ovate-lanceolate

Although Hedera helix and Linaria vulgaris are considered toxic plants, their extracts containing many bioactive compounds are commonly used in the pharmaceutical and cosmetics industries. Plant secretory structures are considered the primary site of synthesis and accumulation of many valuable metabolites. The presence of secretory structures and the location of active substances in H. helix leaf petioles and L. vulgaris inflorescence shoots were detected using light microscopy and histochemical and fluorescence tests. The analyses showed schizogenic secretory ducts in the H. helix petioles and capitate glandular trichomes on the surface of the L. vulgaris shoots. The secretion and secretory cells of the H. helix ducts and the L. vulgaris tri-chomes contained acidic and neutral lipids, sesquiterpenes, polyphenols, tannins, flavonoids, phenolic acids, neutral and acidic polysaccharides, proteins and alkaloids. Data on the

Drugs and cosmetics containing H. helix extracts exert strong anti-inflammatory, antioxidant and antimicrobial effects [Pop et al. 2017, Schulte-Michels et al. 2019. They are dedicated to sensitive skin and are used to treat acne, psoriasis and seborrhoeic dermatitis. They also relieve symptoms of skin irritation, pruritus and genital mycoses [Lutsenko et al. 2010, Ibezim, 2012, Dumitriu et al. 2013. Hedera helix extract positively affects hair growth and is commonly used in foaming agents and body lotions [EMA 2017, Góral andWojciechowski 2020]. It also exhibits conditioning, soothing and toning properties and astringent activity that supports the tightening of skin pores [INCI Beauty 2022]. Hedera helix extract is one of the most effective ingredients of anti-cellulite cosmetics and products to protect stretch mark-prone skin [Hexsel and Soirefmann 2011]. In addition to the antioxidant, anti-inflammatory and antibacterial properties of H. helix leaf extracts, their analgesic, antispasmodic, expectorant and spasmolytic properties are essential for the pharmaceutical industry [Terlecka et al. 2020, Baharara et al. 2021]. Extracts of this raw material are ingredients of syrups and lozenges used in upper respiratory tract infections [Barnes et al. 2020]. They also have anticancer properties [Akhlaghi et al. 2022].
Linaria vulgaris is native to Europe and Asia but is considered an invasive species in the USA and Canada [Sing and Peterson 2011]. This perennial plant is found on grasslands, roadsides, pastures, meadows and fallow lands. It is also a weed of cultivated fields [Saner et al. 1995]. Linaria vulgaris has a 20-90 cm high erect stem with glandular trichomes in the inflorescence part. Its small oblong-lanceolate dense leaves are arranged alternately. The inflorescence is a raceme composed of nectariferous pale yellow snapdragon-like flowers. Its flowering shoots (Herba Linariae) are medicinal and cosmetic raw materials [Chiej 2009]. It contains iridoids, alkaloids, flavonoids, sterols, phenolic and mucilage compounds, organic acids and vitamins [Sokolowska-Wozniak et al. 2003, Vrchovská et al. 2008, Cheriet et al. 2015, Kuang et al. 2018, Sokornova and Matveeva 2021.
