Med. Surg. J. – Rev. Med. Chir. Soc. Med. Nat., Iaşi – 2019 – vol. 123, no. 1
PHARMACY ORIGINAL PAPERS
MORPHOLOGIC PROFILE OF TWO LEUZEA SPECIES HARVESTED FROM WILD FLORA OF EASTERN ROMANIA
A. F. Paduraru1, Oana Cioanca1*, Elvira Gille2, Cristina Iancu1, Flavia Burlec1, Luana Enache1, C. Toma3, Monica Hancianu1 1.“Grigore T. Popa” University of Medicine and Pharmacy, Iasi, Romania National Institute of R&D for Biological Sciences 2. “Stejarul” Biological Research Centre, Piatra Neamt, Romania “Al. I Cuza” University, Iasi, Romania 3. Faculty of Biology *Corresponding author. E-mail: [email protected]
MORPHOLOGIC PROFILE OF TWO LEUZEA SPECIES HARVESTED FROM WILD FLORA OF EASTERN ROMANIA (Abstract): Leuzea carthamoides DC and Leuzea salina are two plant species found in Chinese and Russian traditional medicine for their adaptogen and stress relieving properties. There are still many unknown bioactive compounds that may influence the mechanism of action of these species. Nevertheless, all chemical investigations are directly related to the proper identification of the raw material. Aim: In Romania, it is known that two different species of Leuzea grow in the wild flora, but there is no data that confirms the difference between the species. Moreover, there are specialists that consider that L. carthamoides DC. is a synonym for Rhaponticum carthamoides (Willd) Iljin, which rises a series of questions regarding the botanical belonging. Our aim was to observe and as- sess possible taxonomic differences between the two Leuzea species. Material and meth- ods: The investigations consisted in the morphologic assessment (microscopy, scanning electronic microscopy) of each important structure found in the roots, stems and leaves of both species on plants harvested from Iasi and Neamt counties in 2017. Results: The micro- scopic observations indicated both similar and different structures that are related to the en- vironmental conditions. Some of the observed features can confirm the species and the growth conditions. Conclusions: Although, the results confirmed the taxonomic variability between the investigated samples, further research is still undergoing to assess the chemical and the biological profile based upon the maker compounds important for taxonomy. Key- words: LEUZEA, ROOTS, LEAVES, MICROSCOPY, VARIABILITY, TAXONOMY.
Leuzea species belong to Leuzea genus species. Leuzea carthamoides has a long although, there are researchers that insist history in traditional Siberian medicine that Leuzea and Rhaponticum genus are being used to tone the body and combat synonyms. Moreover, the existent scientific following a long illness (1-3). It is still literature presents mainly Rhaponticum presently used as a remedy for male sexual carthamoides (Willd) Iljin and thus, there dysfunctions. The bioactive compounds of is still great need for more data to confirm this species are ecdysteroids, flavonoids the taxonomic characteristics of Leuzea and polyphenolic acids (4-9). From this
187 A. F. Paduraru et al. species, hydroalcoholic extracts from roots order to identify possible taxonomic simi- administered at a dose of 500 mg / kg body larities and differences. weight in rats increase the locomotor activ- ity, antagonize the narcotic effect of chlo- MATERIAL AND METHODS ralhydrate, increase brain excitability and The plant material was harvested from increase learning memory (4, 7, 10-11). the wild flora in the summer of 2017; Leu- Leuzea carthamoides roots decoction cor- zea carthamoides was collected from Ne- rects depressive status in humans and in- amt county, whereas Leuzea salina raw creases health in patients with somatic material was obtained from Iasi County. gastrointestinal pain. Typically, roots and The sampling of individuals was done for rhizomes are used to prepare extracts used each species within the Biology Faculty, for the adaptogen and tonic qualities, being “Al. I. Cuza” University Iasi. The vegetal incorporated in their entirety or as separate material was represented by the under- fractions in nutraceutical supplements to ground and aerial vegetative organs taken promote muscle development, to treat im- from plants in the antecedent stage. Vege- potence and to eliminate physical and men- tative organs were fixed and conserved in tal fatigue. For this reason, since 1958 70º alcohol. For both samples various types Leuzea has been in the composition of of sections were made using botanical mi- well-known preparations in mixtures with crotome and