Using microphotography technique, the cross-sections of root, aboveground stem and leaf were obtained and investigated for Tribulus terrestris L. (Zygophyllaceae) plants in blossom, collected in June 2016, from the surroundings of Craiova City, Dolj County (southwestern Romania). The polyphenols content of the aerial parts (Tribuli herba) was analyzed by thin layer chromatography. One rutin derivative was identified starting from the 12 peculiar chromatographic bands

Bejenaru, Cornelia

Bejenaru, Ludovic Everard

Bita, Andrei

Mogosanu, George Dan

Annals of the University of Craiova - Agriculture, Montanology, Cadastre Series

Analele Universităţii din Craiova, seria Agricultură – Montanologie – Cadastru (Annals of the University of Craiova - Agriculture,Montanology, Cadastre Series) Vol. XLVI 201619HISTO-ANATOMICAL AND PRELIMINARY TLC INVESTIGATIONS ONTRIBULUS TERRESTRIS L. (ZYGOPHYLLACEAE) SPECIESCORNELIA BEJENARU1, GEORGE DAN MOGOŞANU2*, ANDREI BIŢĂ2,LUDOVIC EVERARD BEJENARU 21Department of Vegetal & Animal Biology, Faculty of Pharmacy, University of Medicineand Pharmacy of Craiova2Department of Pharmacognosy & Phytotherapy, Faculty of Pharmacy, University of Medicineand Pharmacy of Craiova, e-mail: george.mogosanu@umfcv.roKeywords: Tribulus terrestris L., histo-anatomy, polyphenols, thin layer chromatographyABSTRACTUsing microphotography technique, the cross-sections of root, aboveground stemand leaf were obtained and investigated for Tribulus terrestris L. (Zygophyllaceae) plantsin blossom, collected in June 2016, from the surroundings of Craiova City, Dolj County(southwestern Romania). The polyphenols content of the aerial parts (Tribuli herba) wasanalyzed by thin layer chromatography. One rutin derivative was identified starting fromthe 12 peculiar chromatographic bands.INTRODUCTIONTribulus terrestris L., Goat’s head, Devil’s thorn, Zygophyllaceae family, is an annualherbaceous species, widespread mainly in the steppe, pastures, meadows and sandy orsandy-rocky areas in the Central Europe, Mediterranean zone, South East Asia, America,India, Africa, Australia [4].T. terrestris contains various active principles (steroidal saponins, sterols, flavonoids,polysaccharides, alkaloids, tannin) [10, 13] with various pharmacological actions, mainlyaphrodisiac [5], anti-inflammatory [10], antioxidant [8], antimicrobial [11], immunomodulatory,hepatoprotective, hypolipidemic, hypoglicemiant [9, 10].In the specialty papers, there are scarce and incomplete data concerning T. terrestrishisto-anatomy [7, 14].The aim of our paper was the histo-anatomical investigation of the root, abovegroundstem and leaf of T. terrestris and the preliminary analysis of the polyphenols content fromthe aerial parts (Tribuli herba).MATERIAL AND METHODHisto-anatomical investigationThe vegetal material was harvested from T. terrestris plants in blossom, in June 2016,from the surroundings of Craiova City, Dolj County (southwestern Romania).Fixation and preservation of roots, aboveground stems and leaves were achieved in70% ethanol. Cross-sections sections were obtained using botanical razor.After washing with distilled water, the cross-sections were clarified using 10% sodiumhypochlorite solution (Javel water). Then, the clarifying agent was removed by washingwith distilled water. Congo red–chrysoidine mixture (Genevese reagent) was used for thestaining of cross-sections. Depending on the chemical composition of cell membranes, thereactive induced various stains: pink to red for cellulose and mucilage, pale red forcytoplasm, yellow for suberin and brown for lignin [2].Stained and mounted cross-sections were analyzed on a Krüss binocular photonmicroscope (objectives ×4, ×10, ×40) and then photographed using a Sony DSLR-A380digital system adapted to the microscope.The description of microscopic cross-sections was achieved according to classicalauthors [12].Analele Universităţii din Craiova, seria Agricultură – Montanologie – Cadastru (Annals of the University of Craiova - Agriculture,Montanology, Cadastre Series) Vol. XLVI 201620Thin-layer chromatography (TLC) analysisPreliminary analysis of polyphenols was performed on the aerial parts of T. terrestrisspecies (Tribuli herba), using a CAMAG (Muttenz, Switzerland) system, in the followingexperimental conditions [1, 3, 6]: stationary phase TLC silica gel 60 F254 20×10 precoatedglass plates (Merck, Darmstadt, Germany) pre-washed