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Morphoanatomical studies of Uncaria tomentosa and Uncaria guianensis bark and leaves
Phytomedicine 11: 213–223, 2004 http://www.elsevier-deutschland.de/phymed
Morphoanatomical studies of Uncaria tomentosa and Uncaria guianensis bark and leaves M. Gattuso1, O. Di Sapio1, S. Gattuso1, and E. Li Pereyra2 1 2
Cátedra de Botánica, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina Laboratorios Induquímica S.A., Lima, Perú
Summary The genus Uncaria Schreber (Rubiaceae) includes species that are widely distributed in tropical areas. The inner bark of the stems and leaves of two native species of South America, Uncaria tomentosa (Willd. ex Roemer & Schultes) DC., and Uncaria guianensis (Aublet) J. F. Gmelin, “cat’s claw” are used in either folk medicine or in procuring phytotherapeutic drugs. These species contain about sixty active substances which are being tested widely for possible medicinal value. The following applications are considered: peptic ulcer, rheumatism, tumours, antiinflammatory effect, inflammation, diabetes and as general tonic. Currently, Uncaria tomentosa is in demand as tea, tablets or capsules in more than 30 countries outside Perú, as well as in side the country. Pharmacognosy studies are required to determine the comparative morphoanatomical and micrographic features for identification and quality control purposes. Several microscopic parameters, including phloem fibers, calcium oxalate crystals, starch granules, trichomes, and foliar architecture should be considered. The aim of our work is to analyse comparative morphoanatomical and micrographic features which might provide assistance in the identification, analysis and standardization of Uncaria tomentosa (Willd. ex Roemer & Schultes) DC. and Uncaria guianensis (Aublet) J. F. Gmelin stem bark and leaves in order to obtain phytotherapeutic drugs, and of the crude drug as well. Key words: Uncaria tomentosa, Uncaria guianensis, bark, leaves, morphoanatomical studies, micrographic characters, standardization
Introduction Uncaria tomentosa (Willdenow ex Roemer & Schultes) De Candolle and Uncaria guianensis (Aublet) J. F. Gmelin are both known as “cat’s claw” and are native to a number of central and South American countries, (Anderson and Taylor, 1994). These species have been important in traditional healing and are most often prepared as a decoction, through boiling the inner bark or the root bark, or macerated in an alcoholic beverage (Gupta, 1995; Obregón Vilches, 1997). A number of studies have reported on alkaloids and procyanidins (Hemingway and Phillipson, 1974; Montenegro De Matta et al. 1976; Phillipson et al. 1978),
quinovic acid glycosides which have anti-viral activity (Cerri et al. 1988; Aquino et al. 1989; 1991; Yepez et al. 1991) and triterpenes with antitumor activity (Aquino et al. 1990) from these two South American Uncaria species. Research on the steroid fraction was furthermore demonstrated the presence of β-sitosterol, campesterol and traces of cholesterol. β-sitosterol is the principal sterol, which has a moderate anti-inflammatory effect (Senatore et al. 1989). Part of the confusion over the composition of each species may be the result of changes in alkaloid composition of “cat’s claw” over seasons and individual plant life cycles (Laus and Kepplinger, 1994). 0944-7113/04/11/02–03-213 $ 30.00/0
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Only since 1980 have reports appeared on the therapeutic effect of specific “cat’s claw” compounds. Wagner et al. (1985) reported the immune–stimulating activity of Uncaria tomentosa supposedly caused by oxindole alkaloids through a phagocytosis-enhancing effect. Aquino et al. (1989) reported anti-viral activity and anti-inflammatory activity, and later (1991) studied the quinovic acid glycosides of Uncaria tomentosa. Rizzi et al. (1993) showed that “cat’s claw” has significant antimutagenic activities, possibly a result of its anti-oxidant properties, scavenging the single oxygens and other free radicals to which aging and cancer have been attributed. Peruvian researchers have been conducting clinical trials with Uncaria tomentosa and Uncaria guianensis decoctions, pills, or tablets. Costa Fazzi (1989) demonstrated that “cat’s claw” decoction appears to reduce the gastritic consequences of stress effects. Summarizing its therapeutic properties Obregon Vilches (1997) concluded that “cat’s claw” seems to have positive effects in the treatment of arthritis, gastritis, and dermatological infections, as well as infections of the genital canals, asthma, gastric ulcers, diabetes, several kinds of cancers (broncho-pulmonary, gastric and those of the female genital canal), several types of tumors, viral infections, convalescence and general debility. Three patents have thus for been granted; in 1989 (Keplinger et al., U.S. Patent No. 4,844,901); in 1990 (Keplinger et al., U.S. Patent No. 4,940,725); and in 1994 (Keplinger et al., U.S. Patent No. 5,302,611). The patents deal with the extraction process of some of the compounds of “cat’s claw”, which is a discovery that is entirely a result of the intellectual efforts of the patent holders. Currently, “cat’s claw” (especially Uncaria tomentosa) is demanded as tea, tablets or capsules. Unprocessed bark, milled bark, or micropulverized bark of “cat’s claw” require a micrographic reference standard, useful for the quality control of this vegetal drug. The general anatomical studies of the Rubiaceae were carried out by Solereder (1908) and Metcalfe and Chalk (1957). Micrographic references with a light microscope are reported in previous work on the bark of Cinchonae succirubrae (Koch, 1901; Jackson and Snowdon, 1974) and the root of Cephaelis ipecacuanha and Cephaelis acuminata (Jackson and Snowdon, 1974). The aim of our work is to analyze comparative morphoanatomical and micrographic features which might provide further assistance in the identification, analysis and standardization of Uncaria tomentosa (Willd. ex Roemer & Schultes) DC. and Uncaria guianensis (Aublet) J. F. Gmelin stem bark and leaves for the procurement of phytotherapeutic drugs or crude drug.
Material Plant material
• Uncaria tomentosa (Willdenow ex Roemer & Schultes) De Candolle: – Identification: PERÚ. Dpto. Junín: Prov. La Merced: Locality Jardín El Perezoso, E. Cerrate 8455, 27-VII-1982 (USM). Prov. Tarma: Locality Trocha El Perezoso, Cerro de la Merced, R. Ferreyra & C. Díaz 20536, 10-IX-1986, (USM). Dpto. Huanco: Prov. Leoncio Prado: Locality Aucayacu, R. Ferreyra & J. Schunke 19355, 17-I-1981 (USM). Dpto. San Martín, Prov. Tocache: Locality Quebrada Huaquiska, J. Schunke 14215, 9-I-86 (USM). Dpto. Cuzco: Prov. La Convención: Locality Armihuari, H. Beltrán 2604, 29V-1997 (USM). • Uncaria guianensis (Aublet) J. F. Gmelin – Identification: PERÚ. Dpto. Loreto: Prov. Loreto: Locality Reserva Nacional Pacaya, C. del Carpio, O. Tovar & J. Ruíz 1735, 13-XI-1992 (USM); Dpto Madre de Dios: Province Tambopata: Sandoval lake 7 km around Madre de Dios river, H. Beltrán 2009, 2-X1995 (USM); Province Tambopata: Tres Chimbadas lake, P. J. Barbour 5759, 18-VI-1980 (USM); Dpto. San Martín: Prov. Marisol Cáceres: Locality Quebrada Shichimi, J. Schunke 8270, 10-IV-1975 (USM); Dpto. Loreto, Prov. Iquitos: Iquitos-Nauta way, Km 6, M. Rimachi 8220, 2-II-1987 (USM). U. tomentosa and U. guianensis fresh bark and leaves were collected in the “Campo Experimental de la Universidad de Tingo María”, Huanuco, Perú.