shock marrow, as follows: Schisandra chinensis, Panax ginseng, Ara- upper, middle and inferior transversal axial lia mandshurica and Eleutherococcus sections through root, rhizome and stem, senticosus, species with adaptogen proper- also transversal and superficial sections ties (9-10). through the lamina (at the median and lat- Regarding Leuzea salina there is not eral ribs) were obtained. much mentioned in scientific literature and Clarification and coloring according to our observations the plant has of the sections a short and thick rhizome with numerous The sections thus obtained were collect- long, thick, black-and-brown roots. Also, ed and the cellular content was removed by the erect stem is thick, cylindrical, simple, introducing the vegetal material sections finely striated, 60-75 cm tall, while the into a few drops of sodium hypochlorite, basal leaves form long rosettes, but the rest where different time for contact was used of the leaves are petiolate, ovate or elliptic. depending on the vegetative organ from The flowers of magenta color are similar to which they originated (underground organs - Leuzea carthamoides (2-3, 11). 20 minutes, stems - 15 minutes, leaf-10 The purpose of our research is the com- minutes). Superficial sections of the leaf and parative investigation of two species of leaf stalks have not been mapped. The clari- Leuzea, namely a species recognized as a fied sections were processed in conformity medicinal species, Leuzea carthamoides with double coloring technique (iodine DC, and a species about which there is green and red ruthenium), but the superficial almost no data in specialized literature: sections of the leaves and stems were col- Leuzea salina. Our intention was to study ored only once with one of the mentioned the two Leuzea species in terms of morpho- reagents. As a result of the double colora- logical and histological characteristics in tion: the lignified cell walls are colored in
188 Morphologic profile of two Leuzea species harvested from wild flora of Eastern Romania blue, and the non-lignified in violet; the and differences for the investigated sam- suberus is colored green, and the parenchy- ples. Briefly, at the severed level, Leuzea ma and the phloem in red and pink. carthamoides root has a typically second- Thus, the plant material was fixed in ary structure. On the outside of the bark glutaraldehyde, dried at the critical point, there was a very thin periderm with 1-2 metallized and then observed and photo- layers of cork cells. The primary bark is graphed with a photonic microscope (Tes- parenchymal (relatively thick, 10-12 lay- kan Electronic Scanning Microscope). ers), consisting of slightly elongated tan- Reagents and equipment gential cells. The most internal layer is an The used apparatus was first Novex endodermis with cells having lignified and Holland Microscope with Sony Cyber-shot suberized walls. The central cylinder DSC-W730 and then Teskan Electronic showed parenchymal cells, many vascular Scanning Microscope (SEM). bundles (phloem and xylem solitary or Green iodine is obtained from one gram grouped vessels). The secondary fibers of iodine green is dissolved in 100 ml of have fairly numerous pits and were often hexane, whereas red ruthenium is obtained arranged in radar strings which intertwine by dissolving 0.02 g of ruthenium red in the phloem resulting in incomplete rays. 100 ml of distilled water; add a thymol The phloem vessels of variable diameter crystal for preservation. are composed of cells with moderately thick walls irregularly arranged, in incom- RESULTS AND DISCUSSION plete radar areas as shown in the image The results indicated both similarities below (fig. 1).
Fig. 1. Leuzea carthamoides roots and rhizomes – sectional view with detail
thick with parenchymal cells, larger air Our findings indicate that at the sec- cavities surrounded by interfascicular cel- tional level Leuzea salina presents mainly a lular parenchyma are observed between the primary structure, as opposed to what we phloem and xylem fascicles. A detailed have seen in L. carthamoides. The cortical view is presented in the following image parenchyma is very thick, with small cells, (fig 2). including numerous air cavities. The inner At the electronic scanning microscope layer is made of small cells elongated tan- (SEM) we noticed that the cortical cells are gentially, alternating with those of the peri- heavily elongated and all the thin wall (fig. cardium. The central cylinder is relatively 3).