In folk medicine, L. vulgaris is known mainly for its mild laxative properties [Lewkowicz Mosiej 2012]. Its extract, used internally, reduces digestion problems and urinary disorders and exerts choleretic effects. Applied externally, it effectively treats haemorrhoids, venous skin ulcers and festering wounds [Gruenwald et al. 2000, Senderski 2016] as well as cough and asthma [Hua et al. 2002]. The extract also protects against hyperlipidaemia and hepatic steatosis, facilitating the removal of toxins from the organism [Berim andGang 2016, Kuang et al. 2018]. Additionally, it exerts expectorant, strongly diaphoretic, sedative, somniferous, slightly analgesic and diuretic effects [Vrchovská et al. 2008]. Due to the content of alkaloids, the extract may have intoxicating activity, and its higher doses may cause nausea and diarrhoea [Fijałkowski and Chojnacka-Fijałkowska 2009]. Used as a cosmetic ingredient, L. vulgaris extract exhibits antioxidant activity, and therefore it retards skin ageing, softens the skin and enhances skin elasticity [Vrchovská et al. 2008]. Due to its anti-inflammatory, antimicrobial and anti-allergic properties, the extract is used to soothe skin itching, heal irritations and wounds and prevent infections [Kelemen andScedo 2003, Senderski 2016]. The flower and herb of the species are used for the preparation of ointments, infusions and decoctions for such skin problems as psoriasis and acne and in formulations against conjunctivitis and dark under-eye circles [Fijałkowski and Chojancka-Fijałkowska 2009]. Diagnosing the morphological and anatomical traits of medicinal and cosmetic raw materials is crucial for assessing their quality, as the evident increase in the demand for herbal medicines and cosmetics often leads to massive plant harvesting from their habitats, which is inconsistent with the guidelines for obtaining plant material. The plant material may also be incorrectly identified and falsified, thus not meeting quality standards [Sultana et al. 2011, Minarchenko et al. 2021.
While the medicinal and cosmetic properties of H. helix and L. vulgaris extracts have been investigated in detail, the anatomical traits of the raw material and the planta distribution of active compounds are unknown. Therefore, this study focused on analysing . Localisation of active substances in Hedera helix leaves and Linaria vulgaris shoots used in pharmaceutical and cosmetic industry. Acta Sci. Pol. Hortorum Cultus, 22(3), 53-68. https://doi.org/10.24326/asphc.2023.4781 the microstructural traits and the location of active substances in H. helix leaves and L. vulgaris inflorescence shoots, used as raw materials in the pharmaceutical industry and cosmetic formulations. The analyses were carried out with the use of stereoscopic and bright-field light microscopy as well as histochemical and fluorescence assays, which are effective methods for preliminary evaluation of the occurrence of valuable bioactive substances and assessment of the medicinal and cosmetic potential of plant raw material [e.g. Tajik et al. 2019, Konarska andŁotocka 2020]. Moreover, the taxonomic value of some morphological and anatomical traits, which may help evaluate the quality (falsification) of medicinal raw materials, was considered.

MATERIAL AND METHODS
Plant material. On August 10, 2021, older lobate Hedera helix leaves (n = 50) were collected randomly from several specimens growing in Akademicka Street in Lublin, Poland (51°15'44''N, 22°30'48''E). Microscopy and histochemical and fluorescence tests. The study was conducted using light and fluorescence microscopy and histochemical assays detecting the presence of secretory structures and biologically active compounds based on the specific chromogenic reactions or (auto)fluorescence. All images were saved in the .tiff format with a resolution of 300 dpi and compression LZW (Lempel-Ziv-Welch). Corel-DRAW x3 software was used to prepare the images for publication, and Corel PHOTO-PAINT x3 was the integral image processing tool used to enhance the clarity of the images.
Hand-cut sections of fresh H. helix petioles (at their mid-length) and L. vulgaris stems (below the inflorescence) were viewed in a 50% aqueous glycerol solution (as a control) and after application of various histochemical assays and fluorochromes using a Nikon SE 102 light microscope or a Nikon 90i flu-orescence microscope, both equipped with a digital camera (Nikon Fi1) and NIS-Elements Br 2 software. The analyses of the control preparations (n = 10) revealed the location of secretory structures, the number of secretory ducts, and the number of secretory epithelial cells. The inner diameter of the ducts' lumen (n = 20) in H. helix and the height of the secretory trichomes (n = 20) in L. vulgaris were measured.