Methods Both fresh and fixed (formaldehyde, ethanol, acetic acid, water, 2:10:1:3,5) leaves were used. The bark was softened previously by boiling in water with a few drops of detergent. Longitudinal and transverse sections of the bark and transverse sections of the leaves measuring 10 µm each were prepared using a hand microtome, and were stained with Safranin and Fast Green (Dizeo de Strittmatter, 1979). The epidermis was obtained by scraping fixed material and was then stained with diluted Fast Green and Cresyl Violet (Dizeo de Strittmatter, 1980). Sections were mounted in synthetic balsam. The bark was macerated and the leaves were cleared by conventional methods (Boodle, 1916; Dizeo de Strittmatter, 1973). The terminologies proposed by Hickey (1973) and Junikka (1994), respectively, were used for the description of leaf architecture and bark. The distribution of calcium oxalate crystals was observed using a polarizing microscope. Original drawings were made with a drawing device.
Morphoanatomical studies of Uncaria tomentosa Symbols proposed by Metcalfe and Chalk (1957) were used in the diagrams. The black and white photomicrographs were performed using the Axiolab Zeiss Automatic System.
Results Uncaria tomentosa (Willdenow ex Roemer & Schultes) DC., Prodr. 4: 439. 1830
Nauclea tomentosa Willdenow ex Roemer & Schultes; Syst. Veg. 5: 221. 1819. Ourouparia tomentosa (Roemer & Schultes) K. Schumann., Fl. Bras. 6 (6): 132. 1889. Nauclea aculeata Kunth in Humboldt, Bonpland & Kunth, Nov. Gen. Sp. 3: 382. 1819, non Willdenow in Usteri 1793. Uncaria surinamensis Miquel, Linnaea 19:129. 1847. Uncaria surinamensis var. dioica Bremekamp, Rec. Trav. Bot. Neerl. 31: 263. 1934. Liana, up to 20–30 m long, main stem up to 25 cm diameter. Young shoots generally puberulous; thorns curved 30–90º, minutely hirtellous and usually with coarse, subappressed hairs. Stipules obtusely triangular or cordate, subpersistent; ovate leaf blade, elliptic, oblong or obovate densely tomentotellous, domatia, when present, of tuft type, (Fig. 5 A; Fig. 6 B). Inflorescences 1.2–0.3 mm, densely villous outside, densely sericeous inside at base, corolla densely retorsely puberulous or villous outside, glabrous inside, tube 3.5–5.0 mm long, lobes; stamens attached to corolla tube; ovary 2-locular, densely villous. Capsules septicidal, 6–17 mm long, sessile. Seeds oblong with wing often deeply divided. Uncaria tomentosa occurs between 300–800 m above sea level, and is found in closed, mature natural forests of Perú. It is also found in Costa Rica, Colombia, Ecuador, Guatemala, Honduras, Panamá, Surinam, Trinidad and Venezuela. • Common names: “uña de gato”, “garabato amarillo”, “garabato”, “ungananguí”, “samento”, “rangaya”, “tua juncara”, “bejuco de agua”, “cat’s claw”. Uncaria guianensis (Aublet) J. F. Gmelin, Syst Nat. 2: 370 1791.
Ourouparia guianensis Aublet, Hist. Pl. Guiane 177. 1775. Uncaria aculeata (Willdenow) Willdenow, Sp. Pl. 1: 929. 1793. Uncaria spinosa Raeuschel, Nomencl., ed. 3, 55. 1797.
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Liana or scandent shrub. Young glabrous shoots; curved thorns more than 180º, either glabrous or subglabrous, (Fig. 5B; Fig. 7B). Caducous stipules, not seen; leaf blade ovate to elliptic, adaxial surface glabrous, abaxial surface glabrous throughout domatia when present, of pouch to pit type. Inflorescences 1.4–3 cm diameter at anthesis, proximal sparsely hirsute peduncles, dense to distal. Flowers on pedicels, funnel-shaped calix, with obtuse lobes, densely sericeous outside, densely sericeous inside at base; corolla, yellow when young, later turning red, glabrous outside at base, densely sericeous with long, antrorse hairs, glabrous inside, tube 4.1–6.5 mm long, lobes 1.1–3 mm; stamens attached to corolla tube; ovary 2locular, densely sericeous. Septicidal capsules, stipitate, 12–25 × 5–7 mm. Oblong seeds with wing. Uncaria guianensis occurs between an 100 and 500 m above sea level and, is found exclusively within Perú in almost in secondary forests. It is also found in Trinidad, Venezuela, Guyana, Brasil, Bolivia and Paraguay. • Common names: “uña de gato”, “garabato colorado”, “garabato casha”, “ungananguí”, “tambor huasca”, “ancayacu”, “paraguayo”, “ancajsillo”, “cat’s claw”. Common characteristics of the bark of Uncaria tomentosa and Uncaria guianensis
Morphological characteristics The drug used is of the inner bark of the stem obtained by decorticating it in pieces or flat fragments of 6 to 10 cm long, 3 to 6 cm high and 5 to 10 mm thick, which might be accompanied by brownish phellem remaining cells. The outer surface is fibrous and cinnamon in color with longitudinal striation; the inner surface, of the same color, is remarkably fibrous. The fracture is partially laminar, very fibrous and resistant. The bark is odorless with a slightly bitter astringent flavor (Fig. 2A, B). Anatomical characteristics Outer bark: Remaining single rhytidome was present, made up of a single peridermis with 6 to 10 layers of thin-walled tabulated phellem cells. The phelloderm was unstratified. At the boundary between the peridermis and the secondary phloem there was a continuous ring of sclereids (Fig. 1L; Fig. 2G). Inner bark: The cross-sections enabled the distinction of different regions of functional phloem separated by fibers in small clusters of cells and limited by tangential bands of axial parenchyma with abundant calcium oxalate crystals. Longitudinal uniseriate and pluriseriate phloem rays with cell walls of regular contour were observed (Fig. 1A, D; Fig. 2C). The longitudinal
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sections enable the distinction of different rays: heterogeneous, multiseriates and uniseriate. The radial longitudinal section enabled us to determine the phloem radii, which are heterocellular, with 4–5 marginal rows of high upright cells, the multiseriate parts composed of procumbent cells having intercellular spaces (Fig. 1 B; Fig. 2 D). Radius height is 25 to 50 cells. The uniseriate rays are composed of square to upright ray cells. Radius height is 8 to 10 cells (Fig. 1 E). The tangential longitudinal section showed uniseriate parenchyma radii and pluriseriate ones, 4–5 cells wide (Fig. 1 C, F; Fig. 2E). The elements of the sieve tube are broad, presenting a composed, highly developed sieve plate in an inclined position (Fig. 2F).
Macerated bark: The maceration of the bark (Fig. 2H) made the determination of the following cellular elements possible: a) Sclereids: Consisting of polygonal rectangular brachisclereids, 100 µm in length and 40 µm in width; and isodiametric brachisclereids, 50 µm in diameter (Fig. 1K). b) Ray cells: Showing parenchymatous cells of high upright cells and procumbent cells measuring from 22–25 µm to 50–55 µm (Fig. 1J). c) Axial parenchyma: Showing elongated cells measuring 75 µm in height and 25 µm in width (Fig. 1I). d) Phellem cells: in cross-sections, they were rectangular with thin-walled cells and measured 25–30 µm in length and 18–20 mm in width (Fig. 1G). In the superficial view they were polygonal, attaining a diameter of 22–25 µm (Fig. 1H).