189 A. F. Paduraru et al.
The SEM images for Leuzea salina seem different from the other investigated roots are shown in the figure 4. The details species.
Fig. 2. Leuzea salina roots and rhizomes – sectional view with detail
Fig. 3. The root of Leuzea carthamoides seen on the SEM: lateral root to the top (left); cortical cells (right)
Fig. 4. Leuzea salina seen at the scanning electron microscope: rhizoderma (left); the root seen in cross-section (right)
190 Morphologic profile of two Leuzea species harvested from wild flora of Eastern Romania
Considering the rest of the analyzed the hypodermis is composed of collenchyma plant parts, the stem of Leuzea carthamoides within the ribs section. The epidermis exhib- is extremely short, with early primordial its isodiametric cells at the ribs and slightly structure, where the conductor bundles did elongated tangentially to the valleys. All not differentiate, being surrounded by sever- these elements are clearly visible in the al leaves of different size and age, making photographs included below (fig 5). several concentric circles. Nevertheless, the A particular feature that is visible only characteristics of Leuzea salina stem are for L. salina in SEM, is the presence of salt different, the contour of the cross section is glands of particular structure, among the circularly with a thin bark, air cavities and epidermal cells.
Fig. 5. Stem cross-section general view and detail: A - Leuzea carthamoides; B- Leuzea salina
Fig. 6. Salt glands from the epidermis of the stem in Leuzea salina (SEM)
Such elements, which were not ob- their actual role for the plants (12-13). served in L. carthamoides, are largely This is why we need to further investigate discussed in the old scientific literature as the type of the mineral elements that are a characteristic for halophytic plants. secreted. Nevertheless, there is a lack of proper The mature leaf of Leuzea carthamoides information in regards to these glands and has a semicircular cross-sectional shape
191 A. F. Paduraru et al. with a wide and superficial adaxial groove cle. From place to place, stomata are ob- delimited by two latero-adaxial ridges in served. Moreover, the fundamental paren- which homogeneous parenchyma is ob- chyma has cells whose size decreases to- served and a small vascular bundle located wards the inside of the organ. The hypo- in front of a thin cordon of hypodermic dermic layer presents a thin layer of collen- collenchyma. The epidermis exhibits isodi- chyma cells, and in the two latero-adaxial ametric cells with the external wall slightly ridges the collenchyma cells have visibly thicker than the others and with a thin cuti- thickened walls (fig. 7).
Fig. 7. Transverse section through Leuzea carthamoides leaves: petiole (upper images), lamina (lower images)
In the transverse section through the on a spring form, all of which have a pri- limb of Leuzea salina, the medial rib is mary structure. The epidermis presents thick and protruding at the lower face. In slightly elongated cells tangentially to the the thickness of the median rib, the chloro- external wall thicker than the others and plast-free parenchyma predominates in thinly cutinized. From place to place we which there are usually between 10-12 see pluricellular, relatively short salt vascular bunches of different size, arranged glands, nonexistent in Leuzea car-
192 Morphologic profile of two Leuzea species harvested from wild flora of Eastern Romania thamoides. The mesophyll is almost totally cells, so the limb has a bifacial structure palisaded, with relatively short and wide (equiaxial) (fig. 8)
Fig. 8. Cross-section through the leaf of Leuzea salina: median rib (left); mesophyll (right)
Irrespective of size, all vascular bundles with the larger vascular bundles. have pits, tracheid, sieve-tubes elements In the scanning electron microscope, the and intermediary cells. The xylem is epidermal cells on the lower and upper face formed of separate parenchymal cell ves- of the leaf appear irregular with irregularly sels with thick and lignified walls. A pe- curved lateral walls, and among them there ripherical cord of collenchyma elements are stomata, which are elliptical, and the and some mechanical cells with moderately walls defining the ostiole are very thick thick and lignified walls were noted along (fig. 9).
