Latin American Journal of Pharmacy (formerly Acta Farmacéutica Bonaerense) Original Article Received: August 25, 2010 Revised version: September 7, 2010 Accepted: September 11, 2010 Lat. Am. J. Pharm. 30 (4): 673-82 (2011) South American “Marcelas”: Pharmacobotanic Characterization and Quality Control of Achyrocline tomentosa and Gnaphalium cheiranthifolium (Asteraceae) Luis A. DEL VITTO*, Elisa M. PETENATTI & Marta E. PETENATTI Área de Farmacognosia, Herbario/Jardín Botánico, Universidad Nacional de San Luis, Ejército de Los Andes 950, D5700HHW San Luis, Argentina SUMMARY. Achyrocline tomentosa Rusby and Gnaphalium cheiranthifolium Lam. are two of the medicinal plants called “Marcelas” widely distributed in Southern South America, being used in popular and official medicine or even in cosmetic industry and manufacture of bitter beverages. Both are used in ethnomedicine mainly for its digestive properties, such as simple drug or mixed with other herbs. These drugs come from natural populations, and this work provides elements for distinguishing among themselves and other species of both genera by means of exomorphological, anatomical and quantitative micrographic features. It is described and discussed for the first time the stem anatomy of A. tomentosa and the stem and leaf anatomy of G. cheiranthifolium, the macroscopic characteristics of the commercial drugs, the features of dissociated tissue, the relative share of both drugs on the market, and quantitative micrographic parameters of the two species, as a contribution to pharmacobotanic characterization and quality control of these drugs. INTRODUCTION Several species of Gnaphalium and Achyrocline widely distributed in Southern South America are collectively called “Marcela” (in Portuguese, “Macela”). They are important herbs of commerce in Argentina and neighboring countries, used in human medicine and industry for the formulation of herbal medicines and cosmetics, as well as in the development of aperitifs (“amargos”) 1,2. Currently the two genera are placed in the tribe Gnaphalieae Cass. ex Lecoq & Juillet, which alongside Plucheeae Anderb. have been segregated from Inuleae Cass. s.l. 3-6. Morphologically, the species of Achyrocline and Gnaphalium are closely related, although the former are characterized by narrow capitula with less than 25 flowers, functionally female, uniseriate, and glabrous cypselae, while those of Gnaphalium have large, campanulate capitula, with countless multiseriate female marginal flowers and papillate to shortly stipitate-hairy cypselae. Pappus hairs, free at base, distinguished both genera from others of the same tribe as Gamochaeta Wedd. and Lucilia Cass. (sensu Anderberg 5) that have falling and welded together at the base. Achyrocline tomentosa Rusby and Gnaphalium cheiranthifolium Lam. occur with some frequency as substituents or in association with the only officinal recognized entity of this group, Achyrocline satureioides (Lam.) DC. 7. Because of the close taxonomic relationship, the exomorphological resemblance and similar popular uses among different species of “Marcelas”, it is investigated both poorly known pharmacobotany and pharmacognostic aspects of Achyrocline tomentosa and Gnaphalium cheiranthifolium, to characterize and contribute to effective quality control of these herbs. KEY WORDS: Achyrocline tomentosa, Gnaphalium cheiranthifolium, “Marcelas”, Quality control, Herbal remedies. * Author to whom correspondence should be addressed. E-mail: lvitto@unsl.edu.ar ISSN 0326-2383 673 DEL VITTO L.A., PETENATTI E.M. & PETENATTI M.E. MATERIALS AND METHODS Materials Samples were obtained from natural populations, health food stores, pharmacies, local markets and even from the stock of street vendors and/or connoisseurs. Part of the material was designed