Fig. 1. A–L, Common characteristics of the bark of Uncaria tomentosa (Willdenow ex Roemer & Schultes) De Candolle and Uncaria guianensis (Aublet) J. F. Gmelin. A–C: Schematic view of the bark. A: transverse section; B: longitudinal radial section; C: longitudinal tangencial section; D: detail of A; E: detail of B; F: detail of C. G–L: Macerated bark; G: phellem cells in cross section; H: phellem cells in superficial view; I: axial parenchyma cells; J: secondary heterocellular ray cells; K: brachysclereids; L: sclereid ring between phellem and parenchyma. c: calcium oxalate microcrystals, f: phloem fibers, ph: phellem, r: ray, s: sclereid, se: sieve element, sp: sieve plate. Scale bars: 1 to C; 2 to B, F; 3 to A; 4 to E; 5 to D; 6 to G, H, I, J, K, L.
Morphoanatomical studies of Uncaria tomentosa Non-shared characteristics of the bark
Uncaria tomentosa (Willd. ex Roemer & Schultes) DC. a) Phloem fibres: They were located in the inner bark forming tangential bands between functional phloem. Fibre size 1500 to 2000 µm length, 50 µm width (Fig. 3 B; Fig. 4 E). b) Axial and radial parenchyma: including only calcium-oxalate microcrystals (Fig. 3A; Fig. 4 A). c) Axial and radial parenchyma: showing simple or compound starch granules with two, three and occasionally more components. Starch granules sized 8 to 12 µm in diameter (Fig. 3 C; Fig. 4C). Uncaria guianensis (Aublet) J. F. Gmelin a) Phloem fibres: They were located in the inner bark forming tangential bands between functional phloem. Fibre size 800 to 1500 µm length, 20 to 50 µm width (Fig. 3 E; Fig. 4 F). b) Axial and radial parenchyma: in-
Fig. 2. A–H. Common characteristics of the bark of Uncaria tomentosa (Willdenow ex Roemer & Schultes) De Candolle and Uncaria guianensis (Aublet) J. F. Gmelin. A: outer bark surface; B: inner bark surface; C: bark in transverse section; D: bark in longitudinal radial section, showed 4 rows of high upright cells; E: bark in longitudinal tangencial section; F: terminal compound sieve plate; G: sclereid ring between phellem and parenchyma; H: macerated bark elements. p: parenchyma, ph: phellem, r: ray, s: sclereid, sp: sieve plate. Scale bars: A,B: 1,5 cm; C: 100 µm; D: 10 µm; E: 25 µm; F: 10 µm; G: 10 µm; H: 25 µm.
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cluding calcium-oxalate microcrystals and polyhedrical large crystals of calcium-oxalate measuring 40 to 60 µm (Fig. 3D; Fig. 4 B). c) Axial and radial parenchyma: showing simple or compound starch granules with two, three, and occasionally more components. Starch granules sized 4 to 10 µm in diameter (Fig. 3F; Fig. 4D). Characteristics of the leaf
Uncaria tomentosa (Willd. ex Roemer & Schultes) DC. Morphological characteristics
Leaf blade, ovate, elliptic, oblong or obovate (length/ width 1.5–2.2), 6–16 × 2.5–11 cm, broadly cuneate or rounded to truncate or subcordate at base, obtuse to acuminate at apex, 7–10 pairs lateral veins, level to prominulous above, prominent beneath, while tertiary
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veins may be distinguished in its lower part. Adaxial matte surface to subnitid, glabrous or slightly hirtellous on midrib, abaxial surface with the midrib, and sometimes lateral veins as well, most of which are slightly are puberulent or hirtellous and also with coarse, subapressed hairs, very densely tomentose, petiole 0.8–1.5 cm long (Fig. 5 A; Fig. 6A). Anatomical characteristics
• Lamina Surface view of epidermis: The epidermal cells of the adaxial face showed straight, anticlinal walls of 4 to 5 sides. Epidermic abaxial cells presented straight anticlinal walls with unicellular trichomes curved at the apex. The cuticule is smooth. There were stomata only in the inferior epidermis; the stomatal type found was rubiaceous (Fig. 5C, D; Fig. 6E, F).
Cross-sections: It was dorsiventrally compressed. Unstratified adaxial epidermis with conspicuous cuticule, without stomata. Mesophyll was dorsiventral, with only one layer of palisade parenchyma. The spongy parenchyma cells were located next to the abaxial epidermis. Loosely arranged, with many intercellular spaces among them. Mesophyl presented very abundant crystalliferous idioblasts, containing calcium-oxalate microcrystals. The main nerve, very protuberant, in the adaxial and abaxial face, was strengthened by laminar collenchyma. The cylindrical vascular strand enclosed accessory medullary strands. It presented very abundant large schizogenous secretory cavities situated externally to the phloem (at one time described as laticiferous vessels). The lower epidermis with little cells showed a thick, smooth cuticle and stomata located at the same level as the epidermic
Fig. 3. A–F. Non shared characteristics of the bark. AC: Uncaria tomentosa (Willdenow ex Roemer & Schultes) De Candolle. A: microcrystals of calcium oxalate from ray cells from longitudinal radial section; B: phloem fibers; C: starch grains in cells of ray from transverse section. D-F: Uncaria guianensis (Aublet) J. F. Gmelin. D: Microcrystals and polyhedrical large crystals of calcium oxalate from ray cells from longitudinal radial section; E: phloem fibers; F: starch grains in cells of multiseriate ray from transverse section. Scale bars: 1 to A, C; 2 to B; 3 to D, F.
Morphoanatomical studies of Uncaria tomentosa
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cells. There were abundant unicellular trichomes (Fig. 5 G, I; Fig. 6 G, H, I). Foliar architecture: Dense vascularization. The analysis of the foliar architecture made possible the determination of pinnate, camptodromus, brochidodromus venation with an incomplete last marginal venation and simple venules, curved and branched only once. Areolae withouth orientation, rectangular shape. Vascularization terminal tracheids have no sclereids, (Fig. 6C, D).
tion, surrounding the whole perimeter of the petiole. The parenchyma presented very abundant crystalliferous idioblasts, containing calcium-oxalate microcrystals. A main vascular strand exhibiting a shallow open arc usually deep and with its ends inwardly curved, accompanied by smaller accessory bundles towards the wings. It presented large schizogenous secretory cavities situated external to the phloem (Fig. 5E; Fig. 6J).
• Petiole Transverse sections through the distal end, usually seen to be shield-shaped and with a well-developed wing. The epidermis was unstratified, with stomata, thick and smooth cuticle. The petiole surface is provided with a superficial vein-like network containing spiral tracheids, especially on the abaxial surface (Fig. 6K). Laminartype collenchyma was observed in sub-epidermic loca-
Uncaria guianensis (Aublet) J. F. Gmelin
Fig. 4. A–F. Non shared characteristics of the bark. A, C, E: Uncaria tomentosa (Willdenow ex Roemer & Schultes) De Candolle. A: microcrystals (mc); C: starch granules (s); E: sclereids and fibers (f). B, D, F: Uncaria guianensis (Aublet) J. F. Gmelin. B: polyhedrical crystals (pc); D: starch granules (s); F: phloem fibers (f). Scale bars: A: 25 µm; B: 10 µm; C, D: 10 µm; E, F: 100 µm.
Morphological characteristics
Leaf blade ovate to elliptic (length/width 1.7–2.3), 6–16 × 2.5–11 cm, cuneate to rounded at base, acute to acuminate at apex, 4-6 pairs lateral veins, prominulous, distinguishable tertiary veins, adaxial surface glabrous, abaxial surface glabrous throughout. Petiole 0.5–1.5 cm long, glabrous (Fig. 5B; Fig. 7A).
