Download - South American “Marcelas”: Pharmacobotanic Characterization and Quality Control of Achyrocline tomentosa and Gnaphalium cheiranthifolium (Asteraceae)

673ISSN 0326-2383

KEY WORDS: Achyrocline tomentosa, Gnaphalium cheiranthifolium, “Marcelas”, Quality control, Herbal remedies. * Author to whom correspondence should be addressed. E-mail: [email protected]

Latin American Journal of Pharmacy(formerly Acta Farmacéutica Bonaerense)Lat. Am. J. Pharm. 30 (4): 673-82 (2011)

Original ArticleReceived: August 25, 2010

Revised version: September 7, 2010Accepted: September 11, 2010

South American “Marcelas”: Pharmacobotanic Characterizationand Quality Control of Achyrocline tomentosaand 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 medici-nal plants called “Marcelas” widely distributed in Southern South America, being used in popular and of-ficial medicine or even in cosmetic industry and manufacture of bitter beverages. Both are used in eth-nomedicine mainly for its digestive properties, such as simple drug or mixed with other herbs. These drugscome from natural populations, and this work provides elements for distinguishing among themselves andother species of both genera by means of exomorphological, anatomical and quantitative micrographicfeatures. It is described and discussed for the first time the stem anatomy of A. tomentosa and the stem andleaf anatomy of G. cheiranthifolium, the macroscopic characteristics of the commercial drugs, the featuresof dissociated tissue, the relative share of both drugs on the market, and quantitative micrographic pa-rameters of the two species, as a contribution to pharmacobotanic characterization and quality control ofthese drugs.

INTRODUCTIONSeveral species of Gnaphalium and Achyro-

cline widely distributed in Southern SouthAmerica are collectively called “Marcela” (inPortuguese, “Macela”). They are important herbsof commerce in Argentina and neighboringcountries, used in human medicine and industryfor the formulation of herbal medicines and cos-metics, as well as in the development of aperi-tifs (“amargos”) 1,2.

Currently the two genera are placed in thetribe Gnaphalieae Cass. ex Lecoq & Juillet,which alongside Plucheeae Anderb. have beensegregated from Inuleae Cass. s.l. 3-6. Morpho-logically, the species of Achyrocline andGnaphalium are closely related, although theformer are characterized by narrow capitulawith less than 25 flowers, functionally female,uniseriate, and glabrous cypselae, while those ofGnaphalium have large, campanulate capitula,

with countless multiseriate female marginalflowers and papillate to shortly stipitate-hairycypselae. Pappus hairs, free at base, distin-guished both genera from others of the sametribe as Gamochaeta Wedd. and Lucilia Cass.(sensu Anderberg 5) that have falling and weld-ed together at the base.

Achyrocline tomentosa Rusby and Gnapha-lium cheiranthifolium Lam. occur with somefrequency as substituents or in association withthe only officinal recognized entity of thisgroup, Achyrocline satureioides (Lam.) DC. 7.Because of the close taxonomic relationship, theexomorphological resemblance and similar pop-ular uses among different species of “Marcelas”,it is investigated both poorly known pharma-cobotany and pharmacognostic aspects of Achy-rocline tomentosa and Gnaphalium cheiranthi-folium, to characterize and contribute to effec-tive quality control of these herbs.

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DEL VITTO L.A., PETENATTI E.M. & PETENATTI M.E.

MATERIALS AND METHODSMaterials

Samples were obtained from natural popula-tions, health food stores, pharmacies, local mar-kets and even from the stock of street vendorsand/or connoisseurs. Part of the material wasdesigned to prepare herbaria specimens orherbal documentation, another was used in exo-morphological studies and, finally, one thirdwas fixed and preserved in formalin-acetic acid-alcohol (FAA) and used for anatomical studies.The exsiccata examined were as follows: a)Achyrocline tomentosa. Argentina, San Luisprov., 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).

