volatile organic compounds are emitted from petals during floral maturation [2]. In reproductive organs, essential oils are produced by specialized scent glands, i.e., osmophores, located in various floral elements [3,4]. Plants from the family Lamiaceae are characterized by a high content of essential oils [5].
Essential oils are natural multi-component mixtures of products derived from the secondary metabolism in plants. These mixtures and some of their components exhibit a broad spectrum of biological and pharmacological activities. It has been found that the action of whole essential oils is stronger than that of its individual components [6][7][8].
Floral volatile organic compounds play an important role in the interaction between plants and various environmental factors. Terpenoids and benzenoids are important components of these substances [9][10][11]. An important function of floral volatile organic compounds is to attract potential pollinators [1,4,12]. The entomophilic L. album flowers material together with the corolla, long-stalked capitate trichomes and flattened singlecelled mechanical trichomes were found [58].
Studies conducted by other authors on L. album trichomes focused on leaves, stems, and the calyx. The types of trichomes observed on these organs by other authors largely differed from trichomes located on the corolla in this species [59][60][61].
Plants of the Lamiaceae family are known to produce many biologically active secondary metabolites [18,38,62]. In the scientific literature, histochemical analyses were used to determine the distribution of various compounds in the trichomes of many plant species of this family. However, there was no information about the identification and location of biologically active substances in the tissues of L. album flowers, which are a valuable medicinal and pharmaceutical raw material. In our previous study, were detected phenolic acids, flavonoids and tannins in the corollas of this species [58]. The present study is a continuation and extension of previous research and is now focused on other biologically active compounds. The aim of the present study was (i) to conduct morphometric studies of trichomes present on the bilabiate corolla and stamens in L. album subsp. album, (ii) to determine the location of essential oil and iridoids in corolla epidermis cells and different types of trichomes based on histochemical assays, (iii) to perform quantitative and qualitative analysis of essential oil contained in the corolla, and (iv) to conduct qualitative analysis of triterpenes and quantitative analysis of iridoids contained in the corolla.

Corolla
Glandular (capitate and peltate) trichomes and non-glandular trichomes were observed on the corolla. All capitate glandular trichomes on the corolla were short-stalked and had a 1-4 celled head (Figure 1a-c,e). They differed in the number of head-forming cells and sizes. The capitate trichomes with a 1-celled head had a height of ca. 25.5 µm and a 35.6-µm diameter. The capitate trichomes with a 2-and 3-celled head had a similar size, i.e., ca. a height of 27.9 µm and a 43-µm diameter. The capitate trichomes with a 4-celled head were ca. 29.5 µm in height and their diameter was 39.7 µm ( Table 1). The peltate trichomes were composed of one basal cell, a unicellular stalk, and a 4-8-celled head, (Figure 1d,f,g). The trichomes were 29.5 µm high and had a diameter of ca. 80.8 µm ( Table 1).

Stamen
Long-stalked glandular trichomes with a 3-5-celled stalk and a 1-4 celled head were present on the surface of anthers and filaments (Figure 3a-l). The trichomes had a height of 157 µm and a diameter of ca. 21 µm. Additionally, there were unicellular non-glandular trichomes on the anthers with a height of 778.6 µm and a ca. 15-µm diameter (Table 1).