with chloroform–methanol (1:1,v/v); mobile phase chloroform–ethyl acetate–toluene–formic acid–methanol (15:20:10:10:1,in volumes) in a vapor-equilibrated chromatographic tank (20×10 cm twin trough chamber,CAMAG); sample – 20% methanolic extract of Tribuli herba; standards (Merck) – 0.05%methanolic solutions of caffeic acid, chlorogenic acid, quercetin and rutin; migration distance80 mm; sample (1–10 μL) and standards (2 μL) application – CAMAG Linomat 5 semi-automatic system (spray gas nitrogen, dosage speed 150 nL/s and band length 8 mm);detection – CAMAG TLC Scanner 3 photodensitometer, UV 254 nm, without derivatization,deuterium–wolfram lamp, scanning speed 20 mm/s, resolution 100 μm/step; measurementmode – absorption; spectra acquisition, processing and quantification analysis – winCATSver. 1.4.3 software package.RESULTS AND DISCUSSIONSHisto-anatomical investigationRootIn cross-section, the root has circular shape and secondary structure due to the twolateral meristems: phellogen and libero-ligneous cambium. In cross-section, from the outsidetowards the inside of the root, the following histological sequence was evidenced. Suber ismade of 2–3 layers of flattened cells with suberin-impregnated walls. Sometimes suber isexfoliated. Phellogen has circular shape, consisting of two layers of flattened cells with thinand corrugated walls. Phelloderm is made of 2–3 layers of heterodiametric cells, uniformlyplaced, without intercellular spaces. Cortical parenchyma of the primary structure is wellrepresented, consisting of large parenchyma cells, delimiting small intercellular meatus. Atthis level, several bundles of sclerenchyma fibers are distinguished. Conducting tissuesare arranged in two concentric rings, separated by libero-ligneous cambium. Phloem tissueforms a thin, external ring, made of sieve tubes, annex cells and phloem parenchyma.Libero-ligneous cambium has circular shape, consisting of flattened cells with cellulosic thinwalls. Xylem tissue forms the internal ring made of some metaxylem vessels of differentsizes, disseminated into the libriform tissue. In longitudinal-radial sections, metaxylemexhibits reticulate, ringed and spiral thickenings. Protoxylem is pushed toward the centerof the root adjacent to the medullary parenchyma. Medullary rays are multi-cellular,uniseriate, cellulosic (Figure 1).Figure 1. Cross-section through T. terrestris root: (a) suber; (b) phellogen; (c) phelloderm; (d) corticalparenchyma; (e) bundles of sclerenchyma fibers; (f) phloem tissue; (g) libero-ligneous cambium;(h) metaxylem (Congo red–chrysoidine staining, ×100).Analele Universităţii din Craiova, seria Agricultură – Montanologie – Cadastru (Annals of the University of Craiova - Agriculture,Montanology, Cadastre Series) Vol. XLVI 201621Aboveground stemIn cross-section, into the upper third, the aboveground stem has slightly circular sinuousshape and secondary structure generated by the intra- and inter-fascicular libero-ligneouscambium. In cross-section, from the outside towards the inside of the aboveground stem,the following histological sequence was observed. Epidermis consists of a single layer ofisodiametric cells with thin radial walls and slightly thickened tangential external and internalwalls. The external walls are bulged and covered by a thick cuticle with papilliform relief.From point to point, we found unicellular, long and bowed tector hairs, some of themsituated at the rib’s level and implanted in a multi-cellular epidermal pedestal. Hypodermis ismade of a single layer of small cells, uniform in size, alternately stacked with the epidermalcells. Cortical parenchyma is represented by 5–7 layers of large, heterodiametric cells,which generate small intercellular spaces. At this level are evidenced sclerenchyma capsthat protect the conducting fascicles in periphloemic position. The conducting tissues areorganized into multiple collateral-open libero-ligneous fascicles of various sizes. Phloemtissue is represented by sieve tubes, annex cells and phloem parenchyma; at the level ofprimary phloem, it is protected by some sclerenchyma caps. Xylem tissue consists of largecaliber metaxylem vessels scattered in the libriform mass on the internal side of libero-ligneous cambium. At the fascicle base, small diameter protoxylem is accompanied byxylem parenchyma. Libero-ligneous cambium of circular sinuous shape is made of flattenedcells. Medullary rays