The morpho-anatomical study of Solidago virgaurea L. species from the flora of Republic of Moldova

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The morpho-anatomical study of Solidago virgaurea L. species from the flora of Republic of Moldova

Calal Tatiana, Fursenco Cornelia, Ionita Olga, Ghendov Veaceslav

Calal Tatiana. PhD Hab. in biology, head ofpharmacognosy and pharmaceutical botany department, State University of Medicine and Pharmacy „Nicolae Testemitanu”

Fursenco Cornelia. University assistant, State University of Medicine and Pharmacy „Nicolae Testemitanu”

Ionita Olga. PhD in biology, senior research associate, Botanical Garden (Institute) of the Academy of Sciences of Moldova

Ghendov Veaceslav. PhD in biology, head of spontaneous flora and herbarium department, Botanical Garden (Institute) of the Academy of Sciences of Moldova

Summary: This study focuses on the morpho-anatomical evaluation of the sp. Solidago virgaurea L., the only species of g. Solidago spread in the spontaneous flora of the R. of Moldova. The goldenrod plant has been used for centuries in the traditional medicine of European countries as a diuretic and antiflogistic remedy. The main morphological characters of the species and vegetable product Solidaginis virgaureae herba, such as: cylindrical, striated, greenish-yellow (outside) and greenish-white (inside) stem fragments; sessile or short petiolate cauline leaves with the elliptical or lanceolate lamina crossed by a proeminent venation, especially on the lower epidermis; flower heads composed of ray ligulate and tubular yellow florets, were determinated. By the anatomical study of goldenrod, were revealed the structures with diagnostic role in order to identify the species and vegetable product: for leaf - dorsoventral anatomical type, collateral closed vascular bundles, 2 types of protective trichomes (multicellular, conical and multicellular, flabelliform) and secretory hairs (stipe of 1 -2 cells and unicellular gland), amphystomatic leaf with anomocytic stomata; for stem - histological regions on cross-section (epidermis, cortex and central cylinder), 2 types of protective trichomes (multicellular, conical and multicellular, flabeliform), angular collenchyma, collateral open vascular bundles and secretory canals.

Key words: Solidago virgaurea, morphology, anatomy, diagnostic indicator

Introduction

goldenrod leaf macroscopic

The family Asteraceae comprises about 25,000 species, reunited in 1500 genera, including g. Solidago with about 120 species, most of which are native to North America, less to Eurasia, being spread throughout the continents [1,2].

Species belonging to g. Solidago L. are perennials, which develop simple alternate, linear-lanceolate leaves with the entire limb. The plants of this genus have flower heads clustered in simple or paniculate racemes, with ligulate yellow marginal florets and tubular, bisexual disc florets. The fruits represent cylindrical achenes with 8-10 ribs provided with pappus made of serrated bristles [3,4].

The main most studied species are S. virgaurea L., S.gigantea L., S. canadensis L. and S. chilensis M. These species have different origins: S. virgaurea is native to Europe, S. canadensis and S. gigantea - to North America and S. chilensis - to South America [5, 6].

The only species of g. Solidago spread in the spontaneous flora of the R. of Moldova is S. virgaurea, known with the popular romanian common names: varga-de-aur, splinuta, floare-boiereasca, manunchi, splinarita, smeoaica [8]. This species has been used for centuries in the traditional medicine of European countries as a diuretic and antiflogistic remedy [9]. The vegetable product (VP) Solidaginis virgaureae herba, which includes aerial parts from sp. S. virgaurea, is official in the European Pharmacopoeia (8th Edition, 2014), the British Pharmacopoeia (1976), the French Pharmacopoeia (1989), and the Polish Pharmacopoeia (2008) [10]. Phytochemical studies have demonstrated the presence of a wide range of active principles: flavonoids [11], triterpenic saponosides [12], phenolic acids [13], polysaccharides, tannins, essential oils [14]. The totality of these active principles has been studied over time demonstrating important pharmacological actions: urinary antiseptic [15], antiinflammatory [16], antioxidant [17,18], diuretic [19], spasmolytic, antibacterial [16,20,21], antifungal, cytostatic and immunomodulatory [22].