Fluorescence microscopy was used for the localisation of essential oils, flavonoids and phenolic acids after the application of various fluorochromes: Neutral red for essential oils [Kirk 1970], aluminium chloride or magnesium acetate for flavonoids [Charrière-Ladreix 1976] and UV-induced autofluorescence for phenolic acids [Talamond et al. 2015]. Fluorescence was observed using a Cy5 filter set (excitation radiation of 590-650 nm and a barrier filter wavelength 663-738 nm), a FITC filter set (excitation radiation of 465-495 nm and a barrier filter wavelength 515-555 nm) and a TRITC filter set (excitation radiation of 525-565 nm and barrier filter wavelength 555-600 nm). The histochemical and fluorescence methods were applied following standard control procedures suggested by various authors. Each histochemical assay was repeated 6 times to eliminate incorrect interpretation. Similar positive results were obtained 3-5 times.
As shown by the histochemical assays, large droplets of total lipids stained orange or red after the application of Sudan IV ( Fig. 1f-1i) or Neutral red ( Fig. 1j-1l), respectively, were detected in the secretory ducts, epithelial cells and parenchyma cells adjacent to the ducts. Essential oils emitted intense fluorescence after incubation in Neutral red fluorochrome (Fig. 1m). The reaction with Nile blue revealed the acidic and neutral lipids in the secretion (Fig. 1n, 1p, 1q). Acidic lipids were also detected in the non-glandular trichomes (Fig. 1o). Moreover, sesquiterpenes -stained yellow after the application of concentrated sulphuric acid -were visible in the secretion, epithelial cells and surrounding parenchyma cells (Fig. 1r).
The outer cell walls of the epidermis with cuticle, collenchyma and parenchyma cells, fibre-sheathed vascular bundles, and the duct secretion in the H. helix petioles exhibited the presence of phenolic compounds stained blue or navy blue in the reaction with Toluidine blue O ( Fig. 2a-2c) and brown after the use of ferric chloride ( Fig. 2d-2f). The application of potassium dichromate or vanillin-hydrochloric acid revealed the presence of tannins in the collenchyma cells (Fig. 2g) and in the duct secretion, epithelial cells and parenchyma cells located near the secretory ducts (Fig. 2h). In turn, flavonoids contained in the parenchyma and vascular bundle cells exhibited intense fluorescence in the presence of the aluminium chloride and magnesium acetate fluorochromes ( Fig. 2i-2k). Phenolic acids in the duct secretion and parenchyma cells adjacent to the ducts exhibited strong red autofluorescence under UV excitation (Fig. 2l).
Acidic polysaccharides, stained red in the reaction with the Ruthenium red reagent, were detected in all petiole tissues' secretion and cell walls except fibre-sheathed vascular bundles (Fig. 3a, 3b).
In the reaction with Wagner's reagent or Lugol's solution, proteins present in the cuticle, fibre-sheathed vascular bundles and parenchyma cells stained yellow (Figs. 3c-e, 3h, 3i), while brown-stained alkaloids were detected in the epidermis and collenchyma cells and duct secretions (Fig. 3c, f-i).
Linaria vulgaris inflorescence stem. The L. vulgaris inflorescence stem was composed of the uniseriate epidermis with thickened outer and inner walls, several layers of chlorenchyma, uniseriate endodermis, a continuous ring of the secondary vascular tissues with xylem fibres and parenchymatous pith (Fig.  4a). On the stem surface were observed capitate glandular trichomes composed of a 2-4-celled stalk and an oval 2-4-celled head with an average length of 83 µm (ranging from 70 to 94 µm) - Figure 4b, 4c.
The histochemical Sudan IV and Neutral red assays revealed the presence of total lipids in the cuticle, phloem and xylem elements, trichome heads and stalks ( Fig. 4d-4i, 4k), and some epidermal and chlorenchyma cells (Fig. 4j). Fluorescence microscopy revealed bright green fluorescent essential oils in the trichome stalk cells after the application of the Neutral red fluorochrome (Fig. 4l, 4m). Sesquiterpenes in the trichome heads and stalks stained yellow with concentrated sulphuric acid (Fig. 4n, 4o). After the application of Nile blue, acidic lipids were present in all L. vulgaris stem cell layers and in the secretory trichomes stained blue to navy blue ( Fig. 4p-4t).