Histochemistry
The histochemical assays revealed the presence of various substances, i.e., total lipids, acidic lipids, neutral lipids, terpenes, essential oil, and iridoids, in glandular and nonglandular trichomes located on the corolla, in glandular trichomes from stamens, and in corolla papillae (Table 2).
In the fresh control samples, the secretory product was dark yellow in the short capitate and peltate trichomes on the corolla (Figure 1b), light yellow in the long capitate trichomes on the stamen (Figure 3c), and light yellow in the papillae (not shown). The lipids present in the trichomes stained dark blue after application of Sudan Black B (Figure 1e). After the Sudan Red B treatment, there were red-stained total lipids in the heads of glandular trichomes (Figure 1f,g) and in the papillae. In turn, Sudan III stained the trichome cell content orange (Figure 1h-k). The Nile Blue treatment showed the presence of intense, blue-stained acid lipids in all trichome types (Figures 1l,m, 2g-i and 3i-l). Terpenes/essential oils were present in the papillae and in all types of glandular trichomes, which was confirmed by the treatment of the material with Neutral Red, which stained these substances red (Figures 1n and 3d-h), and with the violet-blue staining Nadi reagent (Figures 1o-u and 2d-j). (e,f) Trichomes stained red after Neutral Red treatment (terpenoids/essential oil); (g-i) Trichomes stained blue after Nile Blue application (lipids); (j) Trichome stained dark blue after Nadi reagent treatment (terpenes/essential oil); (k,l) Trichomes stained pink-purple after Godin reagent treatment (iridoids). Scale bars: 500 µm (g), 100 µm (e), 50 µm (b-d,h,i,k,l), 30 µm (a), 20 µm (f), 10 µm (j). The presence of iridoids in the examined corolla structures was possible to detect with the use of Godin reagent, which stained these compounds pink-purple in the short capitate and peltate trichomes (Figure 1v  The fresh and dried flowers of L. album subsp. album yielded 0.044 mL/kg and 0.036 mL/kg of essential oil, respectively. The essential oils detected in the fresh and dried flowers were found to contain 33 and 30 components, which constituted 91.1% and 85.6% of the overall composition of the oil, respectively (Tables 3 and 4). The composition of the essential oil from the fresh flowers was dominated by aldehydes (59.9%) and sesquiterpenes (16.4%), whereas the oil extracted from the dried flowers contained 2.5-fold higher content of sesquiterpenes (42.1%) and alkanes (22%) and over 4.5-fold lower levels of aldehydes (12.9%). Lower contents of alcohols (4.8%), sesquiterpenoids (2.2%), and monoterpenes (1.9%) were determined in the fresh flower oil. Similarly, small amounts of monoterpenes (3.4%), alcohols (2.8%), and sesquiterpenoids (1.3%) were identified in the oil from the dried flowers. Geranial (36.4%) and neral (23.2%) aldehydes, as well as caryophyllene oxide (12.5%) from the class of sesquiterpenes, constituted the highest proportion in the essential oil from the fresh flowers. Branched-chain alkane (21.9%), germacrene D (21.2%), caryophyllene oxide (8.2%), and E-β-caryophyllene (6.6%) sesquiterpenes, as well as geranial aldehyde (8.4%), were the dominant compounds in the oil from the dried flowers. Monoterpenoids, alkybenzenes, ketones, and epoxides were present in both oils in the lowest amounts (<1%) ( Table 3). The identification of the essential oil compounds from L. album subsp. album corolla in the chromatograms is presented in Figures 4 and 5.

High-Performance Thin Layer Chromatography (HPTLC) Analysis of Triterpenes
The densitogram of separated triterpene standards is presented in Figure 6. β-amyrin acetate migrates over the longest distance, a moderate distance is covered by β-amyrin, and oleanolic acid reaches the shortest distance. This order of the spot is in accordance with the physicochemical properties of these triterpenes. β-amyrin acetate is the most non-polar compound, whereas oleanolic acid is the most polar among the investigated products. The intensity of oleanolic acid absorbance is lower in comparison to the other triterpenes.          β-amyrin and β-amyrin acetate were determined in all extracts. The content of the former compound in the methanolic extract from L. album subsp. album flowers (Figure 7) was almost the same as in the standard in terms of the zone area or height. The content of the latter triterpene in the extract was lower in comparison with the standard concentration. The best solvent to extract both compounds (β-amyrin and β-amyrin acetate) was ethyl acetate (Figure 8), whereas the poorest extraction was achieved when heptane was used ( Figure 9). The zone of oleanolic acid was hard to detect in all extracts due to co-elution with other compounds.
To solve this problem, further experiments were undertaken after choosing the wavelength of oleanolic acid.
The identification of oleanolic acid in the chromatogram of the ethanolic extract from L. album subsp. album is presented in Figure 10. The chromatographic systems developed for triterpenes were used for quantitative analysis of these compounds in plant raw materials. The chemical structures of the isolated triterpenes are shown in Table 5. We decided to use the spectrometric method for quantitative analysis of iridoids (aucubin) in the L. album subsp. album extract. These compounds were expressed as aucubin ( Table 5).
The average content of iridoid aucubin in the L. album subsp. album flower extract determined with the use of the calibration curve was 0.0015 ± 4.3 × 10 −4 mg/mL (Table 6).