MethodsPermanent preparations were obtained by

dehydration with an increasing series of ethanol,infiltration and paraffin embedding, cutting bymicrotome, dewaxing, staining with safranin-fastgreen 8 and mounting in DPX. The leaves werediaphanized 9 to apply quantitative micrograph-ic 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, andother parameters with 20x, making at least 20repetitions per parameter and species. Addition-ally, primary standard material was powderedand treated with Jeffrey reagent 14 to comparethe dissociated tissues of both species. Micro-scopic observations and measurements weremade by optical microscope (DMRB; Leitz Wet-zlar), and macromorphological ones by stere-omicroscope (M-10; Leica Microsystems GmbH).Photomicrographs were obtained by digitalcamera (EC-3; Leica Microsystems Ltd.) and im-ages capture system (LAS EZ software v. 1.7.1;Leica Microsystems Ltd.). Histological prepara-tions and genuine drug standards are depositedin the Herbarium at the Universidad Nacional deSan Luis (UNSL).

RESULTS AND DISCUSSIONAchyrocline tomentosa Rusby (Fig. 1)

H.H. Rusby, Bull. New York Bot. Gard. 4(14): 388. 1907.- R.A. Giangualani, Darwiniana20 (3-4): 557-9. 1976.- A.L. Cabrera, Fl. Prov. Ju-juy 10: 272-4. 1978.- L. Ariza-Espinar in Barbozaet al., Fl. Med. Prov. Córdoba (Argentina): 305.

2006.- N.D. Bayón, Inuleae, in S.E. Freire et al.,Darwiniana 44 (2): 424. 2006. Synonyms: Achy-rocline 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 remarksGiangualani 19 synonymized Achyrocline to-

mentosa with A. rupestris Cabrera, but Freire 20

interpreted that the vigorous plants, denselywoolly, with sparsely branched stems andbroader leaves match the type of A. tomentosa,while the small specimens, densely branchedfrom the base, with small leaves and loose todense pubescence, living above 2,900 m asl, re-semble the type of A. rupestris, revalidatingtherefore 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 steepslopes.

ExomorphologyStems cylindrical, thin, little to much

branched, loosely leafy to the apex, denselywoolly-pubescent (Fig. 1, C). Leaves alternate,simple, long-attenuated into the pseudopetiole,up to 25 mm, blades lanceolate, oblanceolate tonarrowly elliptic, (8-) 50-120 (-135) x (1.5 -) 2-15(-35) mm, margin entire or slightly erose, some-times revolute; discolor or gently discolor, up-per surface subglabrous to densely lanuginous,and lower surface greyish-tomentose, denselywoolly.

Capitula turbinate, very numerous, arrangedin dense clusters in turn grouped in corymbi-form cymes (Fig. 1, B-C). Involucre subglabrous,cylindrical-fusiform, 2.5-4 mm, yellow whitish orbrownish yellow, with 8-9 bracts, the outer 2.5-3.5 mm, median 3-3.5 mm (both with densewoolly pubescence of non-glandular trichomesinterspersed with short glandular), the inner 2.5-3.5 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 andanthers 1-1.5 mm, and radial florets (3-) 4 (-5),carpelates. Ovary 0.5 mm, style 2-3 mm, stigmat-ic branches 0.5 mm. Fruits ellipsoid, 0.7-1.1 mm,glabrous, finely striated. Pappus hair-like, of thesame length of the corolla.

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Stem anatomy (Fig. 1, H-I)Primary stem cross-section shows a 1-layered

epidermis, cortex consisting of several layers ofangular collenchyma with few interruptions of

Figure 1. Achyrocline tomentosa. A, plant habit in Central Sierras of San Luis, Argentina. B-C, detail of capitules-cences and flowering tops. D-E, cross-section of leaf showing midrib (D) and upper surface cells with thicken-ing cuticle (E). F-G, pubescence on lower leaf surface, with non-glandular flagelliform trichomes (F) and glan-dular biseriates (G). H-I, primary stem cross-section, with detailed pith cells (I). J, appearance of the commercialpowdered 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 µmfor 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.

spongy chlorophyllic parenchyma and a con-spicuous endodermis of large, rectangular cellswith lipidic inclusions. The vascular bundles arecollateral, forming a discontinuous cylinder (a

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typical eustele) around the pith which consistsof isodiametric cells with starchy contents. Thestem anatomy is described for the first time forthis species.