Purpose of this paper - the morpho-anatomical study of the plants of sp. S. virgaurea in order to elucidate the specific structural indicators with diagnostic character in the identification of the species and its vegetable product.

Materials and Methods

Plants of sp. S. virgaurea, collected in the flowering phase, preserved in the Herbarium of the Botanical Garden (Institute) of the ASM, herborised during the 1970-1995 period, from different ecological areas and localities of the Republic of Moldova, have served as botanical material for the morpho-anatomical study (tab. 1). Also, were used the goldenrod plants, collected from the Landscape Reserve " Suta de Movile" (Glodeni district), during the flowering period (august 2017).

Table 1

The macroscopic study was based on the morphological characters of the species and vegetable product (VP) Solidaginis virgaureae herba. The main indicators used for species analysis include: plant height, shape and relief of the rhizome and stem surface, type of the leaf and its arrangement on the stem, size and configuration of the leaf blade, type of the inflorescence and fruit. The macroscopic examination of VP Soli- daginis virgaureae herba comprises the morphological characteristics of each organ, the measurement of its component parts, the organoleptic samples (color, smell, taste).

The microscopic study was performed on clarified superficial preparations of the leaf, flowers/

Results and discution

Morphology of the sp. S. Virgaurea

Figure 1. Plants of S. virgaurea: A - radiate flower heads (ligulate ray florets and tubular disc florets), B - cauline leaves, C - basal leaves, D - oblique rhizome

Perennial herbaceous plant, which develops underground an oblique, cylindrical, knotless rhizome - the result of many scars (fig. 1D). The stem is erect, can reach a height of 100 cm, the upper part being branched and pubescent. The leaves are alternately arranged, slightly pubescent on the upper and pubescent on the underside. The basal ones are ovate or ovate-elliptic, with winged petiole and acute apex, and the upper leaves - linear-lanceolate or elliptic, shortened petio- late with serrated or entire margin (fig. 1B,C). The flowerheads are of the radiate type - with distinct ray and rescence bracts and on cross sections through leaf lamina and stem according to the classical techniques [23,24]. The anatomical study was performed at the Department of pharmacognosy and pharmaceutical botany, SUMPh "Nicolae Testemitanu", using the binocular optical microscope Micros (Austria), with a digital camera, coupled to the computer, at 4x, 10x and 40x objective magnification.

The results of the determinations were statistically processed using the Microsoft Excel application of the Office 2007.

Disc yellow florets. The flower heads are clustered in a simple or paniculate branched raceme. Radial florets are ligulate, female, and central - tubular, bisexual (fig. 1A). The receptacle is flat and smooth. Fruit - cylindrical achene (cypsela), which has 8-10 ribs, provided with hair-like calyx (pappus).

Macroscopic characters of Solidaginis virgaureae herba

Figure 2. Vegetable product Solidaginis virgaurea.e herha

Vegetable product Solidaginis virgaureae herha represents the flowering aerial parts of sp. S. virgaurea, being a combination of dried fragments of stems, leaves, flower heads, separate ray and disc flowers and involucre bracts (fig 2). The stem is cylindrical, fistulous, striated, glabrous or slightly pubescent with a diameter of 0.5-1 cm. The outer surface of the stem is greenish-yellow, and inside - greenish-white. The basal leaves are oblong-lanceolate or wide-elliptical, with the serrated margin, tapered at the base into a long, winged petiole; the cauline leaves are alternate, smaller in size compared to basal ones, elliptical or lanceolate, with an entire or slightly toothed margin, being sessile or short petiolate. The leaf surfaces are glabrous or only slightly pubescent, with a prominent reticulate venation, especially on the lower surface. The flowers are grouped into heterogeneous flower heads with a diameter of 0.5 cm. The flower heads form a tightly pyramidal panicle. The involucre consists of 2-4 rows of bracts with the outer surface glabrous or slightly hairy. Each flower head consists of 6-12 female, ligulate florets with a length of 0.3-0.5 cm, approximately twice as long as the bracts and 10-30 hermaphrodite, tubular florets of 0.20.3 cm in length. Both types of florets are yellow. The ovary is inferior, with a ribbed, brown surface; it is accompanied by a whitish pappus. The smell is specific, the taste - mildly astringent.