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Anatomical characteristics
• Lamina Surface view of epidermis: The epidermal cells of the adaxial face showed straight thick anticlinal walls of 4 to 5 sides. Epidermic abaxial cells presented ondulate anticlinal walls without trichomes. The cuticule is smooth. There were stomata only in the inferior epidermis and the stomatal type was rubiaceous (Fig. 7E, F). Cross-section: It was dorsiventrally compressed. Unstratified adaxial epidermis with a thin cuticule, without stomata or trichome. Mesophyll was dorsiventral, with 1–2 layers of palisade parenchyma. The spongy parenchyma cells were located next to the abaxial epidermis. Loosely arranged, with many intercellular spaces. Mesophyl presented few crystalliferous idioblasts, containing microcrystals, and few polyhedrical crystals of calcium oxalate. Palisade and
spongy parenchyma cells were filled with red-brown unidentified contents. The main vascular strands strengthened by laminar collenchyma are concave but with a few small accessory strands in the concavity. They presented few schizogenous secretory cavities situated external to the phloem. The lower epidermis with little cells showed a thin, smooth cuticle and stomata located at the same level as the epidermic cells (Fig. 5 H, J; Fig. 7G, H). Foliar architecture: The analysis of the foliar architecture made possible the determination of pinnate, camptodromus, brochidodromus venation with an incomplete last marginal venation and simple venules, curved and branched only once. Well-developed areolae without orientation, polygonally shaped. Terminal tracheids present a welldeveloped sclereid sheath (Fig. 7 C, D).
Fig. 5. A–J. Characteristics of the leaf. A, C, D, E, G, I: Uncaria tomentosa (Willdenow ex Roemer & Schultes) De Candolle. A: leaf blade, thorns curved and stipules; C–D: epidermis, superficial view; C: abaxial; D: adaxial; E, G, I, transverse sections of leaf: E: petiole; G: main nerve; I: mesophyll. B, F, H, J: Uncaria guianensis (Aublet) J. F. Gmelin. B: leaf blade, thorns curved and stipules; F, H, J: transverse sections of leaf; F, petiole; H: main nerve; J: mesophyll. Scale bars: A, B: 1,5 cm; C: 10 µm; D: 25 µm; E, F, G, H: 250 µm; I, J: 150 µm.
Morphoanatomical studies of Uncaria tomentosa • Petiole Transversely through the distal end section usually shield-shaped and with well-developed wing. The epidermis was unstratified, with stomata, thick and smooth cuticle. Beneath the epidermis, a few layers of small, rounded parenchymatous cells were filled with red-brown unidentified contents. The parenchyma presented few crystalliferous idioblasts, containing calcium-oxalate microcrystals. Loosely arranged, with many intercellular spaces. The central parenchymatous region presented the cells walls variably and unevenly thickened. Main vascular strand exhibiting a shallow open arc usually deep and with its ends inwardly curved, accompanied by smaller accessory bundles towards the wings. It presented large schizogenous secretory cavities situated external to the phloem (Fig. 5F; Fig. 7 I, J).
Fig. 6. A–K. Uncaria tomentosa (Willdenow ex Roemer & Schultes) De Candolle, leaf. A: leaf blade; B: thorns curved and stipules; C: last marginal venation and areolae; D: terminal tracheids, without sclereids; E–F: superficial view of epidermis, E: adaxial; F: abaxial; G–K: tranverse sections of the leaf; G: leaf diagrams; H: detail of main nerve; I: detail of mesophyll; J–K: transverse sections of petiole; J: petiole diagrams; K: detail of J. Scale bars: 1 to A, B; 2 to G, J; 3 to D, E, F, H, I, K; 4 to C.
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Discussion The expansion of the market for phytodrugs underscores the importance of pharmacognostic studies of those medicinal plant species used in traditional or popular medicineal systems which may become undistrially important in the near future. These data are required not only for identification procedures that guarantee the utilization of the appropriate raw material, but also for quality-control standards demanded by health legislation in western countries. Studies on botanical description, including macroand microscopic parameters, are useful tools to determine the anatomic parameters to be considered as characteristic of a certain plant and to avoid falsifications or adulterations in the process of commercialization of raw materials.
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Acknowledgements
Collaboration from Subprogram X, Fine Pharmaceutical Chemistry, RIPROFITO (Iberoamerican Network of Phytopharmaceutical Products), a part of the Iberoamerican Program of Science and Technology for Development (CYTED), is gratefully acknowledged. This work was supported in part by a grant to project N° 19/B058 of Universidad Nacional de Rosario (Argentina) to MG.
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Aquino R, De Simone F, Vincieri F, Pizza C, Gacs-Baltz E (1990) New polyhydroxylated triterpenes from Uncaria tomentosa. J Nat Prod 53 (3): 559–564 Aquino R, De Feo V, De Simone F, Pizza C, Cirino G (1991) Plant metabolites: New compounds and antiinflamatory activity of Uncaria tomentosa. J Nat Prod 54 (2): 453–458 Boodle LA (1916) A method of macerating fibers. Roy Bot Gard Kew, Bull Misc Inform 4: 108–110 Cerri R, Aquino R, De Simone F, Pizza C (1988) New quinovic acid glycosides from Uncaria tomentosa. J Nat Prod 51 (2): 257–261 Costa Fazzi M (1989) Evaluación de la Uncaria tomentosa (uña de gato) en la prevención de úlceras gástricas de stress producidas experimentalmente en ratas. Facultad de Medicina. Tesis de Bachiller en Medicina, Universidad Peruana Cayetano Heredia, Lima (Perú) . Dizeo de Strittmatter C (1973) Nueva técnica de diafanización. Bol Soc Argent Bot 15 (1): 126–129
Fig. 7. A–J. Uncaria guianensis (Aublet) J. F. Gmelin, leaf. A: leaf blade; B: thorns curved and stipules; C: last marginal venation and areolae; D: terminal tracheids, with sclereids; E-F: superficial view of epidermis, E: adaxial; F: abaxial; G-J: tranverse sections of the leaf; G: leaf diagrams; H: detail of G; I-J transverse sections of petiole; I: petiole diagrams; J: detail of I. e: cell walls variably and unevenly thickened. Scale bars: 1 to A, B; 2 to G, I; 3 to D, E, F, H, J; 4 to C.