Trichomes
The histochemical assays used in the present work detected lipids, essential oils, terpenoids, and iridoids in the glandular trichomes of L. album subsp. album corollas. Since we have not found such information in the literature so far, we present these data for the first time to fill the gap in the knowledge of this issue. Our previous study showed the presence of phenolic compounds: tannins, phenolic acids, and flavonoids in trichomes of this species [58]. The present study is a continuation and extension of our previous research.
The results indicate a heterogenous composition of the secretion in the peltate and capitate trichomes present on L. album subsp. album corollas. Based on histochemical reactions, the presence of lipids was detected in the cells of all types of glandular trichomes. However, the most intense color reaction was observed in the case of the peltate trichomes (Sudan Red B), which indicates the highest lipid content in the essential oil. The results of the present study largely confirm the findings reported by some authors, who observed the greatest amounts of essential oil contained in peltate trichomes [1,64].
The histochemical assays revealed a less intense reaction to the presence of lipophilic substances in the capitate trichomes of the L. album subsp. album corollas than in the peltate trichomes. Similarly, other studies reported that capitate trichomes in species from the family Lamiaceae produced fewer lipophilic compounds [65,66].
The presence of terpenes/essential oil was detected with the histochemical method (Nadi reagent) in all glandular trichomes of the L. album subsp. album corolla. Trichomes of many other species of this family have also been found to produce terpene-rich essential oils [67][68][69]. Terpenes present in essential oil are probably responsible for the pharmacological activity/medicinal properties and protective function in plants from the family Lamiaceae [68].
Previous studies suggest that different types of trichomes serve specific functions [70,71]. This hypothesis is confirmed by the results of the investigation on trichomes in Hyssopus officinalis subsp. aristatus (Lamiaceae) conducted by Venditti et al. [72]. The authors showed that short-stalked capitate trichomes produced polysaccharides, only terpenes were present in medium-stalked capitate trichomes, and a complex mixture of phenolic and terpenoid compounds was contained in peltate trichomes. Diversity in the substances produced in different types of glandular trichomes was also observed in Stachys annua subs. annua [73]. In this species, the presence of lipids, terpenes, and polyphenols was detected in peltate trichomes, lipids and terpenes were mainly produced by short-stalked capitate trichomes, and lipids exclusively were contained in long-stalked capitate trichomes.
In plants from other families, different types of trichomes may specialize in the production of specific secretion. In Lippia scaberrima (Verbenaceae), terpenoids were determined as the major components contained in large peltate trichomes and phenolic compounds dominated in small capitate trichomes [74]. In turn, Sacchetti et al. [75] showed that diterpenes were produced in secretory trichomes with a 2-celled head in Calceolaria adscendens (Scrophullariaceae), and secretory trichomes with an 8-celled head in C. volckmanni produced triterpenes. The dependence of the chemical composition of glandular trichome secretion on the morphology of the secretory head has been suggested in investigations of Cordia verbenacea (Boraginaceae) conducted by Vantrella and Marinho [76]. The authors have found that the secretion of globular trichomes consists mainly of essential oil, whereas the secretion of reniform trichomes is dominated by flavonoids.
As shown by our previous observations, short-stalked capitate trichomes with a 2-celled head in L. album flowers function non-synchronously [58]. The capitate trichomes located next to each other in L. album subsp. album were in different stages of metabolic/secretory activity: some stained intense blue in the reaction with Toluidine Blue O (phenolic compounds), other trichomes stained green-blue, and some did not stain at all, which suggested that they were in the post-secretory phase or degenerated. Similar results in a study of capitate trichomes with a 2-celled head present on Euphrasia stricta (Orobanchaceae) leaves were reported by Haratym and Weryszko-Chmielewska [77]. This phenomenon seems to be one of the causes of the differences in the amounts of various substances in different types of trichomes.
Mechanical protection is most often reported as the function of non-glandular trichomes, which are referred to as tector or clothing trichomes [78,79]. However, the histochemical assays used in the present study revealed the presence of lipids, terpenes, and iridoids in the cells of the non-glandular trichomes in the L. album corollas. In a previous study, we also detected tannins, flavonoids, and pectins in this type of trichomes [58]. Bioactive compounds were also found in non-glandular trichomes of several other Lamiaceae and Verbenaceae species [80]. Similar results were reported in studies of non-glandular trichomes from Dracocephalum moldavica leaves, which contained various types of metabolites: phenolic compounds, flavonoids, lipids, and terpenes [69]. These data indicate that nonglandular trichomes not only play a passive protective role but also can actively influence the biotic elements of the environment.