The morphological values of the herborised plant leaves differ as shown in the table 2:

Table 2. Morphological characteristics of goldenrod leaf from different localities

Note: n - deviation

Microscopic characters of Solidaginis virgaureae herba

Microscopy of the leaf (superficial view). Superficial preparations of basal, middle and apical leaves of goldenrod, cleared with chloralhydrate or 3% NaOH were performed.

The analysis of superficial preparations of the leaf shows that both epidermises are single-layered, composed of well wrapped cells. The cells of the upper epidermis of the leaf are polygonal, isodiametric, with slightly sinuous and uniformly thickened external walls. Stomata are present on both epidermises (am- phistomatic leaf), but numerically, more on the abaxial surface. For the upper epidermis, it is specific the ano- mocytic type of stomata: the guard cells are surrounded by 4-5 subsidiary cells of the same shape as other epidermal cells (fig. 3).

Figure 3. Superficial view of goldenrod leaf (upper epidermis): A (10*), B (40*); 1 - stoma, 2 - cell wall with punctuations

Numerous, surrounded by rule of 3-5 cells, moreover there are 4 subsidiary cells - anomotetracytic stoma type (fig. 4A,B).

The lower epidermis consists of isodiametric cells with an evident sinuous outline; occasionaly, there is a moniliform thickening of the cell walls.

Figure 4. Superficial view of goldenrod leaf (lower epidermis): A (10*), B (40*); 1 - stomata, 2 - flabelliform trichome 3 - sinuous cell walls 4 - striated cuticle

Often, we notice the striated cuticular ornamentation on the abaxial surface of the epidermis.

More evident stripes, there are present on the subsidiary stomata cells (fig. 4C). Both epidermises develop secretory trichomes and two types of protective (covering trichomes): conical and flabelliform (fig. 5A,B,C).

Figure 5. Superficial view of goldenrod leaf (lower epidermis): A (10*), B, C (40*); 1 - flabelliform trichomes, 2 - protective multicellular trichome with the broken apical cell

For the goldenrod leaves, it were found the following specific trichomes:

- multicellular protective trichomes describing a conical shape, consisting of a single row of 2-7 cells; the cell walls are thin, with a smooth or slightly wavy surface. They are frequent on the both surfaces of the leaf blade (fig. 6B,C,D,E), mainly on the veins and leaf margins (fig. 6 B). Often, the apical cell of the trichome is curved or may easily break, only the basal cells of the trichomes remaining on the leaves (fig. 6E). Usually, around the trichomes, the epidermal cells are radially disposed, forming a basal rosette (fig. 6D,E). The basal cells of the trichomes are filled with a brownish-red content (fig. 6E,F);

- uniseriate multicellular trichomes consisting of 2-3 cells, the apical one can be elongated to the filiform, for this reason, these trichomes are also found in the literature review [6,25,26] as flabelliform, moniliform or flagellate trichomes. These ones are pennant-like, with 1-3 thin basal cells, provided with a filamentally elongated end-shaped cell in the form of a flagella or a ribbon. The connection between the basal cells and the or less rounded cell. This type of trichomes are more common on the lower epidermis of S. virgaurea middle leaves. Resembling the conical multicellular trichomes, we notice the basal rosette from epidermal cells around flabelliform trichomes along with the striped cuticle (fig. 5B,C);

- the secretory trichomes that are short, with a stalk formed of 1-2 cells and a unicellular head, representing the apical secretory cell. This type of trichomes was found more common on the upper epidermis of the apical leaves (fig. 6A). The secretory trichomes are less numerous compared to the covering ones.

Flagella apical cell is maintained by an enlarged, more.

Figure 6. Superficial view of goldenrod leaf: A, B, C, D, E (10*), F (40*); 1 - secretory trichomes, 2 - multicellular protective trichomes on the leaf margin, 3 - multicellular protective trichomes on the veins, 4 - curved multicellular protective trichomes, 5 - protective trichome with the broken apical cell, 6 - basal cells of the trichome filled with a brownish-red content

Figure 7. Cross-section view of goldenrod leaf A (10*), B, C (40*) - transversal section of leaf lamina; 1 - upper epidermis, 2 - palisade tissue, 3 - spongy tissue, 4 - lower epidermis.