The cuticle, fibres' cell walls, trichome heads and stalks stained navy blue or turquoise after the application of Toluidine blue O, indicating the presence of phenolics (Fig. 5a-5g), while polyphenols contained in trichome cells stained brown in the reaction with iron chloride (Fig. 5h, 5i). Tannins were detected using potassium dichromate or vanillin-hydrochloric acid in the cells of trichome heads and stalks, cuticles, and vascular tissue elements, especially fibres ( Fig. 5j-5m). Flavonoids in the cuticle, epidermis, trichome heads and stalks, and duct secretions emitted intense fluorescence light in the reaction with aluminium chloride or magnesium acetate fluorochromes ( Fig. 5n-5q). In turn, intense autofluorescence of phenolic acids was visible in trichome heads (Fig. 5r).

DISCUSSION
Plant secretory structures are regarded as the main sites of the synthesis and accumulation of biologically active substances used in the pharmaceutical and cos-a-g -ruthenium red; h-l -Wagner's reagent; m, n -Lugol's solution a-g -acidic polysaccharides present in the walls of all cell layers (a-c) and in the trichome head and stalk cells (d-g); h-j -alkaloids present in the epidermis and vascular elements; k -alkaloids in the trichome head cells and proteins in the trichome stalk cells; l-n -proteins in the fibres (m) and glandular trichomes (l, n); h, i, m -starch present in the chlorenchyma cells. Cu -cuticle, Ep -epidermis, Ch -chlorenchyma, En -endodermis, Ph -phloem, Xy -xylem, F -fibres, P -parenchyma, Hc -head cells, Sc -stalk cells. Scale bars: a, h -100 µm; b, m -50 µm; c-g, i-l, n -30 µm Fig. 6. Fresh cross-sections across the Linaria vulgaris inflorescence stem subjected to histochemical tests . Localisation of active substances in Hedera helix leaves and Linaria vulgaris shoots used in pharmaceutical and cosmetic industry. Acta Sci. Pol. Hortorum Cultus, 22(3), 53-68. https://doi.org/10.24326/asphc.2023.4781 metic industry [e.g. Haratym et al. 2020, Sulborska et al. 2020.
External to the vascular bundles in the petioles of H. helix, there were 7-9 larger and 3-5 smaller schizogenic secretory ducts, usually surrounded by 4-9 secretory epithelial cells. It supports the data by Săvulescu and Luchian [2009], who showed that these cells had thin walls, prominent nuclei, and dense cytoplasm. As shown by Amini et al. [2019], H. helix petioles have 9 to 12 secretory ducts, whereas their number in H. pastuchovii petioles is lower, i.e. from 6 to 8. The presence of secretory ducts is a general characteristic of other araliacean species [Plunkett et al. 2018]. The present study showed the presence of non-glandular stellate trichomes on the H. helix petiole surface and druses in many petiole cells. Similarly, other authors observed numerous oxalate crystals in the leaf cells in this taxon [Săvulescu andLuchian 2009, Amini et al. 2019]. The presence of non-glandular stellate trichomes on the leaf surface of this species was also described by Kamińska et al. [2018]. The authors found that the compounds contained therein may cause skin reactions -lesions. As Hart and Henwood [2006] and Wen [2011] demonstrated, the indumentum in the araliaceans often comprises simple or multicellular stellate trichomes. As suggested by Amini et al. [2019] and Lestari and Elya [2019], the presence of secretory ducts, calcium oxalate crystals, and non-glandular trichomes has great diagnostic importance in Hedera spp. and the family Araliaceae. These traits may be helpful for the evaluation of the quality (falsification) of medicinal raw materials and in taxonomy Domaciuk 2018, Haratym et al. 2020].
The approx. 80 µm long multicellular capitate glandular trichomes on the surface of the Linaria vulgaris inflorescence stems were composed of several stalk cells and a 2-4-celled head. Similar spherical glandular trichomes with a similar length were observed by Jachuła et al. [2018] on the pedicel and sepals of this species. Capitate glandular trichomes with varying distribution, density, height and number of trichome stalk and head cells have also been identified on stems, leaves, bracts, inflorescences, flowers and capsules in other Linaria species, e.g. L. aeruginea, L. arabiniana, L. depauperata and L. supina [Segarra and Mateu 2001]. As suggested by these authors, the morphological traits of glandular trichomes are essential diagnostic and taxonomic traits of the Linaria spp.