Leaf anatomy (Fig. 1, D-G)Cross-section of leaf shows an uniseriate epi-

dermis, whose anticlinal cell walls are straight inthe upper leaf surface, while in the abaxial sur-face are wavy. Cuticle on upper side is thickand striated while the lower one is thinner.Leaves are hypostomatic, with anomocytic andraised stomata only in the abaxial epidermalside. Hair-covering is formed by abundant bothnon-glandular (flagelliform) and glandular(vesiculate) trichomes. Mesophyll structure is bi-facial, with one or two layers of palisadeparenchyma and a multilayered loose spongyparenchyma. Main veins are poorly printed onthe epiphyll, but the midrib (and to a lesser de-gree lateral veins) is very prominent towards thehypophyll; the veins are protected by angularcollenchyma next to both epidermal surfaces;the fibrovascular bundle is collateral. Overall,these results are consistent with those of Amat15.

Chorology and HabitatIt lives in forest environments, wet slopes,

sandy or rocky soils, between 500 and 3,000 masl in the North, center and Mesopotamia of Ar-gentina and adjacent areas of Bolivia, Chile andParaguay and Southern Brazil, in the Altoandi-na, Yungas, Puna, Chaco, Monte and Paranensebiogeographical provinces. In Argentina it de-scends from the Andes foothills of Jujuy, Salta,Tucumán, Catamarca, La Rioja, San Juan andMendoza provinces, toward temperate moun-tains (Sierras of San Luis and Córdoba), andthrough the plains reaches Corrientes and Mi-siones provinces.

PhenologyIn temperate zones it presents a winter break

with senescence and fall of the foliage and frostloss of shoots; the woody bases generate newshoots next spring. In subtropical environments,the plants remain active most of the year. Itblooms in summer and fruits ripen at the end ofthat season, spreading up into fall. Share withother species of Gnaphalium and Achyroclinethe dissemination strategy (anemochory) andthe response to unfavorable environmental con-ditions.

Ethnomedical usesThe “flowers” (truly, capitula and more ap-

propriately capitulescences) and the floweringtops are used in folk medicine as digestive 16-22;have also been used as an antiemetic, antitus-sive/against catarrh/ expectorant and against flu18,23. According to the analyzed samples, it isless used than A. satureioides, maybe due to itslower 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. Cabr-era, Revista Mus. La Plata, n.s. Bot. 4 Sec Bot 17:162-164. 1941.- A.L. Cabrera, Fl. Prov. BuenosAires 6: 165-166. 1963.- A.L. Cabrera, RevistaMus. Argent. Ci. Nat. Bernardino Rivadavia,Bot. 2 (5): 313. 1961.- A.L. Cabrera, Fl. Il. EntreRíos, 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. Com-puestas (=Asteraceae) Prov. Buenos Aires, Ar-gent.: 60. 2000. Synonyms: Gnaphalium cit-rinum Hook. & Arn., Bot. Beechey Voy. 1: 31.1830.- Gnaphalium paniculatum Bertero exColla, Mem. Acad. Sci. Torino 38: 17, tab. 26.1835.- Gnaphalium valdivianum Philippi, Lin-naea 29: 6. 1857.- Gnaphalium araucanumPhilippi, Anales Univ. Chile 43: 502. 1873.-Gnaphalium acutifolium Philippi, Anales Univ.Chile 90: 12. 1895.- Pseudognaphalium cheiran-thifolium (Lam.) Hilliart & B.L. Burtt, Bot. J.Linn. Soc. 82 (3): 25. 1981.- Gnaphalium andi-cola Philippi, Anales Univ. Chile 90: 17. 1895.Common names: In the Northeast of Argentinait is marketed under the name “Marcelita”, e.g.in Corrientes city (R. Martínez Crovetto # 10768in scheda, CTES), a name which this surveyhave corroborated in other markets recently. Onthe 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 Argentinaand some Uruguay markets it is called “Marcelamacho” or “Marcelote”, presumably to differenti-ate by size from species of Achyrocline, and al-so is called “Vira-vira” 18.