to prepare herbaria specimens or herbal documentation, another was used in exomorphological studies and, finally, one third was fixed and preserved in formalin-acetic acidalcohol (FAA) and used for anatomical studies. The exsiccata examined were as follows: a) Achyrocline tomentosa. Argentina, San Luis prov., L.A. Del Vitto & E.M. Petenatti # 8121 (UNSL), and b) Gnaphalium cheiranthifolium. Argentina, San Luis prov., L.A. Del Vitto, E.M. Petenatti & M.E. Petenatti # 9195 (UNSL). Methods Permanent preparations were obtained by dehydration with an increasing series of ethanol, infiltration and paraffin embedding, cutting by microtome, dewaxing, staining with safranin-fast green 8 and mounting in DPX. The leaves were diaphanized 9 to apply quantitative micrographic techniques, determining the stomatal number (SN) 10 on both epidermal faces, stomatal index (SI) 11, palisade ratio (PR) 12, vein-islet number (VIN) 13 and veinlet termination number (VTN) 14; SN was measured with 40x objective, and other parameters with 20x, making at least 20 repetitions per parameter and species. Additionally, primary standard material was powdered and treated with Jeffrey reagent 14 to compare the dissociated tissues of both species. Microscopic observations and measurements were made by optical microscope (DMRB; Leitz Wetzlar), and macromorphological ones by stereomicroscope (M-10; Leica Microsystems GmbH). Photomicrographs were obtained by digital camera (EC-3; Leica Microsystems Ltd.) and images capture system (LAS EZ software v. 1.7.1; Leica Microsystems Ltd.). Histological preparations and genuine drug standards are deposited in the Herbarium at the Universidad Nacional de San Luis (UNSL). RESULTS AND DISCUSSION Achyrocline tomentosa Rusby (Fig. 1) H.H. Rusby, Bull. New York Bot. Gard. 4 (14): 388. 1907.- R.A. Giangualani, Darwiniana 20 (3-4): 557-9. 1976.- A.L. Cabrera, Fl. Prov. Jujuy 10: 272-4. 1978.- L. Ariza-Espinar in Barboza et al., Fl. Med. Prov. Córdoba (Argentina): 305. 674 2006.- N.D. Bayón, Inuleae, in S.E. Freire et al., Darwiniana 44 (2): 424. 2006. Synonyms: Achyrocline polycephala Rusby, Bull. New York Bot. Gard. 4 (14): 388. 1907. Common names: “Marcela”, “Uira”, “Uira-uira” 16-18, “Vira-vira” (M. Saldías & L. Orozco # 4207 in scheda, USZ, CTES). Taxonomic remarks Giangualani 19 synonymized Achyrocline tomentosa with A. rupestris Cabrera, but Freire 20 interpreted that the vigorous plants, densely woolly, with sparsely branched stems and broader leaves match the type of A. tomentosa, while the small specimens, densely branched from the base, with small leaves and loose to dense pubescence, living above 2,900 m asl, resemble the type of A. rupestris, revalidating therefore this taxon. Habit and bioforms (Fig. 1, A) Chamaephytes (subshrubs) from (7-) 50-100 (-150) cm tall, with ascending or erect branches, but the lower ones often decumbent on steep slopes. Exomorphology Stems cylindrical, thin, little to much branched, loosely leafy to the apex, densely woolly-pubescent (Fig. 1, C). Leaves alternate, simple, long-attenuated into the pseudopetiole, up to 25 mm, blades lanceolate, oblanceolate to narrowly elliptic, (8-) 50-120 (-135) x (1.5 -) 2-15 (-35) mm, margin entire or slightly erose, sometimes revolute; discolor or gently discolor, upper surface subglabrous to densely lanuginous, and lower surface greyish-tomentose, densely woolly. Capitula turbinate, very numerous, arranged in dense clusters in turn grouped in corymbiform cymes (Fig. 1, B-C). Involucre subglabrous, cylindrical-fusiform, 2.5-4 mm, yellow whitish or brownish yellow, with 8-9 bracts, the outer 2.53.5 mm, median 3-3.5 mm (both with dense woolly pubescence of non-glandular trichomes interspersed with short glandular), the inner 2.53.5 mm long, with some glandular trichomes, sometimes