Morphoanatomical studies of Uncaria tomentosa Dizeo de Strittmatter C (1979) Modificación de una de coloración de Safranina Fast-green. Bol Soc Argent Bot 18: 121–122 Dizeo de Strittmatter C (1980) Coloración con Violeta de Cresyl. Bol Soc Argent Bot 19 (1-2): 273–276 Gupta M (1995) Uncaria tomentosa (Willd. ex Roemer & Schultes) DC and Uncaria guianensis (Aublet) J. F. Gmelin en 270 Plantas Medicinales Iberoamericanas, (Gupta, M. ed.), Santafé de Bogotá (Colombia), pp 485–487 Hemingway SR, Phillipson JD (1974) Alkaloids from S. American species of Uncaria (Rubiaceae). J Pharm Pharmacol 26: 113 Hickey LJ (1973) Classification of the architecture of Dicotyledons leaves. Am J Bot 60: 17–33 Jackson BP, Snowdon D (1974) Powdered Vegetable Drug. Standley Thornes Publishers London, pp 14–15, 160–161 Junikka L (1994) Survey of english macroscopic bark terminology. IAWA Journal 15 (1): 3–45 Keplinger K, Wagner H, Kreutzkamp B (1989) Oxindole alkaloids having properties stimulating the immunologic system. United States Patent Number 4.844.901 Keplinger K, Wagner H, Kreutzkamp B (1990) Oxindole alkaloids having properties stimulating the immunologic system. United States Patent Number 4.940.725 Keplinger K, Keplinger D (1994) Oxindole alkaloids having properties stimulating the immunologic system and preparation containing the same. United States Patent Number 5.302.611 Koch L (1901) Die Mikroskopische Analyse der DROGENPULVER, Leipzig-Verlag von Gebruder Borntraeger Taf. III, pp 68–74 Laus G, Keplinger D (1994) Separation of stereoisomeric oxindole alkaloids from Uncaria tomentosa by high performance liquid chromatography J Chromatogr 662: 243–249 Metcalfe CR, Chalk L (1957) Anatomy of the Dicotyledons I, Oxford, London: Clarendon Press, pp 759–776
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Montenegro De Matta S, Delle Monache F, Ferrari F, MariniBettolo GB (1976) Alkaloid and procyanidins of an Uncaria sp. from Perú. Il Farmaco 31: 527–535 Obregón Vilches, L (1997) Uña de gato. Género Uncaria, estudios botánicos, químicos y farmacológicos de Uncaria tomentosa y Uncaria guianensis, (Instituto de Fitoterapia Americano ed.) Lima (Perú), pp 53–57 Phillipson JD, Hemingway SR, Ridsdale CE (1978) Alkaloids of Uncaria, Part V, Their occurrence and Chemotaxonomy. Lloydia 41 (6): 503–570 Rizzi R, Re F, Bianchi A, De Feo V, De Simone F, Bianchi L, Stivala A (1993) Mutagenic and antimutagenic activities of Uncaria tomentosa and its extracts. J Ethnopharmacol 38: 63–77 Senatore A, Cataldo A, Iaccarino FP, Elberti MG (1989) Ricerche fitochjmjche e biologiche sull Uncaria tomentosa. Boll Soc It Biol Sper 656: 517–520 Solereder H (1908) Systematic anatomy of the Dicotyledons I, Oxford, London Clarendon Press, pp 444–456 Wagner H, Kreutzkamp B, Jurcic K (1985) Alkaloids of Uncaria tomentosa and their phagocytosis enhancement activity. Planta Med 51: 419–423 Yépez M, Lock de Ugaz O, Alvarez ACM, De Feo V, Aquino, R, De Simone F, Pizza C (1991) Quinovic acid glycosides from Uncaria guianensis. Phytochemistry 30 (5): 1635–1637
Address M. Gattuso, Botánica, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531 (2000) Rosario, Argentina Fax: ++54-341-4375315; e-mail: [email protected]; [email protected]
Morphoanatomical studies of Uncaria tomentosa and Uncaria guianensis bark and leaves M. Gattuso1, O. Di Sapio1, S. Gattuso1, and E. Li Pereyra2 1 2
Cátedra de Botánica, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Argentina Laboratorios Induquímica S.A., Lima, Perú
Summary The genus Uncaria Schreber (Rubiaceae) includes species that are widely distributed in tropical areas. The inner bark of the stems and leaves of two native species of South America, Uncaria tomentosa (Willd. ex Roemer & Schultes) DC., and Uncaria guianensis (Aublet) J. F. Gmelin, “cat’s claw” are used in either folk medicine or in procuring phytotherapeutic drugs. These species contain about sixty active substances which are being tested widely for possible medicinal value. The following applications are considered: peptic ulcer, rheumatism, tumours, antiinflammatory effect, inflammation, diabetes and as general tonic. Currently, Uncaria tomentosa is in demand as tea, tablets or capsules in more than 30 countries outside Perú, as well as in side the country. Pharmacognosy studies are required to determine the comparative morphoanatomical and micrographic features for identification and quality control purposes. Several microscopic parameters, including phloem fibers, calcium oxalate crystals, starch granules, trichomes, and foliar architecture should be considered. The aim of our work is to analyse comparative morphoanatomical and micrographic features which might provide assistance in the identification, analysis and standardization of Uncaria tomentosa (Willd. ex Roemer & Schultes) DC. and Uncaria guianensis (Aublet) J. F. Gmelin stem bark and leaves in order to obtain phytotherapeutic drugs, and of the crude drug as well. Key words: Uncaria tomentosa, Uncaria guianensis, bark, leaves, morphoanatomical studies, micrographic characters, standardization
Introduction Uncaria tomentosa (Willdenow ex Roemer & Schultes) De Candolle and Uncaria guianensis (Aublet) J. F. Gmelin are both known as “cat’s claw” and are native to a number of central and South American countries, (Anderson and Taylor, 1994). These species have been important in traditional healing and are most often prepared as a decoction, through boiling the inner bark or the root bark, or macerated in an alcoholic beverage (Gupta, 1995; Obregón Vilches, 1997). A number of studies have reported on alkaloids and procyanidins (Hemingway and Phillipson, 1974; Montenegro De Matta et al. 1976; Phillipson et al. 1978),
quinovic acid glycosides which have anti-viral activity (Cerri et al. 1988; Aquino et al. 1989; 1991; Yepez et al. 1991) and triterpenes with antitumor activity (Aquino et al. 1990) from these two South American Uncaria species. Research on the steroid fraction was furthermore demonstrated the presence of β-sitosterol, campesterol and traces of cholesterol. β-sitosterol is the principal sterol, which has a moderate anti-inflammatory effect (Senatore et al. 1989). Part of the confusion over the composition of each species may be the result of changes in alkaloid composition of “cat’s claw” over seasons and individual plant life cycles (Laus and Kepplinger, 1994). 0944-7113/04/11/02–03-213 $ 30.00/0
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Only since 1980 have reports appeared on the therapeutic effect of specific “cat’s claw” compounds. Wagner et al. (1985) reported the immune–stimulating activity of Uncaria tomentosa supposedly caused by oxindole alkaloids through a phagocytosis-enhancing effect. Aquino et al. (1989) reported anti-viral activity and anti-inflammatory activity, and later (1991) studied the quinovic acid glycosides of Uncaria tomentosa. Rizzi et al. (1993) showed that “cat’s claw” has significant antimutagenic activities, possibly a result of its anti-oxidant properties, scavenging the single oxygens and other free radicals to which aging and cancer have been attributed. Peruvian researchers have been conducting clinical trials with Uncaria tomentosa and Uncaria guianensis decoctions, pills, or tablets. Costa Fazzi (1989) demonstrated that “cat’s claw” decoction appears to reduce the gastritic consequences of stress effects. Summarizing its therapeutic properties Obregon Vilches (1997) concluded that “cat’s claw” seems to have positive effects in the treatment of arthritis, gastritis, and dermatological infections, as well as infections of the genital canals, asthma, gastric ulcers, diabetes, several kinds of cancers (broncho-pulmonary, gastric and those of the female genital canal), several types of tumors, viral infections, convalescence and general debility. Three patents have thus for been granted; in 1989 (Keplinger et al., U.S. Patent No. 4,844,901); in 1990 (Keplinger et al., U.S. Patent No. 4,940,725); and in 1994 (Keplinger et al., U.S. Patent No. 5,302,611). The patents deal with the extraction process of some of the compounds of “cat’s claw”, which is a discovery that is entirely a result of the intellectual efforts of the patent holders. Currently, “cat’s claw” (especially Uncaria tomentosa) is demanded as tea, tablets or capsules. Unprocessed bark, milled bark, or micropulverized bark of “cat’s claw” require a micrographic reference standard, useful for the quality control of this vegetal drug. The general anatomical studies of the Rubiaceae were carried out by Solereder (1908) and Metcalfe and Chalk (1957). Micrographic references with a light microscope are reported in previous work on the bark of Cinchonae succirubrae (Koch, 1901; Jackson and Snowdon, 1974) and the root of Cephaelis ipecacuanha and Cephaelis acuminata (Jackson and Snowdon, 1974). The aim of our work is to analyze comparative morphoanatomical and micrographic features which might provide further assistance in the identification, analysis and standardization of Uncaria tomentosa (Willd. ex Roemer & Schultes) DC. and Uncaria guianensis (Aublet) J. F. Gmelin stem bark and leaves for the procurement of phytotherapeutic drugs or crude drug.