are wide and lignified, with the increasing move towards secondarystructure. Medullary parenchyma is well developed, made of large cells with thin, cellulosicwalls, of meatus type (Figures 2 and 3).Figure 2. Cross-section through T. terrestris aboveground stem: (a) epidermis; (b) tector hair;(c) hypodermis; (d) cortical parenchyma; (e) sclerenchyma cap; (f) phloem tissue; (g) libero-ligneouscambium; (h) metaxylem; (i) medullary ray; (j) protoxylem; (k) medullary parenchyma (Congo red–chrysoidine staining, ×100).Figure 3. Cross-section through T. terrestris aboveground stem: (a) cortical parenchyma;(b) sclerenchyma cap; (c) phloem tissue; (d) libero-ligneous cambium; (e) metaxylem; (f) libriformtissue; (g) medullary ray; (h) medullary parenchyma (Congo red–chrysoidine staining, ×100).Analele Universităţii din Craiova, seria Agricultură – Montanologie – Cadastru (Annals of the University of Craiova - Agriculture,Montanology, Cadastre Series) Vol. XLVI 201622Leaf’s limbIn cross-section, from the outside towards the inside of leaf’s limb, the followinghistological sequence is observed. Upper epidermis is made of a single layer of elongatedheterodiametric cells with thickened tangential external and internal walls and thin radialwalls. A thin cuticle covers the epidermis. Rare unicellular tector hairs, long and with sharppeak, are found at the main rib level. Mesophyll is uniform, consisting of a single layer ofelongated cells, without intercellular spaces, ordered in palisade, rich in chloroplasts, underthe upper epidermis and of two layers of smaller cells, slightly elongated, also withoutintercellular spaces and rich in chloroplasts, above the lower epidermis. The conductingtissue is represented by libero-ligneous fascicles evenly distributed into the mesophyll. Onthe outside, an assimilatory fascicular sheath protects the libero-ligneous fascicles. Lowerepidermis is made of a single layer of oval cells, with thin radial walls and slightly thickenedexternal and internal walls. At this level, stomata are observed from point to point. The leafhas hypostomatic bifacial equifacial structure (Figures 4 and 5).Figure 4. Cross-section through T. terrestris leaf’s limb: (a) upper epidermis; (b) tector hair;(c) palisade parenchyma; (d) libero-ligneous conducting fascicle; (e) assimilatory fascicularsheath; (f) lower epidermis (Congo red–chrysoidine staining, ×100).Figure 5. Cross-section through T. terrestris leaf’s limb: (a) upper epidermis; (b) palisadeparenchyma; (c) libero-ligneous conducting fascicle; (d) assimilatory fascicularsheath; (e) lower epidermis (Congo red–chrysoidine staining, ×100).TLC analysisThe experimental data on the preliminary TLC analysis of polyphenols from Tribuliherba are highlighted in Figures 6–8. One rutin derivative (Rf 0.05, 161.86 mg/100 g of driedvegetal product) was identified starting from the 12 peculiar chromatographic bands.Analele Universităţii din Craiova, seria Agricultură – Montanologie – Cadastru (Annals of the University of Craiova - Agriculture,Montanology, Cadastre Series) Vol. XLVI 201623Figure 6. TLC chromatogram of polyphenols from Tribuli herba methanolic extract (UV 254 nm,without derivatization). From left to right: first five applications – sample (1–5 μL); subsequentfour applications – standards (2 μL); last five applications – sample (6–10 μL).Figure 7. Densitogram of polyphenols (UV 254 nm) separated from Tribuli herba methanolic extract.Figure 8. Rutin derivative in situ UV spectra of standard and compound separated fromthe analyzed sample.Analele Universităţii din Craiova, seria Agricultură – Montanologie – Cadastru (Annals of the University of Craiova - Agriculture,Montanology, Cadastre Series) Vol. XLVI 201624CONCLUSIONSThe histo-anatomical investigation of the root, aboveground stem and leaf of Tribulusterrestris species and the preliminary TLC analysis of Tribuli herba polyphenols wereaccomplished. The root has circular shape and secondary structure. 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HISTO-ANATOMICAL AND PRELIMINARY TLC INVESTIGATIONS ON TRIBULUS TERRESTRIS L. (ZYGOPHYLLACEAE) SPECIES

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HISTO-ANATOMICAL AND PRELIMINARY TLC INVESTIGATIONS ON TRIBULUS TERRESTRIS L. (ZYGOPHYLLACEAE) SPECIES

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Annals of the University of Craiova - Agriculture, Montanology, Cadastre Series