Microscopy of the leaf (cross-section view). The leaf in cross-section has a dorsoventral (bifacial) structure. Upper and lower epidermis were distinguished, including well developed mesophyll, differentiated into palisade parenchyma and spongy parenchyma (fig. 7A,B,C).

The upper epidermis cells are polygonal, singlelayered, well wrapped and covered with a layer of cuticle. The lower epidermis consists of a row of polygonal cells smaller in size compared to those that form the upper one. The mesophyll of the leaf has a bifacial structure: the palisade parenchyma occurs in the upper part of the leaf blade, while the spongy parenchyma occupies the abaxial area of the leaf. Palisade chlorenchyma consists of two layers of living, cylindrical-elongated cells, incorporating a large number of chloroplasts with small intercellular spaces (fig. 7A,B). The spongy tissue occupies the largest area of the leaf mesophyll, being located beneath the lower epidermis, it consists of oval or lobed cells with large intercellular spaces. The number of chloroplasts is relatively reduced in contrast to palisade mesophyll (fig. 7A,C).

Figure 8. Cross-section view of goldenrod stem: A (4*), Lugol solution; B (10*); C (10*), Methylene blue solution; 1 - epidermis, 2 - angular collenchyma, 3 - cortical parenchyma, 4 - endodermis, 5 - phloem, 6 - cambium, 7 - xylem, 8 - vascular bundles, 9 - medullary parenchyma, 10 - secretory canals

Microscopy of the stem (cross-section view). Analysis of the microscopic images shows that stem in cross-section has a polygonal contour. The epidermis is single-layered, cutinized, composed of well packed, polygonal cells (fig. 8B). Below the epidermis, we mention the angular collenchyma with cell thickening at intercellular contact points, followed by the cortical parenchyma, comprising several layers of large, oval- spherical cells (fig. 8B,C). At the border between cortical parenchyma and endodermis, secretory canals are present (fig. 8D). The vascular cylinder is made up of roundly arranged collateral open vascular bundles, situated very close to each other. The medullary parenchyma is well developed, consisting of parenchymatous cells of the fundamental tissue (fig. 8A).

Microscopy of the flower (superficial view). The ligulate ray florets show the epidermis consisting of rectangular-shaped cells with thin cell walls without intercellular spaces. The cells are rich in spherical, globular chromoplasts (fig. 9A,B).

The tubular floret shows the epidermis formed by cells of the same shape as those of the ray floret epidermis, although, we mention the elongation of the cells and thickening of their walls as well as the formation of cuticle strips. Chromoplasts are present abundantly, usually in microscopic view, they look like forming groups. On the stigma of the tubular floret, there are numerous pollen grains of spherical shape with irregular surface (fig. 9C,D). At the base of the floret there is attached the pappus, made up of numerous bristles. The pappus is abundant, being composed of multiseriate bristles with the marginal cells overlapping outwards (fig. 9E,F).

Figure 9. Superficial view of goldenrod ligulate floret A, B (40*) and tubular floret C (40*), D (10*), E (4*), F (10*); 1 - orange, spherical chromoplasts, 2 - ligulate floret epidermis cells, 3 - tubular floret epidermis cells, 4 - spherical pollen grains with irregular surface, 5 - pappus at the base of tubular floret, 6 - multiserriate, imbricated pappus bristles

Microscopy of the bract (superficial view). The bract epidermises consist of polygonal, well packed cells, usually with the sinuous walls. The anomocytic type of stomata is distinguished - stoma guard cells are surrounded by 4-6 subsidiary cells of the same shape as the other epidermal cells (fig. 10A). On the margin of the bracts, we noted 2 types of protective trichomes: multicellular, conical in shape or curved trichomes and multicellular, flabelliform ones (fig. 10B,C). Also, there is the evidence of multicellular, biseriate, secretory trichomes (fig.10C).