Material Plant material
• Uncaria tomentosa (Willdenow ex Roemer & Schultes) De Candolle: – Identification: PERÚ. Dpto. Junín: Prov. La Merced: Locality Jardín El Perezoso, E. Cerrate 8455, 27-VII-1982 (USM). Prov. Tarma: Locality Trocha El Perezoso, Cerro de la Merced, R. Ferreyra & C. Díaz 20536, 10-IX-1986, (USM). Dpto. Huanco: Prov. Leoncio Prado: Locality Aucayacu, R. Ferreyra & J. Schunke 19355, 17-I-1981 (USM). Dpto. San Martín, Prov. Tocache: Locality Quebrada Huaquiska, J. Schunke 14215, 9-I-86 (USM). Dpto. Cuzco: Prov. La Convención: Locality Armihuari, H. Beltrán 2604, 29V-1997 (USM). • Uncaria guianensis (Aublet) J. F. Gmelin – Identification: PERÚ. Dpto. Loreto: Prov. Loreto: Locality Reserva Nacional Pacaya, C. del Carpio, O. Tovar & J. Ruíz 1735, 13-XI-1992 (USM); Dpto Madre de Dios: Province Tambopata: Sandoval lake 7 km around Madre de Dios river, H. Beltrán 2009, 2-X1995 (USM); Province Tambopata: Tres Chimbadas lake, P. J. Barbour 5759, 18-VI-1980 (USM); Dpto. San Martín: Prov. Marisol Cáceres: Locality Quebrada Shichimi, J. Schunke 8270, 10-IV-1975 (USM); Dpto. Loreto, Prov. Iquitos: Iquitos-Nauta way, Km 6, M. Rimachi 8220, 2-II-1987 (USM). U. tomentosa and U. guianensis fresh bark and leaves were collected in the “Campo Experimental de la Universidad de Tingo María”, Huanuco, Perú.
Methods Both fresh and fixed (formaldehyde, ethanol, acetic acid, water, 2:10:1:3,5) leaves were used. The bark was softened previously by boiling in water with a few drops of detergent. Longitudinal and transverse sections of the bark and transverse sections of the leaves measuring 10 µm each were prepared using a hand microtome, and were stained with Safranin and Fast Green (Dizeo de Strittmatter, 1979). The epidermis was obtained by scraping fixed material and was then stained with diluted Fast Green and Cresyl Violet (Dizeo de Strittmatter, 1980). Sections were mounted in synthetic balsam. The bark was macerated and the leaves were cleared by conventional methods (Boodle, 1916; Dizeo de Strittmatter, 1973). The terminologies proposed by Hickey (1973) and Junikka (1994), respectively, were used for the description of leaf architecture and bark. The distribution of calcium oxalate crystals was observed using a polarizing microscope. Original drawings were made with a drawing device.
Morphoanatomical studies of Uncaria tomentosa Symbols proposed by Metcalfe and Chalk (1957) were used in the diagrams. The black and white photomicrographs were performed using the Axiolab Zeiss Automatic System.
Results Uncaria tomentosa (Willdenow ex Roemer & Schultes) DC., Prodr. 4: 439. 1830
Nauclea tomentosa Willdenow ex Roemer & Schultes; Syst. Veg. 5: 221. 1819. Ourouparia tomentosa (Roemer & Schultes) K. Schumann., Fl. Bras. 6 (6): 132. 1889. Nauclea aculeata Kunth in Humboldt, Bonpland & Kunth, Nov. Gen. Sp. 3: 382. 1819, non Willdenow in Usteri 1793. Uncaria surinamensis Miquel, Linnaea 19:129. 1847. Uncaria surinamensis var. dioica Bremekamp, Rec. Trav. Bot. Neerl. 31: 263. 1934. Liana, up to 20–30 m long, main stem up to 25 cm diameter. Young shoots generally puberulous; thorns curved 30–90º, minutely hirtellous and usually with coarse, subappressed hairs. Stipules obtusely triangular or cordate, subpersistent; ovate leaf blade, elliptic, oblong or obovate densely tomentotellous, domatia, when present, of tuft type, (Fig. 5 A; Fig. 6 B). Inflorescences 1.2–0.3 mm, densely villous outside, densely sericeous inside at base, corolla densely retorsely puberulous or villous outside, glabrous inside, tube 3.5–5.0 mm long, lobes; stamens attached to corolla tube; ovary 2-locular, densely villous. Capsules septicidal, 6–17 mm long, sessile. Seeds oblong with wing often deeply divided. Uncaria tomentosa occurs between 300–800 m above sea level, and is found in closed, mature natural forests of Perú. It is also found in Costa Rica, Colombia, Ecuador, Guatemala, Honduras, Panamá, Surinam, Trinidad and Venezuela. • Common names: “uña de gato”, “garabato amarillo”, “garabato”, “ungananguí”, “samento”, “rangaya”, “tua juncara”, “bejuco de agua”, “cat’s claw”. Uncaria guianensis (Aublet) J. F. Gmelin, Syst Nat. 2: 370 1791.
Ourouparia guianensis Aublet, Hist. Pl. Guiane 177. 1775. Uncaria aculeata (Willdenow) Willdenow, Sp. Pl. 1: 929. 1793. Uncaria spinosa Raeuschel, Nomencl., ed. 3, 55. 1797.
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Liana or scandent shrub. Young glabrous shoots; curved thorns more than 180º, either glabrous or subglabrous, (Fig. 5B; Fig. 7B). Caducous stipules, not seen; leaf blade ovate to elliptic, adaxial surface glabrous, abaxial surface glabrous throughout domatia when present, of pouch to pit type. Inflorescences 1.4–3 cm diameter at anthesis, proximal sparsely hirsute peduncles, dense to distal. Flowers on pedicels, funnel-shaped calix, with obtuse lobes, densely sericeous outside, densely sericeous inside at base; corolla, yellow when young, later turning red, glabrous outside at base, densely sericeous with long, antrorse hairs, glabrous inside, tube 4.1–6.5 mm long, lobes 1.1–3 mm; stamens attached to corolla tube; ovary 2locular, densely sericeous. Septicidal capsules, stipitate, 12–25 × 5–7 mm. Oblong seeds with wing. Uncaria guianensis occurs between an 100 and 500 m above sea level and, is found exclusively within Perú in almost in secondary forests. It is also found in Trinidad, Venezuela, Guyana, Brasil, Bolivia and Paraguay. • Common names: “uña de gato”, “garabato colorado”, “garabato casha”, “ungananguí”, “tambor huasca”, “ancayacu”, “paraguayo”, “ancajsillo”, “cat’s claw”. Common characteristics of the bark of Uncaria tomentosa and Uncaria guianensis
Morphological characteristics The drug used is of the inner bark of the stem obtained by decorticating it in pieces or flat fragments of 6 to 10 cm long, 3 to 6 cm high and 5 to 10 mm thick, which might be accompanied by brownish phellem remaining cells. The outer surface is fibrous and cinnamon in color with longitudinal striation; the inner surface, of the same color, is remarkably fibrous. The fracture is partially laminar, very fibrous and resistant. The bark is odorless with a slightly bitter astringent flavor (Fig. 2A, B). Anatomical characteristics Outer bark: Remaining single rhytidome was present, made up of a single peridermis with 6 to 10 layers of thin-walled tabulated phellem cells. The phelloderm was unstratified. At the boundary between the peridermis and the secondary phloem there was a continuous ring of sclereids (Fig. 1L; Fig. 2G). Inner bark: The cross-sections enabled the distinction of different regions of functional phloem separated by fibers in small clusters of cells and limited by tangential bands of axial parenchyma with abundant calcium oxalate crystals. Longitudinal uniseriate and pluriseriate phloem rays with cell walls of regular contour were observed (Fig. 1A, D; Fig. 2C). The longitudinal
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sections enable the distinction of different rays: heterogeneous, multiseriates and uniseriate. The radial longitudinal section enabled us to determine the phloem radii, which are heterocellular, with 4–5 marginal rows of high upright cells, the multiseriate parts composed of procumbent cells having intercellular spaces (Fig. 1 B; Fig. 2 D). Radius height is 25 to 50 cells. The uniseriate rays are composed of square to upright ray cells. Radius height is 8 to 10 cells (Fig. 1 E). The tangential longitudinal section showed uniseriate parenchyma radii and pluriseriate ones, 4–5 cells wide (Fig. 1 C, F; Fig. 2E). The elements of the sieve tube are broad, presenting a composed, highly developed sieve plate in an inclined position (Fig. 2F).