With the use of the histochemical assays, several groups of bioactive substances, i.e. lipid compounds, phenolic compounds, polysaccharides, proteins, and alkaloids, were identified in the secretion, secretory epithelial cells of H. helix ducts, and secretory trichomes of L. vulgaris. Neutral lipids, essential oil, and sesquiterpenes were present in the secretory structures of both species. The presence of essential oil in H. helix leaves was also confirmed by histochemical studies conducted by Kamińska et al. [2018], while Jachuła et al. [2018] demonstrated essential oil content in L. vulgaris trichomes. As reported by Gruenwald et al. [2000] and Wichtl [2004], essential oil extracted from H. helix leaves contained germacrene B, β-elemene, γ-elemene, methyl ethyl ketone, methyl isobutyl ketone, trans-2-hexenal, trans-2-hexanol, germacrene D, β-caryophyllene, sabinene, α-, β-pinene, limonene and furfural. No information on the composition of L. vulgaris essential oil is available in the literature. In turn, essential oils extracted from the herb of other Linaria species were found to contain 2-methoxy-4-vinylphenol, α-terpinene and 3,5-dimethylphenyl isocyanate [Nafis et al. 2021]. Sesquiterpenes, which were identified in the H. helix petioles and L. vulgaris stems, are standard essential oils components exhibiting anti-inflammatory, antiparasitic, anti-carcinogenic, anti-diabetic, anti-proliferative, and antimicrobial properties [Chadwick et al. 2013, Bartikova et al. 2014. The present study also showed the presence of acidic lipids in the H. helix ducts and the L. vulgaris secretory trichomes. Unsaturated fatty acids, i.e. petroselinic, oleic and vaccenic acids, representing the group of acidic lipids, were identified in H. helix extracts by Lognay et al. [1987], whereas linoleic acid was detected in L. vulgaris extracts by Ucciani et al. [1994].
The fluorescence and histochemical tests confirmed the presence of phenolic compounds (flavonoids, tannins, and phenolic acids) in the cells and secretory ducts of H. helix petioles and in the tissues and the secretory trichomes of L. vulgaris stems. As suggested by Wichtl [2004] and Bezruk et al. [2020b], quercetin and kaempferol, i.e. substances responsible for the anti-arthritic, antioxidant, and antimicrobial activity, are the essential flavonoids of H. helix extracts [Pop et al. 2017, Shokry et al. 2022. In turn, L. vulgaris extracts have been found to contain linarin, neo-linarin, pectolin, and isolinarin A and B, which have mainly anti-inflammatory and antipyretic properties [El'kina et al. 2014, Kuang et al. 2018. As demonstrated by other researchers, many Linaria species are a rich source of valuable flavonoids [e.g. Hanfer et al. 2019, Ahmed-Chaouch et al. 2021. Intensive autofluorescence of phenolic acids was observed in the secretory structures of both examined taxa. Wichtl [2004] reported the presence of caffeic, dihydroxy-benzoic, chlorogenic, neo-chlorogenic, dihydroxybenzoic, protocatechuic, and p-coumaric acids in H. helix extracts and protocatechuic, gallic, ρ-hydroxybenzoic, vanillic, salicylic, caffeic, ρ-coumaric, ferulic, homoprotocatechuic, ohydroxyphenylacetic, gluco-syringic, and ρ-methoxybenzoic acids in L. vulgaris herb [Hua et al. 2000, Sokolowska-Wozniak et al. 2003]. As Wang et al. [2019] show, phenolic acids exhibit strong antibacterial, antioxidant, and anti-inflammatory properties. Tannins were another group of phenolic compounds detected in the H. helix and L. vulgaris raw material. These metabolites were located in the peripheral layers, and secretory ducts of the H. helix petioles and in the secretory trichomes and vascular bundles of the L. vulgaris stems. The presence of tannins in the epidermis of H. helix leaves was also confirmed by histochemical studies [Kamińska et al. 2018] and phytochemical analyses [Suica-Bunghez et al. 2020]. Like other phenolic compounds, tannins have many pharmaceutical and cosmetic applications due to their antibacterial, antiviral, antioxidant, antitumour, and anti-oncogenic activities [Pizzi 2019 and references therein].