with a few flagelliform trichomes. Flowers 4-6 in each head, the disk florets 1-2, perfect, with tubular corolla of 2-3.5 mm and anthers 1-1.5 mm, and radial florets (3-) 4 (-5), carpelates. Ovary 0.5 mm, style 2-3 mm, stigmatic branches 0.5 mm. Fruits ellipsoid, 0.7-1.1 mm, glabrous, finely striated. Pappus hair-like, of the same length of the corolla. Latin American Journal of Pharmacy - 30 (4) - 2011 Stem anatomy (Fig. 1, H-I) Primary stem cross-section shows a 1-layered epidermis, cortex consisting of several layers of angular collenchyma with few interruptions of spongy chlorophyllic parenchyma and a conspicuous endodermis of large, rectangular cells with lipidic inclusions. The vascular bundles are collateral, forming a discontinuous cylinder (a Figure 1. Achyrocline tomentosa. A, plant habit in Central Sierras of San Luis, Argentina. B-C, detail of capitulescences and flowering tops. D-E, cross-section of leaf showing midrib (D) and upper surface cells with thickening cuticle (E). F-G, pubescence on lower leaf surface, with non-glandular flagelliform trichomes (F) and glandular biseriates (G). H-I, primary stem cross-section, with detailed pith cells (I). J, appearance of the commercial powdered drug. K-N, dissociated commercial drug (milled flowering tops) with pieces of whip-like trichomes, epidermis, fibers and parenchyma. The bar is about 10 cm for A, 2.5 cm for C, 2 cm for J, 1.5 cm for B, 950 µm for F, 550 µm for D, 330 µm for H, 200 µm for I, K and L, 180 µm for E and G, 100 µm for N and 60 µm for M. 675 DEL VITTO L.A., PETENATTI E.M. & PETENATTI M.E. typical eustele) around the pith which consists of isodiametric cells with starchy contents. The stem anatomy is described for the first time for this species. Leaf anatomy (Fig. 1, D-G) Cross-section of leaf shows an uniseriate epidermis, whose anticlinal cell walls are straight in the upper leaf surface, while in the abaxial surface are wavy. Cuticle on upper side is thick and striated while the lower one is thinner. Leaves are hypostomatic, with anomocytic and raised stomata only in the abaxial epidermal side. Hair-covering is formed by abundant both non-glandular (flagelliform) and glandular (vesiculate) trichomes. Mesophyll structure is bifacial, with one or two layers of palisade parenchyma and a multilayered loose spongy parenchyma. Main veins are poorly printed on the epiphyll, but the midrib (and to a lesser degree lateral veins) is very prominent towards the hypophyll; the veins are protected by angular collenchyma next to both epidermal surfaces; the fibrovascular bundle is collateral. Overall, these results are consistent with those of Amat 15. Chorology and Habitat It lives in forest environments, wet slopes, sandy or rocky soils, between 500 and 3,000 m asl in the North, center and Mesopotamia of Argentina and adjacent areas of Bolivia, Chile and Paraguay and Southern Brazil, in the Altoandina, Yungas, Puna, Chaco, Monte and Paranense biogeographical provinces. In Argentina it descends from the Andes foothills of Jujuy, Salta, Tucumán, Catamarca, La Rioja, San Juan and Mendoza provinces, toward temperate mountains (Sierras of San Luis and Córdoba), and through the plains reaches Corrientes and Misiones provinces. Phenology In temperate zones it presents a winter break with senescence and fall of the foliage and frost loss of shoots; the woody bases generate new shoots next spring. In subtropical environments, the plants remain active most of the year. It blooms in summer and fruits ripen at the end of that season, spreading up into fall. Share with other species of Gnaphalium and Achyrocline the dissemination strategy (anemochory) and the response to unfavorable environmental conditions. 