Macerated bark: The maceration of the bark (Fig. 2H) made the determination of the following cellular elements possible: a) Sclereids: Consisting of polygonal rectangular brachisclereids, 100 µm in length and 40 µm in width; and isodiametric brachisclereids, 50 µm in diameter (Fig. 1K). b) Ray cells: Showing parenchymatous cells of high upright cells and procumbent cells measuring from 22–25 µm to 50–55 µm (Fig. 1J). c) Axial parenchyma: Showing elongated cells measuring 75 µm in height and 25 µm in width (Fig. 1I). d) Phellem cells: in cross-sections, they were rectangular with thin-walled cells and measured 25–30 µm in length and 18–20 mm in width (Fig. 1G). In the superficial view they were polygonal, attaining a diameter of 22–25 µm (Fig. 1H).
Fig. 1. A–L, Common characteristics of the bark of Uncaria tomentosa (Willdenow ex Roemer & Schultes) De Candolle and Uncaria guianensis (Aublet) J. F. Gmelin. A–C: Schematic view of the bark. A: transverse section; B: longitudinal radial section; C: longitudinal tangencial section; D: detail of A; E: detail of B; F: detail of C. G–L: Macerated bark; G: phellem cells in cross section; H: phellem cells in superficial view; I: axial parenchyma cells; J: secondary heterocellular ray cells; K: brachysclereids; L: sclereid ring between phellem and parenchyma. c: calcium oxalate microcrystals, f: phloem fibers, ph: phellem, r: ray, s: sclereid, se: sieve element, sp: sieve plate. Scale bars: 1 to C; 2 to B, F; 3 to A; 4 to E; 5 to D; 6 to G, H, I, J, K, L.
Morphoanatomical studies of Uncaria tomentosa Non-shared characteristics of the bark
Uncaria tomentosa (Willd. ex Roemer & Schultes) DC. a) Phloem fibres: They were located in the inner bark forming tangential bands between functional phloem. Fibre size 1500 to 2000 µm length, 50 µm width (Fig. 3 B; Fig. 4 E). b) Axial and radial parenchyma: including only calcium-oxalate microcrystals (Fig. 3A; Fig. 4 A). c) Axial and radial parenchyma: showing simple or compound starch granules with two, three and occasionally more components. Starch granules sized 8 to 12 µm in diameter (Fig. 3 C; Fig. 4C). Uncaria guianensis (Aublet) J. F. Gmelin a) Phloem fibres: They were located in the inner bark forming tangential bands between functional phloem. Fibre size 800 to 1500 µm length, 20 to 50 µm width (Fig. 3 E; Fig. 4 F). b) Axial and radial parenchyma: in-
Fig. 2. A–H. Common characteristics of the bark of Uncaria tomentosa (Willdenow ex Roemer & Schultes) De Candolle and Uncaria guianensis (Aublet) J. F. Gmelin. A: outer bark surface; B: inner bark surface; C: bark in transverse section; D: bark in longitudinal radial section, showed 4 rows of high upright cells; E: bark in longitudinal tangencial section; F: terminal compound sieve plate; G: sclereid ring between phellem and parenchyma; H: macerated bark elements. p: parenchyma, ph: phellem, r: ray, s: sclereid, sp: sieve plate. Scale bars: A,B: 1,5 cm; C: 100 µm; D: 10 µm; E: 25 µm; F: 10 µm; G: 10 µm; H: 25 µm.
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cluding calcium-oxalate microcrystals and polyhedrical large crystals of calcium-oxalate measuring 40 to 60 µm (Fig. 3D; Fig. 4 B). c) Axial and radial parenchyma: showing simple or compound starch granules with two, three, and occasionally more components. Starch granules sized 4 to 10 µm in diameter (Fig. 3F; Fig. 4D). Characteristics of the leaf
Uncaria tomentosa (Willd. ex Roemer & Schultes) DC. Morphological characteristics
Leaf blade, ovate, elliptic, oblong or obovate (length/ width 1.5–2.2), 6–16 × 2.5–11 cm, broadly cuneate or rounded to truncate or subcordate at base, obtuse to acuminate at apex, 7–10 pairs lateral veins, level to prominulous above, prominent beneath, while tertiary
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veins may be distinguished in its lower part. Adaxial matte surface to subnitid, glabrous or slightly hirtellous on midrib, abaxial surface with the midrib, and sometimes lateral veins as well, most of which are slightly are puberulent or hirtellous and also with coarse, subapressed hairs, very densely tomentose, petiole 0.8–1.5 cm long (Fig. 5 A; Fig. 6A). Anatomical characteristics
• Lamina Surface view of epidermis: The epidermal cells of the adaxial face showed straight, anticlinal walls of 4 to 5 sides. Epidermic abaxial cells presented straight anticlinal walls with unicellular trichomes curved at the apex. The cuticule is smooth. There were stomata only in the inferior epidermis; the stomatal type found was rubiaceous (Fig. 5C, D; Fig. 6E, F).
Cross-sections: It was dorsiventrally compressed. Unstratified adaxial epidermis with conspicuous cuticule, without stomata. Mesophyll was dorsiventral, with only one layer of palisade parenchyma. The spongy parenchyma cells were located next to the abaxial epidermis. Loosely arranged, with many intercellular spaces among them. Mesophyl presented very abundant crystalliferous idioblasts, containing calcium-oxalate microcrystals. The main nerve, very protuberant, in the adaxial and abaxial face, was strengthened by laminar collenchyma. The cylindrical vascular strand enclosed accessory medullary strands. It presented very abundant large schizogenous secretory cavities situated externally to the phloem (at one time described as laticiferous vessels). The lower epidermis with little cells showed a thick, smooth cuticle and stomata located at the same level as the epidermic
Fig. 3. A–F. Non shared characteristics of the bark. AC: Uncaria tomentosa (Willdenow ex Roemer & Schultes) De Candolle. A: microcrystals of calcium oxalate from ray cells from longitudinal radial section; B: phloem fibers; C: starch grains in cells of ray from transverse section. D-F: Uncaria guianensis (Aublet) J. F. Gmelin. D: Microcrystals and polyhedrical large crystals of calcium oxalate from ray cells from longitudinal radial section; E: phloem fibers; F: starch grains in cells of multiseriate ray from transverse section. Scale bars: 1 to A, C; 2 to B; 3 to D, F.