Fig. 9. Epidermis (left) and stomata (right) from Leuzea salina in SEM view
CONCLUSIONS ducted for the rhizomes, roots and the aeri- In the present paper we presented the al part of two Leuzea species. Our conclu- results of the histoanatomical studies con- sion is that the cross sections of each ana-
193 A. F. Paduraru et al. lyzed sections sustain that the species are ences between the two species remains to related and justify the inclusion in the same be further investigated. Also, Leuzea sa- genus. Nevertheless, the noted differences lina, unknown for its bioactive potential especially in terms of tissue type and cer- may also become interesting as a supplier tain elements found only in Leuzea salina, of ecdysteroid compounds. suggest that the investigated species are different. Such features important for taxo- CONFLICT OF INTEREST nomic classification at the generic level The authors declare that the research represent the novelty of our research. How- was conducted in the absence of any com- ever, the chemical composition and the mercial or financial relationships that could pharmacological action of the extracts that be construed as a potential conflict of in- follow closely the similarities and differ- terest.
REFERENCES
1. Holub J. Contribution to the taxonomy in nomenclature of Leuzea DC and Rhaponticum auct. Folia Geobotanica et Phytotaxonomica. 1973; 8: 377-395. 2. Nistreanu A. Farmacognozie. Chisinau: Tipografia Centrala 2001; 594-597. 3. Savulescu T, Grintescu I, Gusuleac M, Prodan I. Flora Republicii Populare Romane, Bucuresti: Editura Acad. Republicii Populare Romane,1964; 951-952. 4. Biskup E, Szynklarz B, Golebiowski M, Borsuk K, Stepnowski P, Lojkowska E. Composition and biological activity of Rhaponticum carthamoides extracts obtained from plants collected in Poland and Russia. J Med Res 2013; 7(11): 687-695. 5. Janovska D, Kloucek P, Urban J, Vanek T, Rada V, Kokoska L. Susceptibility of some clinical iso- lates of Staphyloccoccus aureus to fractions from the aerial parts of Leuzea carthamoides. Biologia 2008; 63(5): 607-609. 6. Kokoska L, Janovska D. Chemistry and pharmacology of Rhaponticum carthamoides: a review. Phytochemistry 2009; 70: 842-855. 7. Budesinsky M, Vokcac, Harmatha J, Cvacka J. Additional minor ecdysteroid components of Leuzea carthamoides. Steroids 2008; 73: 502-514. 8. Koleckar V, Opletal L, Macakova K, Jahodar L, Jun D, Kunes J, Kuca K. New antioxidants flovonoid isolated from Leuzea carthamoides. J Enzyme Inhib. Med Chem 2010; 25(1): 143-145. 9. Borovikova EB, Shangaraeva GS, Baltaev U. Rhapisterone D-20 acetat from the seeds of Leuzea carthamoides. Chem Nat Compd 1999; 35(2): 184-186. 10. Prodius PA, Manukhina EB, Bulanov AE, Wikmann G, Malyshev IYu. Adaptogen ADAPT modu- lates stress-induced HSP70 synthesis and improves organism’s resistence to heat shock. Bull Exper Biol & Med 1997; 6: 548-550. 11. Sticher O, Heilmann J, Zundorf I. Hansel/ Sticher Pharmakognosie Phytopharmazie, ed. X, Stuttgart: WVG, 2015, 106-108. 12. Helder RJ. The loss of substances by cells and tissues (salt glands). In: Ruhland W. (Ed.): Handbuch der Pflanzenphysiologie, Springer, Berlin-Gottingen-Heidelberg 1956, vol. 2, 468-488. 13. Thomson WW. The Structure and Function of Salt Glands. In: Poljakoff-Mayber A, Gale J (eds) Plants in Saline Environments. Ecological Studies (Analysis and Synthesis) Berlin, Heidelberg: Springer 1975, vol. 15.
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