676 Ethnomedical uses The “flowers” (truly, capitula and more appropriately capitulescences) and the flowering tops are used in folk medicine as digestive 16-22; have also been used as an antiemetic, antitussive/against catarrh/ expectorant and against flu 18,23. According to the analyzed samples, it is less used than A. satureioides, maybe due to its lower population frequency. Gnaphalium cheiranthifolium Lam. (Fig. 2) J.-B. Monnet de Lamarck, Encycl. Méthod. 2: 752. 1786.- A.P. De Candolle, Prodr. 6 : 223. 1838.- A.L. Cabrera, Revista Centr. Estud. Agron. Vet. Buenos Aires 22 (139): 37. 1929.- A.L. Cabrera, Revista Mus. La Plata, n.s. Bot. 4 Sec Bot 17: 162-164. 1941.- A.L. Cabrera, Fl. Prov. Buenos Aires 6: 165-166. 1963.- A.L. Cabrera, Revista Mus. Argent. Ci. Nat. Bernardino Rivadavia, Bot. 2 (5): 313. 1961.- A.L. Cabrera, Fl. Il. Entre Ríos, Argent 6: 317. 1974.- A.L. Cabrera, Fl. Prov. Jujuy 10: 287. 1978.- O.M. Hilliard & B. L. Burtt, Bot. J. Linn. Soc. 82: 181. 1981.- S.E. Freire, Fl. Fanerog. Argent., 280. Asteraceae, Parte 2. Fasc. 14: 33. 1995.- A.L. Cabrera et al., Cat. Il. Compuestas (=Asteraceae) Prov. Buenos Aires, Argent.: 60. 2000. Synonyms: Gnaphalium citrinum Hook. & Arn., Bot. Beechey Voy. 1: 31. 1830.- Gnaphalium paniculatum Bertero ex Colla, Mem. Acad. Sci. Torino 38: 17, tab. 26. 1835.- Gnaphalium valdivianum Philippi, Linnaea 29 : 6. 1857.- Gnaphalium araucanum Philippi, Anales Univ. Chile 43 : 502. 1873.Gnaphalium acutifolium Philippi, Anales Univ. Chile 90: 12. 1895.- Pseudognaphalium cheiranthifolium (Lam.) Hilliart & B.L. Burtt, Bot. J. Linn. Soc. 82 (3): 25. 1981.- Gnaphalium andicola Philippi, Anales Univ. Chile 90: 17. 1895. Common names: In the Northeast of Argentina it is marketed under the name “Marcelita”, e.g. in Corrientes city (R. Martínez Crovetto # 10768 in scheda, CTES), a name which this survey have corroborated in other markets recently. On the coast of Buenos Aires province it is called “Yateí-caá” (fide R. Carnevali # 3428 in scheda, CTES), while in Ouro Preto (Minas Gerais, Brazil) its common name is “Marcela” (fide A. Schinini & S. Ferrucci # 24595 in scheda, CTES) or “Macela”. In other parts of Argentina and some Uruguay markets it is called “Marcela macho” or “Marcelote”, presumably to differentiate by size from species of Achyrocline, and also is called “Vira-vira” 18. Latin American Journal of Pharmacy - 30 (4) - 2011 Figure 2. Gnaphalium cheiranthifolium. A, plant habit in a Prosopis forest at Los Puquios, San Luis. B-C, detail of capitulescences. D-E, leaf midrib cross-section (D) and a portion of the blade (E). F, lower epidermis with sunken stoma.- G, developing trichome. H-I, glandular trichomes (H, uniseriate; I, biseriate). J, primary stem cross-section. K, whip-like trichome. L, the drug as presented in the street market. M-P, dissociated raw drug (flowering tops) with vessels, fibrotracheids, fibers, sclereids, pieces of glandular and non-glandular trichomes, etc. (O-R), glandular trichomes (O), and stem epidermis (P). The bar is about 10 cm for A and L, 5 cm for B, 2 cm for C, 1,300 µm for K, 1,000 µm for D, 450 µm for E and J, 450 µm for H and P, 320 µm for O, 170 µm for F and G, 100 µm for I, and 40 µm for M and N. 677 DEL VITTO L.A., PETENATTI E.M. & PETENATTI M.E. Taxonomic remarks Covas 24 suggested the synonymy between G. cheiranthifolium and G. gaudichaudianum, based on populations of the Argentinean province of La Pampa. Along this work, has not been found intergradation of characters between the two taxa in center and North of Argentina, therefore does not adhere to this view until conclusive genetic and/or molecular evidences. In Tropicos database 25 three subordinate taxa are considered as valid, as follows: fma. citrinum (Hook. & Arn.) Kuntze, Revis. Gen. pl. 3 (3): 151, 1898 (basionym: Gnaphalium citrinum Hook. & Arn.); var. multiflorum J. Koster, Blumea 5 (3): 655, 1945; and var. paniculatum Skottsb., Köngl. Svenska Vetenskapsakad. Handl. 