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cells. There were abundant unicellular trichomes (Fig. 5 G, I; Fig. 6 G, H, I). Foliar architecture: Dense vascularization. The analysis of the foliar architecture made possible the determination of pinnate, camptodromus, brochidodromus venation with an incomplete last marginal venation and simple venules, curved and branched only once. Areolae withouth orientation, rectangular shape. Vascularization terminal tracheids have no sclereids, (Fig. 6C, D).
tion, surrounding the whole perimeter of the petiole. The parenchyma presented very abundant crystalliferous idioblasts, containing calcium-oxalate microcrystals. A main vascular strand exhibiting a shallow open arc usually deep and with its ends inwardly curved, accompanied by smaller accessory bundles towards the wings. It presented large schizogenous secretory cavities situated external to the phloem (Fig. 5E; Fig. 6J).
• Petiole Transverse sections through the distal end, usually seen to be shield-shaped and with a well-developed wing. The epidermis was unstratified, with stomata, thick and smooth cuticle. The petiole surface is provided with a superficial vein-like network containing spiral tracheids, especially on the abaxial surface (Fig. 6K). Laminartype collenchyma was observed in sub-epidermic loca-
Uncaria guianensis (Aublet) J. F. Gmelin
Fig. 4. A–F. Non shared characteristics of the bark. A, C, E: Uncaria tomentosa (Willdenow ex Roemer & Schultes) De Candolle. A: microcrystals (mc); C: starch granules (s); E: sclereids and fibers (f). B, D, F: Uncaria guianensis (Aublet) J. F. Gmelin. B: polyhedrical crystals (pc); D: starch granules (s); F: phloem fibers (f). Scale bars: A: 25 µm; B: 10 µm; C, D: 10 µm; E, F: 100 µm.
Morphological characteristics
Leaf blade ovate to elliptic (length/width 1.7–2.3), 6–16 × 2.5–11 cm, cuneate to rounded at base, acute to acuminate at apex, 4-6 pairs lateral veins, prominulous, distinguishable tertiary veins, adaxial surface glabrous, abaxial surface glabrous throughout. Petiole 0.5–1.5 cm long, glabrous (Fig. 5B; Fig. 7A).
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Anatomical characteristics
• Lamina Surface view of epidermis: The epidermal cells of the adaxial face showed straight thick anticlinal walls of 4 to 5 sides. Epidermic abaxial cells presented ondulate anticlinal walls without trichomes. The cuticule is smooth. There were stomata only in the inferior epidermis and the stomatal type was rubiaceous (Fig. 7E, F). Cross-section: It was dorsiventrally compressed. Unstratified adaxial epidermis with a thin cuticule, without stomata or trichome. Mesophyll was dorsiventral, with 1–2 layers of palisade parenchyma. The spongy parenchyma cells were located next to the abaxial epidermis. Loosely arranged, with many intercellular spaces. Mesophyl presented few crystalliferous idioblasts, containing microcrystals, and few polyhedrical crystals of calcium oxalate. Palisade and
spongy parenchyma cells were filled with red-brown unidentified contents. The main vascular strands strengthened by laminar collenchyma are concave but with a few small accessory strands in the concavity. They presented few schizogenous secretory cavities situated external to the phloem. The lower epidermis with little cells showed a thin, smooth cuticle and stomata located at the same level as the epidermic cells (Fig. 5 H, J; Fig. 7G, H). Foliar architecture: The analysis of the foliar architecture made possible the determination of pinnate, camptodromus, brochidodromus venation with an incomplete last marginal venation and simple venules, curved and branched only once. Well-developed areolae without orientation, polygonally shaped. Terminal tracheids present a welldeveloped sclereid sheath (Fig. 7 C, D).
Fig. 5. A–J. Characteristics of the leaf. A, C, D, E, G, I: Uncaria tomentosa (Willdenow ex Roemer & Schultes) De Candolle. A: leaf blade, thorns curved and stipules; C–D: epidermis, superficial view; C: abaxial; D: adaxial; E, G, I, transverse sections of leaf: E: petiole; G: main nerve; I: mesophyll. B, F, H, J: Uncaria guianensis (Aublet) J. F. Gmelin. B: leaf blade, thorns curved and stipules; F, H, J: transverse sections of leaf; F, petiole; H: main nerve; J: mesophyll. Scale bars: A, B: 1,5 cm; C: 10 µm; D: 25 µm; E, F, G, H: 250 µm; I, J: 150 µm.
Morphoanatomical studies of Uncaria tomentosa • Petiole Transversely through the distal end section usually shield-shaped and with well-developed wing. The epidermis was unstratified, with stomata, thick and smooth cuticle. Beneath the epidermis, a few layers of small, rounded parenchymatous cells were filled with red-brown unidentified contents. The parenchyma presented few crystalliferous idioblasts, containing calcium-oxalate microcrystals. Loosely arranged, with many intercellular spaces. The central parenchymatous region presented the cells walls variably and unevenly thickened. Main vascular strand exhibiting a shallow open arc usually deep and with its ends inwardly curved, accompanied by smaller accessory bundles towards the wings. It presented large schizogenous secretory cavities situated external to the phloem (Fig. 5F; Fig. 7 I, J).
Fig. 6. A–K. Uncaria tomentosa (Willdenow ex Roemer & Schultes) De Candolle, leaf. A: leaf blade; B: thorns curved and stipules; C: last marginal venation and areolae; D: terminal tracheids, without sclereids; E–F: superficial view of epidermis, E: adaxial; F: abaxial; G–K: tranverse sections of the leaf; G: leaf diagrams; H: detail of main nerve; I: detail of mesophyll; J–K: transverse sections of petiole; J: petiole diagrams; K: detail of J. Scale bars: 1 to A, B; 2 to G, J; 3 to D, E, F, H, I, K; 4 to C.
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Discussion The expansion of the market for phytodrugs underscores the importance of pharmacognostic studies of those medicinal plant species used in traditional or popular medicineal systems which may become undistrially important in the near future. These data are required not only for identification procedures that guarantee the utilization of the appropriate raw material, but also for quality-control standards demanded by health legislation in western countries. Studies on botanical description, including macroand microscopic parameters, are useful tools to determine the anatomic parameters to be considered as characteristic of a certain plant and to avoid falsifications or adulterations in the process of commercialization of raw materials.
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Acknowledgements
Collaboration from Subprogram X, Fine Pharmaceutical Chemistry, RIPROFITO (Iberoamerican Network of Phytopharmaceutical Products), a part of the Iberoamerican Program of Science and Technology for Development (CYTED), is gratefully acknowledged. This work was supported in part by a grant to project N° 19/B058 of Universidad Nacional de Rosario (Argentina) to MG.
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Fig. 7. A–J. Uncaria guianensis (Aublet) J. F. Gmelin, leaf. A: leaf blade; B: thorns curved and stipules; C: last marginal venation and areolae; D: terminal tracheids, with sclereids; E-F: superficial view of epidermis, E: adaxial; F: abaxial; G-J: tranverse sections of the leaf; G: leaf diagrams; H: detail of G; I-J transverse sections of petiole; I: petiole diagrams; J: detail of I. e: cell walls variably and unevenly thickened. Scale bars: 1 to A, B; 2 to G, I; 3 to D, E, F, H, J; 4 to C.
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Address M. Gattuso, Botánica, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Suipacha 531 (2000) Rosario, Argentina Fax: ++54-341-4375315; e-mail: [email protected]; [email protected]