Essential Oil
Significantly low amounts of essential oil were extracted from the fresh and dried L. album subsp. album flowers (0.044 mL/kg and 0.036 mL/kg, respectively). The level of essential oil in fresh flowers was 22% higher than the content in the dried material. Similarly, half the amount of essential oil from dried vs. fresh L. album raw material was obtained by Morteza-Semnani et al. [26]. In turn, Flamini et al. [5] demonstrated considerable variability of essential oil yields in the range of 0.01-0.31% in different Lamium species. Previous studies demonstrated that the most volatile components of essential oil evaporate during drying, which may reduce the amount of extracted oil [81,82]. Moreover, some components of essential oil can be decomposed through oxidation and hydroperoxidation at elevated temperatures [83]. Drying at increased temperatures may also result in more intense degradation of the cell wall and plasma membrane, which may exert an effect on plasma membrane permeability [84]. In contrast, greater amounts of essential oil from dried flowers were extracted by, e.g., Zheljazkov et al. [85], Mashkani et al. [86], and Silva et al. [87].
In the present study, 33 and 30 compounds were identified in the oil extracted from the fresh and dried raw material, respectively. Other investigators reported the presence of a 4-fold higher number (130) [88] or by an over 30% higher number (43) [26] of components in L. album essential oil. In turn, Alpieva et al. [29] and Mickiene et al. [27] identified significantly fewer components (12 and 22, respectively).
The composition of the oil extracted from the dried L. album subsp. album flowers differed considerably from the composition of the oil from the fresh material. Aldehydes, which accounted for almost 60% of all essential oil components, constituted the main group of compounds in the essential oil extracted from the fresh flowers; they were followed by sesquiterpenes in this type of essential oil. The dried flower oil was dominated by sesquiterpenes (42%), alkanes (22%), and aldehydes (12.9%). Many authors reported similar results of the differences in the composition of essential oil, depending on the extraction method used. They suggest that the composition of oil from the same species largely depends on the mode of raw material processing, i.e., drying and distillation methods [81,86,[89][90][91]. Moreover, the differences in the qualitative and quantitative composition of essential oils derived from the same taxon can also be related to other factors, i.e., seasonal variations, plant development phases and age, chemotypes, soil, light intensity, and water availability [88,[92][93][94][95][96][97]. As reported by Salman et al. [98], changes in the amount and composition of essential oils extracted from various taxa from the family Lamiaceae are associated with genetic and environmental factors, which also determine the genetic expression and thus influence the chemism of oil components.
In the group of sesquiterpenes, the essential oil from the dried L. album subsp. album flowers was dominated by germacrene D (21%). As reported by Yordanova et al. [18], the content of this compound in L. album essential oil may vary substantially (from 6.9% to 46.7%). The content of germacrene D detected by various authors in oil extracted from flowers, bracts, and leaves of four other Lamium species, i.e., L. purpureum, L. hybridum, L.bifidum, and L. amplexicaule, was lower than in the L. album subsp. album flowers [5]. In turn, Nickavar et al. [105] reported that germacrene D was the main compound in the essential oil from L. amplexicaule aerial parts, and its content was similar to that detected in the L. album subsp. album material in the present study. As indicated by a study conducted by Hu et al. [106], germacrene has antitumoral activity against various types of cancer and may potentially serve as an antineoplastic drug. Moreover, the antioxidant potential of germacrene D has been supported by various studies [107,108].
Considerable amounts of caryophyllene oxide sesquiterpene were detected in the oil from the fresh and dried L. album subsp. album flowers (12.5% and 8.2%, respectively). Large amounts of this compound have also been identified in oil from L. maculatum aerial parts [109]. This component exhibits antimicrobial [85], antifungal [110], and insecticidal properties [111,112] and induces apoptosis of neoplastic cells [113].
E-β-caryophyllene is another sesquiterpene present in the oil extracted in the present study from the dried L. album subsp. album flowers (6.6%). The presence of β-caryophyllene in the oil from L. album flowers was also reported by Kapchina-Toteva et al. [88] and Yordanova et al. [18] as well as Flamini et al. [5] in other Lamium species. As shown by Yordanova et al. [18], the content of β-caryophyllene in L. album oil may vary from 1.1% to 13%, depending on plant growth conditions. As demonstrated by various researchers, caryophyllene is attributed a number of biological activities such as anti-inflammatory, anticancer, antibacterial, antioxidant, and local anesthetizing properties. It can also serve as a stimulant for the immune system [18,[114][115][116].