51 (9): 5, 1914 (basionym: Gnaphalium paniculatum Bert. ex Colla?). Until conclusive studies are conducted on this group, the validity of infraspecific taxa is subject to reasonable doubt, mainly because their wide distribution, which may show quite pronounced local variations. Habit and bioforms (Fig. 2, A-B) Hemicryptophytes to chamaephytes (shortlived perennial herbs), from 20-80 (-100) cm tall, completely gray- or albo-tomentose. Exomorphology Aerial parts with dense pubescence with different types of trichomes: I) single-celled (papillae) with distended cuticle; II) glandular generally biseriate, with 2-7-celled stalk and cell wall thick and smooth, with ovate-globose apical cell of the same thickness as the basal; III) non-glandular long, with 2 (-3) basal wider cells with one terminal elongated, curled, flagelliform (whip-shaped) cell (Fig 2, K) that frequently is lost to maturity, which gives a subglabrous appearance to leaves and stems. Stems erect, simple or branched, leafy towards the apex, densely albo-tomentose with non-glandular hairs mixed with glandular ones, especially towards the apex. Leaves alternate, simple, (8-) 60-80 (-120) x (2-) 3-8 (-15) mm, initially in a basal rosette, oblanceolate-spatulate, obtuse, and basal leaves oblanceolate, lanceolate to linear-lanceolate or even linear, long attenuate and acute in apex, sessile, somewhat decurrent at the base (thus stems narrowly winged), margin entire or sometimes slightly curly, concolorous, densely albotomentose to woolly on both sides, with long glandular hairs interspersed in the epiphyll. Capitula numerous, hemispherical to cam678 panulate, 4.5-6 mm high x 5-6 mm dia., clustered in dense terminal glomerules, which sometimes are solitary or more frequently grouped in dense corymbiform cymes (Fig. 2, BC). Involucre multibracteate, scarious. Phyllaries in 4-5 series, citrine; outer ovate, acute, the inner lanceolate to obovate or oblong, obtuse at the apex, with broad scarious margin. Marginal florets carpellate (functionally female), very numerous, corolla filiform; disk florets 10-15, perfect (functionally hermaphrodites), corolla tubular. Cypselae smooth to finely tuberculate, glabrous, 0.8-0.9 mm. Papus with free hairs, something barbelate at the base. Stem anatomy (Fig. 2, J) Cross-section shows a terete to subcircular structure, the epidermis is 1-layered, with a thick and smooth cuticle, some papillae and stomata at the same level as other epidermal cells; the dense indumentum is composed of glandular and non-glandular trichomes of the types described above. Cortex of 2-3 collenchymatous layers, interrupted by groups of chlorenchymatic cells, contacting with 1-layered conspicuous endodermis, composed of tabular cells with thick walls, in which it has not been observed Casparian strips. The vascular bundles are collateral, arranged in an eustele, with about 20 side bundles of various sizes separated by medullary rays, each protected externally by a sclerenchymatic cap contacting the endodermis. The pith is solid, with large isodiametric, starchy, parenchymatic cells of polyhedral cross section; the larger ones are placing toward the center. Stem anatomy is described for the first time for this species. Leaf anatomy (Fig. 2, D-I) Cross-section shows the epidermis is uniseriate on both sides; adaxial epidermal cells are polygonal, with their anticlinal walls straight and covered with relatively thick cuticle, while those of abaxial surface are irregular, with wavy anticlinal walls, and covered with a thin cuticle. The leaves are hypostomatic, because the upper epidermis has no stomata, whereas the lower one has a large number of stomata, anomocytic because the surrounding (4-5) cells not differ from other epidermal cells; these characters are shared by other species of the genus; unlike Achyrocline species, in G. cheiranthifolium stomata are located at the same level as other epidermal cells, or just sunk. Both epidermal sides have glandular and non-glandular trichomes of types described above, although Latin American Journal of Pharmacy - 30 (4) - 2011 more abundantly in the lower face. Mesophyll structure is isobilateral, with 2 layers of palisade chlorenchyma to the epiphyll, and only one of short, tabular cells to the hypophyll, while the center is occupied by 2-3 layers of spongy parenchyma. Chlorenchymatic cells bear abundant lipidic globules. Vascular bundles are collateral; the side veins are totally immersed in the mesophyll, surrounded by a conspicuous parenchymatic sheath; the midrib is prominent toward the hypophyll, and is protected by caps of angular collenchyma in contact with both epidermal sides. Leaf anatomy of this entity is reported for the first time in this study. Chorology and habitat It is widespread in South America, from Southern Brazil and Bolivia, Uruguay to central Argentina and Chile, between sea level and 2,500 m asl, in loose soils, sandy to rocky, in the biogeographical provinces Yungas, Chaqueña, Monte, Pampeana, Patagónica and their ecotones. It has been documented in the Argentinean provinces of Buenos Aires, Córdoba, Corrientes, Entre Ríos, Jujuy, La Pampa, La Rioja, Mendoza, Neuquén, Río Negro, Salta, San Luis, Santa Cruz, Santa Fe and Tucumán. Phenology Similar to that of the preceding species, except plants behave as biennials in places of greatest height and coldest winters. Ethnomedical uses Leaves, stems and “flowers” (in fact, capitulescences) are used in folk medicine, especially as a diaphoretic / febrifuge, antitussive and especially as a depurative 18,26; the infusion as an emmenagogue and abortifacient 18; its use as a digestive and hepatic 18 probably due to their morphological similarity with G. gaudichaudianum and some species of Achyrocline. Leaf Architecture Venation of both species is pinnate, camptodrome, with a prominent, straight midrib, and 24 basal lateral primary veins emerging from the base of the blade as ramifications of the midrib, running nearly parallel to it, diverging progressively and more forward curve inward and finally anastomose with secondary veins, these arise from the midrib and in the same way as the lateral primary run nearly parallel, diverging then with an angle of 50 °. The tertiary veins, which are reticulate, and the higher-order ones decrease progressively in importance to the margin, with arches from second to fourth order. The last marginal venation is generally buttonhole. It has some intersecondary veins, too. Divergence angles of the ramifications of 3rd. order and above are acute, but little below 80 °. The areoles are highly developed, from quadrangular to polygonal, and the venules (veinlet termination in each areola) vary from simple to 1-2 branched. The differences between the two species are reduced to the basal lateral primary veins arising from the petiole vascular bundle in A. tomentosa, keeping parallel to the midrib by a short length, diverging at an angle up to 20 º, while in G. cheiranthifolium at least two lateral veins are already present in the decurrence of the leaf on the stem, accompanying to the midrib by longer distance, and finally separating it with a more acute angle of divergence that in A. tomentosa. To the present knowledge, this is the first description of leaf architecture of these entities, according the nomenclature of Hickey 27. Macroscopic features of the commercial drugs (Figs. 1, J and 2, L) For both species, the drug of the commerce consists of the flowering tops, and often only the inflorescences (capitula) grouped in capitulescences, always dried; depending on the degree of milling the drug can be made up mostly of single flowers isolated, accompanied by fragments of peduncles, receptacles and phyllaries. Its general color is pale yellow (when prevail capitula) to grayish or whitish (when prevail stem and leaf), the smell is highly aromatic and the taste is bitter. The appearance is that of disordered clusters of foliar and flowering elements, matted by the abundance of the long flagelliform trichomes that form the most conspicuous pubescence of both species. Participation of these species in the market By means of the sampling of “Marcelas” in herbal shops, pharmacies and local markets, as well as in the stock maintained by street vendors and connoisseurs, in more important cities and towns in Argentina and some bounding countries, the authors have recorded the percentage share of both species in the herbal market in the region. Has been determined in this work that Acyrocline tomentosa, Gnaphalium cheiranthifolium and mixtures thereof include approximately 10 % of the herbs marketed un- 679 DEL VITTO L.A., PETENATTI E.M. & PETENATTI M.E. der the collective nouns “Marcela”, “Marcela macho”, “Marcelita”, and “Vira-vira”, and phonetic spelling variations recorded in the whole country, that botanically correspond mostly to species of the genera Gnaphalium and Achyrocline. From more than 300 samples obtained from various Argentine markets and neighboring countries under the above vulgar mentioned names, both in simple formulations as mixtures of herbs, 33 of them correspond to the species studied, or their associations, and the relative percentage share is shown in Table 1. Quantitative micrographic parameters Values, expressed in Table 2, are useful in raw drug identification and processed herbal medicines obtained from them, especially when the material is milled or reduced to powder. As observed in other species of Achyrocline and Gnaphalium, the leaves of both species studied are hypostomatic, therefore abaxial epidermal surface showed between 12 to 13.5 Proven botanical identity Simple stomata per mm2 in A. tomentosa, with a rate close to 14 stomata, while in G. cheiranthifolium these values reach 16 to 17 stomata per mm2, with a stomata index near 15. The veinlet termination number ranged between 4 and 5 for A. tomentosa, varying between 19.5 and 21.2 in samples of G. cheiranthifolium, while the veinislet number per mm2 was between 6.7 and 8.3 in A. tomentosa, and between 14 and 15 in G. cheiranthifolium. Finally, the palisade ratio showed an average of almost 5 parenchymatic cells by each epidermal cell in A. tomentosa, and ca. 7 parenchymatic cells by each epidermal cell in G. cheiranthifolium. In brief, the stomata numbers and rates did not differ significantly in samples of the two species, whereas the veinlet termination number, vein-islet number and to a lesser extent, the palisade ratio were useful to distinguish (with highly significant differences) among the samples of the two species. These parameters are reported for the first time for both species. %* Claimed identity Commercial procedure Achyrocline tomentosa 15.6 “Marcela” H,P,M/F,V/C Gnaphalium cheiranthifolium 25 “Marcela” “Marcelita” M/F,V/C Mixture Achyrocline tomentosa + A. satureioides 18.7 “Marcela” H Gnaphalium cheiranthifolium + Achyrocline satureioides 9.4 “Wira-wira” H,P,M/F Gnaphalium cheiranthifolium + G. gaudichaudianum 31.3 “Marcela macho” H,V/C “Vira-vira” H,M/F,V/C Table 1. Botanical identity, claimed identity (common names), and commercial source of samples of Achyro- cline tomentosa and Gnaphalium cheiranthifolium. Ref.: H = Herbal shop; P = Pharmacy; M/F = Market or Fair; V/C = Vendor or Connoisseur; * On the total sample. Parameter Achyrocline tomentosa Gnaphalium cheiranthifolium Upper epidermal surface Stomatal number . mm–2 0 0 Lower epidermal surface Stomatal number . mm–2 Stomatal index 12.9 ± 0.7 (13.2) 13.8 (14.4) 16.6 ± 0.5 (14.7) 15.1 (15.6) 4.5 ± 0.5 7.5 ± 0.8 (4.7) 4.9 (5.2) 20.3 ± 0.9 14.6 ± 0.5 (6.7) 6.8 (7.2) Veinlet termination number . mm–2 Vein-islet number . mm–2 Palisade ratio Table 2. Quantitative micrographic parameters of samples of Achyrocline tomentosa and Gnaphalium cheiranthifolium. 