The use of Ruthenium red helped indicate mucilages from the group of acidic polysaccharides in the H. helix duct secretions and the L. vulgaris trichome heads. The presence of mucilage in H. helix leaves was also confirmed by Huang et al. [2016], who demonstrated that pectic rhamnogalacturonan-I polysaccharide domains held together by a nanospherical arabinogalactan protein molecule were its significant components. H. helix mucilage nanoparticles have protective activity against UV radiation; hence, they can be used as components of photoprotective products and cosmetic fillers or as nanocarriers in drug delivery systems. Plant mucilages are also used as cosmetics stabilisers and pharmaceutical agents in ther-apies for various metabolism-related diseases. They have natural antimicrobial activity and can also be a matrix for introducing other antimicrobial agents in manufacturing food, cosmetics and healthcare materials [Dybka-Stępień et al. 2021].
The presence of alkaloids in the H. helix and L. vulgaris raw material was confirmed by the histochemical assays based on the use of Wagner's reagent and Lugol's solution. Nitrogen-supplying amino acids, which may be provided by protein decomposition in the organs of both plants, are precursors required for the biosynthesis of alkaloids. The presence of the alkaloid emetine in some H. helix varieties was confirmed by Mahran et al. [1975] and Alkattan et al. [2021]. Emetine exerts an expectorant, emetic, and protozoicidal effect; however, its high doses accumulate in the body and may cause damage to the stomach or liver and even cardiac arrest [Gaillard et al. 2003, ChemIDplus 2022. Other researchers detected the presence of two quinazoline alkaloids with spasmolytic and peristaltic activity, i.e. linarinic acid and peganine (vasicine) in L. vulgaris herb [Hua et al. 2002, Ma et al. 2008. Peganine also exerts a cardiac-depressant effect and uterine-stimulant activity [Rajani et al. 2008]. In a previous study, quinazoline alkaloids were also demonstrated to have potential anti-inflammatory and anti-allergic activities [Chandrika et al. 2008]. Sing and Peterson [2011] and Hanfer et al. [2019] reported high content of tricyclic quinazoline alkaloids in Linaria tingitana and L. dalmatica organs.

CONCLUSIONS
The secretory structures in the H. helix petioles and the L. vulgaris inflorescence shoots were the sites of accumulating many valuable bioactive substances with cosmetic and medicinal potential. The secretions of the H. helix secretory ducts and the L. vulgaris trichomes contained acidic and neutral lipids, essential oil, sesquiterpenes, polyphenols, flavonoids, phenolic acids, tannins, neutral and acidic polysaccharides, proteins, and alkaloids. Some metabolites were present not only in the secretory structures but also in other layers of cells in the H. helix petioles, and L. vulgaris stems. The presence of secretory ducts, glandular and non-glandular trichomes, oxalate crystals and other . Localisation of active substances in Hedera helix leaves and Linaria vulgaris shoots used in pharmaceutical and cosmetic industry. Acta Sci. Pol. Hortorum Cultus, 22 (3), 53-68. https://doi.org/10. 24326/asphc.2023.4781 anatomical traits of the cosmetic and medicinal raw material of both species have taxonomic value for the identification of the analysed genera and families and may be helpful in the evaluation of the quality (falsification) of these raw materials.

SOURCE OF FUNDING
Poland's Ministry of Sciences and Higher Education as part of the statutory activities of the Department of Botany and Plant Physiology, University of Life Sciences in Lublin (project LKR/S/49/2022BiO).