Triterpenes
Some of the triterpenes identified in the L. album flowers in the present study have been described earlier in this species by other authors, e.g., oleanolic acid and β-amyrin [35][36][37]. In turn, the presence of β-amyrin acetate in the flowers of this species was demonstrated in this study for the first time. We also found that ethyl acetate was the best solvent for the extraction of β-amyrin and β-amyrin acetate. In turn, Paduch et al. [37] reported that heptane extracts contained the largest amounts of β-amyrin in comparison with methanol and ethyl acetate extracts.
Using modern techniques of ultrasonic extraction, Wójciak-Kosior et al. [36] determined the optimal conditions for extraction of high concentrations of oleanolic acid. Investigations of the pharmacological activity of oleanolic acid have demonstrated that this compound may be applied in post-Helicobater pylori infection treatment [119]. The antimicrobial activity of oleanolic acid and β-amyrin acetate has been shown to be associated with moderate anti-adhesion properties [31]. Some authors presented oleanolic acid as a potential anti-diabetic compound in plant extracts [120].

Iridoids
The presence of iridoids in the glandular and non-glandular trichomes from the L. album subsp. album corollas was demonstrated using histochemical assays and quantified as aucubin in phytochemical analyses of the flower extracts. Aucubin in the extract from the L. album subsp. album corollas were identified for the first time in the present study. Only Adema [47] detected lamioside, i.e., an iridoid classified as an aucubin-like compound, in the leaves of L. album and other Lamium species, e.g., L. garganicum, L. longiflorum, L. amplexicaule, L. purpureum, and L. maculatum. Many researchers have described the presence of other iridoids in L. album organs, e.g., caryoptoside, lamalbid, and secoiridoid alboside B [127], lamalbid, caryoptoside, shanzhiside methyl ester, and barlerin [44,45], lamiusides A, B, and C and 6"-O-β-D-glucopyranosylmartynoside [50], and lamalbid and shanzhiside methyl ester [128,129]. As reported by these authors, most of these nonvolatile glycoside compounds exhibit numerous therapeutic properties. As shown by Zhang et al. [130], aucubin has anti-inflammatory, antioxidative, and antiapoptotic properties and is able to reduce the degree of liver ischemia-reperfusion injury significantly. In turn, the results reported by Zhou et al. [131] indicate an inhibitory effect of aucubin on the proliferation and differentiation of fibroblasts, thereby providing protection against pulmonary fibrosis. In turn, the impact of the presence of aucubin on the effectiveness of cosmetic macroemulsions has been shown by Dąbrowska and Nowak [132]. It was found to diminish transepidermal water loss, increase skin hydration, and improve the values of surface evaluation of living skin and skin macrorelief parameters.

Plant Material
Corollas of L. album subsp. album with stamens collected in the initial period of anthesis from plants growing in the Botanical Garden of Maria Curie-Skłodowska University in Lublin, Poland (51 • 26 20.7 N, 22 • 51 37.8 E) were used in the morphological, anatomical, and phytochemical analyses. The anatomical investigations were carried out in May 2019 and 2020, whereas the phytochemical study was carried out in May 2020. The botanical identification was made by comparison with authentic samples deposited in the Department of Botany and Plant Physiology, University of Life Sciences in Lublin and, additionally, by taxonomy specialist Professor Bożena Denisow, who confirmed the correctness of the identification of the taxon. Moreover, botanical identification of plants was performed using "Illustrierte Flora von Mittel Europa" [133] and "A taxonomic revision of Lamium (Lamiaceae)" [134]. Preliminary observations of the morphology and distribution of glandular and non-glandular trichomes were performed with the use of a stereoscopic microscope STM 800 (Microlab).

Light Microscopy-Handmade Preparations
Corollas and stamens were collected from fresh material of 50 randomly chosen specimens. Using razor blades, handmade cross-sections were made in three areas on the corolla: (i) the edge of the middle part of the upper lip, (ii) the lower part of the upper lip (near the site of fusion with the lower lip), and (iii) the widest point of the corolla tube throat ( Figure 11). The plant sections were de-aerated by embedding in a water-glycerine mixture in a 1:1 ratio for 24 h; next, glycerine preparations were made. The stamens were transferred directly on a glass slide and sealed in glycerin (POCH, Gliwice, Poland). The slides were viewed under a light microscope (Nikon, Tokyo, Japan). All types of trichomes present on the abaxial and adaxial corolla surfaces and on the stamens were measured. The height (n = 30) and diameter (n = 30) at their widest point were measured. Photographic documentation was prepared using a Coolpix 4500 (Nikon, Tokyo, Japan) camera coupled to an Eclipse 400 (Nikon) light microscope.