680 Latin American Journal of Pharmacy - 30 (4) - 2011 Achyrocline tomentosa Gnaphalium cheiranthifolium Bioform, functional type and general appearance Chamaephytes (subshrubs), branched, green-grayish to green-yellowish Hemicryptophytes (biennial herbs) to chamaephytes (short-lived perennials), shortly branched at the base, albo-tomentose to yellowish Trichomes Short vesiculate glandular trichomes; non-glandular flagelliform (whip-like); short non-glandular Long glandular uniseriate and biseriate trichomes; non-glandular flagelliform (whip-like) Nomophylls Shortly decurrent, blade broadly lanceolate, oblanceolate to narrowly elliptical, 50-120 x 2-15 mm, discolor (upper surface yellowish-green and lower surface albo-tomentose) Long decurrent, blade linear-lanceolate, 60-80 x 3-8 (15) mm, subconcolor, greyish-tomentose on both sides Capitulescences Several-headed dense glomerules, arranged in corymbs or cymes terminal and/or axillary Few-headed dense glomerules, apical Capitula Very numerous, narrowly cylindric to subfusiforms, pauci-(4-6) flowered Few, large (4-6 mm long x 5-6 mm dia.), campanulate to hemispheric, pluriflora Phyllaries 8-9 phyllaries in 3 series, whitish-yellow or brownish, the outer woolly and sparsely glandular, the inner only glandular Numerous, in 4-5 series, scarious, citrine bordered and trichomes non-glandular and glandular Ray florets Few, (3) 4 (5), carpelate Very numerous (>30-35), carpelate Disk florets 1-2, perfect, tubulose 10-15, perfect, tubulose Cypselae Broadly ellipsoidal to ovoid, subglabrous, striated, pappus 1-seriate with bristles simple, capillary, strigose Glabrous, smooth to finely tuberculate, pappus 1-seriate with hairs simple, slender, barbelate at base Upper epidermal side Polygonal cells with anticlinal walls straight; cuticle thick and striated Cells with anticlinal walls somewhat sinuous Lower epidermal side Anticlinal cell walls windings Cells larger, anticlinal walls very curvy Mesophyll structure Bifacial Isobilateral Divergence of basal lateral primary veins Angle ca. 20° Angle < 20° Stomata Anomocytic, raised Anomocytic, from slightly elevated to something depressed Stomatal number.mm–2 12.9 – 13.6 (hypophyll) 16.1 – 17.1 Palisade ratio 4.7 – 5.2 6.7 – 7.2 Vein-islet number.mm–2 6.7 – 8.3 14.1 – 15.1 Veinlet termination number.mm–2 4–5 19.4 – 21.2 Table 3. Diacritic morphoanatomic and micrographic characters for Achyrocline tomentosa and Gnaphalium cheiranthifolium. 681 DEL VITTO L.A., PETENATTI E.M. & PETENATTI M.E. Analysis of dissociated tissues (Figs. 1, K-N, and 2, M-P) Authentic material of the two species has shown cellular elements common to both, as scalariform vessels, fibrotracheids, fibers, sclereids, and the remains of whip-shaped trichomes and parenchyma, while the diacritical elements were adaxial epidermal cells with straight anticlinal walls, and wavy in the abaxial epidermal cells, conspicuously raised stomata, short vesiculate glandular trichomes and short non-glandular biseriate trichomes for Achyrocline tomentosa, while that G. cheiranthifolium abaxial epidermal cells show anticlinal walls somewhat sinuous, and very curvy in the adaxial epidermal cells; stomata from slightly elevated to sunken in the epidermis; and long biseriate glandular trichomes. 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