Figure 10. Superficial view of goldenrod flower head bracts: A (40*), B (10*), C (40*); 1 - stomata, 2 - subsidiary cells, 3 - multicellular flabelliform trichomes, 4 - multicellular curved protective trichomes, 5 - biseriate secretory trichome, 6 - broken secretory trichome

Thus, this was the first local study focused on performing the morphological and anatomical investigation of sp. S. virgaurea from flora of the R. of Moldova and its VP Solidaginis virgaureae herba. The most important morphological indicators that play a special role in the identification of the VP are the following ones: striated, fistulous stem; oblong-lanceolate or wide-elliptical basal leaves with serrated margin, tapered into a winged petiole; elliptical or lanceolate cauline leaves with the entire or fine-serrated margin, sessile or short petiolate; glabrous or slightly pubescent surface of the leaves; proeminent reticulate venation, especially on the lower surface; ray ligulate florets and tubular ones grouped in flower heads and separate florets; involucre of 2-4 rows of bracts, with the smooth surface and serrated margin. The morphological traits of goldenrod occurring in the R. of Moldova are in accordance with those described by Dobjanschi et al. (2005) for the goldenrod from the flora of Romania [27].

The anatomical characteristics that are of the greatest interest for the sure identification of sp. S. vir- gaurea, mentioned in this study, include: amphisto- matic leaf, anomocytic stomata, the presence of multicellular (conical and flabelliform) trichomes and secretory ones on the both epidermises, dorsoventral leaf structure, collateral open vascular bundles and secretory canals in the stem. The type of anomocytic stomata described for the leaf are in agreement with Szymura and Wolsky (2011). In their study, also, it is mentioned that anomotetracytic stomata were the type most frequent in other Solidago taxa occuring in Poland [25]. According to Metcalfe and Chalk (1950), in Aster- aceae, the stomata are commonly anomocytic and an- isocytic [28]. The taxonomical value of trichome characteristics and trichome distribution in genera of the Asteraceae family is shown by the Sasikala and Narayanan (1998) [29]. The presence of two types of multicellular trichomes (conical and flabelliform) on goldenrod leaf was mentioned by Dobjanschi et al. (2005),

Szymura and Wolsky (2011) [25,27]. Trichomes of the same both types were shown by Buinov (2013) on S. dahurica leaf, by Douglas et al. (2016) on S. chilensis leaf, by Fedotova (2012) on S. caucasica leaf [30,5,31]. They vary in form, size, distribution and abundance. The dorsoventral mesophyll structure of the European native S. virgaurea, is noted by Szymura and Wolsky (2011), being different from Solidago taxa of American origin [25]. The presence of secretory canals on the border between cortical parenchyma and endodermis of the goldenrod stem is in accordance with Dobjanschi et al. (2005) [27].

Conclusions

The macroscopic study of Solidaginis virgureae herba highlights the following morphological distictive indicators: cylindrical, fistulous, striated, greenish-yellow (outside) and greenish-white (inside) stem fragments; sessile or short petiolate cauline leaves with the elliptical or lanceolate lamina crossed by a pro- eminent venation, especially on the lower epidermis; flower heads composed of ray ligulate and tubular yellow florets. The anatomical study of goldenrod occurring in the R. of Moldova reveals structures with diagnostic character in order to identify the species and its VP: 2 types of protective trichomes (multicellular, conical and multicellular, flabelliform) and secretory hairs (stripe of 1-2 cells and unicellular gland), amphysto- matic leaf with anomocytic stomata, dorsoventral anatomical type of the leaf, collateral closed vascular bundles in the leaf; the presence of angular collenchyma, collateral open vascular bundles and secretory canals in the stem. The following histological zones were identified on cross-section of the stem: single-layered epidermis, cortex including angular collenchyma, parenchyma of the bark followed by endodermis.

Note the presence of secretory canals in the bark, central cylinder with collateral open vascular bundles in cycle, separated by medular parenchymal rays.

The anatomical traits of goldenrod that are taxonomically useful for identifying the species and vegetable products are: for the leaf - anomocytic type of stomata, two types of multicellular protective trichomes (conical and flabelliform), secretory trichomes, trichomes basal cells with a brown-reddish content, bifacial anatomical structure; for the stem - single-layered epidermis, angular collenchyma, collateral open vascular bundles, presence of secretory canals; for flowers - irregularly shaped spherical pollen grains on tubular flowering stigma, pappus composed of seriated bristles at the base of tubular floret; for bracts - two types of protective trichomes (conical and flabelliform) and biseriate secretory trichomes.

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