Scanning Electron Microscopy (SEM) Preparations
Stamens and fragments of corollas of L. album subsp. album collected from the same sites as described above were fixed in a 4% solution of glutaraldehyde in 0.1 M phosphate buffer (pH 7.0) for 12 h at 4 • C. Next, the plant samples were rinsed with the same buffer four times at 20-min intervals and subsequently fixed in 1% osmium tetroxide (w/v) (Johnson Matthey, Royston, UK) for 1 h and dehydrated in an ethanol series (30,50,70,90, 95% (v/v) and immersed in anhydrous ethanol (POCH, Gliwice, Poland) twice for 30 min). Then, the plant material was critical-point dried in liquid CO 2 using a K850 (Emitech, Laughton, East Sussex, UK) Critical Point Dryer. The next stage consisted of spraying the samples with gold (thickness of the gold layer of 20 nm) using a sputter coater K550X (Emitech, Ashford, UK). The material was viewed under a Tescan Vega II LMU (Tescan, Brno, Czech Republic) scanning electron microscope at an accelerating voltage of 30 kV [135]. Microphotographs were taken.

Essential Oil Isolation
The essential oils were obtained from 115 g of fresh and 103 g of dried L. album subsp. album corollas. The drying of the plant material was conducted in a forced air convection oven for two days at 34 • C. The essential oil of dead-nettle was extracted by hydrodistillation method [146]. Both fresh and dried corollas were distilled in 400 mL of water for 3 h in Deryng-type apparatus. The volume of the essential oil was measured; next, the oil was dried over anhydrous sodium sulfate and stored at 4 • C until gas chromatographic determination of its composition.

Gas Chromatography-Mass Spectrometry (GC-MS)
The chemical composition of the essential oil was analyzed using the GC-MS instrument TRACE GC ULTRA with a POLARIS mass spectrometer Q (Thermo-Finnigan, San Jose, CA, USA). An Equity-5 fused-silica capillary column (30 m × 0.25 mm, 0.25 µm df) (Supelco, Bellefonte, PA, USA) was used. Helium (grade 5.0) was used as a carrier gas. A split-splitless injector was operated in the split mode 1:20 for all chromatographic runs. The temperature of the injector as well as the transfer line temperature was 280 • C. The following temperature programs were applied: 1 min at 35 • C and then a linear temperature increase up to 280 • C at the rate of 4 • C/min. The mass spectrometer was operated in the EI mode at 70 eV; the manifold temperature was 220 • C. The mass spectra were measured in the range 35-400 amu.
Qualitative analysis was carried out via MS spectra, these were compared with the spectra library by means of the NIST MS Search Program (NIST 2.0, 2001), Wiley, and own library, as well as with data available in the literature [63,[147][148][149]. Identity of the compounds was confirmed by their retention indices, whereas the retention indices were determined in relation to a homologous series of n-alkanes (C 7 -C 30 ) under the same opaerationg conditons.

Ethanolic Extract Preparation for Triterpene Analysis
The ethanolic extract was prepared according to the Polish Pharmacopoeia V monography on natural products [150]. The flowers of L. album subsp. album were dried in the shade and air conditions. Next, 25 g of finely divided raw material were filtered through a sieve with 0.315-mm pores. An amount of 0.5 g of L. album subsp. album sample was weighed and transferred onto a stoppered conical glass flask insert; 50 mL of ethanol was poured into the flask. Next, the flask was placed in an ultrasonic bath (Bandelin, Sonorex, Germany) at 15 • C. The ultrasonic extraction was performed with the use of ultrasounds two times for 15 min. Then, the extract was filtered through a sintered glass funnel (Kisker Biotech GMbh&Co, Steinfurt, Germany). The extract was stored in the refrigerator before further investigations.

Preparation of Organic Extracts for Identification of Triterpenes
Extracts were prepared by heating 20 g of plant material with 300 mL of the solvent (heptane or methanol, or ethyl acetate) for 5 h at 60 • C in reflux conditions. After this time, the extracts were concentrated under reduced pressure at 30 • C. The final volume of the extract was 100 mL.

Chromatographic High-Performance Thin Layer Chromatography (HPTLC) Analysis of Some Triterpenes
The chromatographic analysis was performed on 100 × 100 mm precoated glass plates with a 0.25-mm layer of silica-HPTLC Kieselgel Si 60 F254 (Merck, Darmstadt, Germany). Before use, the plates were washed with methanol and dried for 20 min at room temperature for activation. The triterpene standards (Sigma Chemical Co, St. Louis, MO, USA) were dissolved in methanol (0.01% w/v). Then, 5-µL samples of the triterpene standards (oleanolic acid, β-amyrin, and β-amyrin acetate) were spotted using an AS 30 automatic applicator (Desaga CD 60, Heidelberg, Germany) under nitrogen at 2.5 atm of pressure. Chromatograms were developed in horizontal DS chambers (DS II chamber, Chromdes, Lublin, Poland) on a distance of 85 mm. All chromatographic eluent compositions were chosen experimentally. The mixture of n-hexane: dichloromethane: methanol: distilled water (5.0:6.0:0.4:0.1 v/v) turned out to be the best mobile phase for triterpenes. The separated triterpene zones were detected after the derivatization with the mixture of anise aldehyde, glacial acetic acid, methanol, and concentrated sulfuric acid (VI) (0.5:10:85:5 mL).
After spraying the derivative reagent solution and drying, the plates were heated at 100 • C in the oven for 5-10 min. The triterpene zones were detected at VIS or UV (366 nm) light. 4.5.6. Selection of the Analytical Wavelength for Oleanolic Acid As mentioned earlier, there were problems with the detection of oleanolic acid in the extracts and we had to decide to choose a characteristic wavelength for this compound. Therefore, in the next stage of our experiment, to enhance both the measurement accuracy and detection limit, we applied 0.1 mm of the densitometer slit. The oleanolic acid zone on the chromatographic plate was scanned in the range of 300-680 nm of the light spectrum. The range wavelength of 500-620 nm was promising. Figure 12 shows the selection of the analytical wavelength for oleanolic acid-560 nm. This wavelength was chosen for further investigations.

Spectrophotometric-Quantitative Analysis of Iridoids
Quantitative analysis of aucubin was performed according to Polish Pharmacopoeia VI [146] and literature data [151].
Aucubin quantitative analysis was performed with the use of the spectrophotometric method. To build the calibration curve, aucubin stock solutions were prepared with the following concentrations: 0.015 mg/mL, 0.0075 mg/mL, and 0.00375 mg/mL). An amount of 0.5 mL from each solution was transferred to three test tubes. Then, 1 mL of methanol, 2 mL of a 4-dimethylaminebenzaldehyde solution containing 0.5 mol/L of hydrochloric acid (Ehrlich reagent), and 1.5 mL of distilled water were added to the test tube. The mixture was heated for 3 min in a boiling water bath (EkoTerm TW 20, Julabo, Schwalbach, Germany). Next, the test tube contents were transferred to a 10-mL flask and diluted to the volume with distilled water. After 15 min, the absorbance of the blue solutions was measured using a Genesys 20 spectrophotometer (Thermo Fisher Scientific, Spectronic, Texas City, TX, USA) at 590 nm as the analytical wavelength. The experiments were triplicated. The average results were applied to prepare the calibration curve ( Figure 13).

Conclusions
To sum up, we conducted a complete pharmagnostical analysis of the corolla in L. album subsp. album to determine the distribution, content, and composition of essential oils as well as phytochemical analyses of triterpenes and iridoids. We have shown that these active substances are mainly located in corolla epidermis cells and various types of thrichomes. The content of essential oils in fresh and dried flowers is similar and relatively low. Three triterpenoids were identified in the flowers, i.e., oleanolic acid, β-amyrin, and β-amyrin acetate. The presence of β-amyrin acetate was demonstrated for the first time. Similarly, the presence of iridoid aucubin in the extract from L. album subsp. album corollas were identified for the first time. L. album is a common plant in the flora of Europe and a large part of Asia. It is characterized by abundant and long flowering, which lasts from April to September in Poland; hence, its flowers are a valuable and easily available raw material with nutritional, antioxidant, and anti-allergic effects. Flowers containing various active compounds, e.g., lipophilic compounds and iridoids, are not only used in phytotherapy and cosmetology but can also be recommended as potential supplements in human nutrition and tea additives or food decorations. The consumption of a variety of natural compounds is consistent with the principles of a healthy lifestyle.