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Figure 2. Gnaphalium cheiranthifolium. A, plant habit in a Prosopis forest at Los Puquios, San Luis. B-C, detailof capitulescences. D-E, leaf midrib cross-section (D) and a portion of the blade (E). F, lower epidermis withsunken stoma.- G, developing trichome. H-I, glandular trichomes (H, uniseriate; I, biseriate). J, primary stemcross-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, 2cm 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 Fand G, 100 µm for I, and 40 µm for M and N.

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Taxonomic remarksCovas 24 suggested the synonymy between

G. cheiranthifolium and G. gaudichaudianum,based on populations of the Argentineanprovince of La Pampa. Along this work, has notbeen found intergradation of characters be-tween the two taxa in center and North of Ar-gentina, therefore does not adhere to this viewuntil conclusive genetic and/or molecular evi-dences.

In Tropicos database 25 three subordinatetaxa are considered as valid, as follows: fma. cit-rinum (Hook. & Arn.) Kuntze, Revis. Gen. pl. 3(3): 151, 1898 (basionym: Gnaphalium citrinumHook. & Arn.); var. multiflorum J. Koster,Blumea 5 (3): 655, 1945; and var. paniculatumSkottsb., Köngl. Svenska Vetenskapsakad. Han-dl. 51 (9): 5, 1914 (basionym: Gnaphalium pan-iculatum Bert. ex Colla?). Until conclusive stud-ies are conducted on this group, the validity ofinfraspecific taxa is subject to reasonable doubt,mainly because their wide distribution, whichmay show quite pronounced local variations.

Habit and bioforms (Fig. 2, A-B)Hemicryptophytes to chamaephytes (short-

lived perennial herbs), from 20-80 (-100) cm tall,completely gray- or albo-tomentose.

ExomorphologyAerial parts with dense pubescence with dif-

ferent types of trichomes: I) single-celled (papil-lae) with distended cuticle; II) glandular gener-ally biseriate, with 2-7-celled stalk and cell wallthick and smooth, with ovate-globose apical cellof the same thickness as the basal; III) non-glan-dular long, with 2 (-3) basal wider cells withone terminal elongated, curled, flagelliform(whip-shaped) cell (Fig 2, K) that frequently islost to maturity, which gives a subglabrous ap-pearance to leaves and stems. Stems erect, sim-ple or branched, leafy towards the apex, dense-ly albo-tomentose with non-glandular hairsmixed with glandular ones, especially towardsthe apex.

Leaves alternate, simple, (8-) 60-80 (-120) x(2-) 3-8 (-15) mm, initially in a basal rosette,oblanceolate-spatulate, obtuse, and basal leavesoblanceolate, lanceolate to linear-lanceolate oreven linear, long attenuate and acute in apex,sessile, somewhat decurrent at the base (thusstems narrowly winged), margin entire or some-times slightly curly, concolorous, densely albo-tomentose to woolly on both sides, with longglandular hairs interspersed in the epiphyll.

Capitula numerous, hemispherical to cam-

panulate, 4.5-6 mm high x 5-6 mm dia., clus-tered in dense terminal glomerules, whichsometimes are solitary or more frequentlygrouped in dense corymbiform cymes (Fig. 2, B-C). Involucre multibracteate, scarious. Phyllariesin 4-5 series, citrine; outer ovate, acute, the in-ner lanceolate to obovate or oblong, obtuse atthe apex, with broad scarious margin. Marginalflorets carpellate (functionally female), very nu-merous, corolla filiform; disk florets 10-15, per-fect (functionally hermaphrodites), corolla tubu-lar. 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 athick and smooth cuticle, some papillae andstomata at the same level as other epidermalcells; the dense indumentum is composed ofglandular and non-glandular trichomes of thetypes described above. Cortex of 2-3 collenchy-matous layers, interrupted by groups ofchlorenchymatic cells, contacting with 1-layeredconspicuous endodermis, composed of tabularcells with thick walls, in which it has not beenobserved Casparian strips. The vascular bundlesare collateral, arranged in an eustele, with about20 side bundles of various sizes separated bymedullary rays, each protected externally by asclerenchymatic cap contacting the endodermis.The pith is solid, with large isodiametric,starchy, parenchymatic cells of polyhedral crosssection; the larger ones are placing toward thecenter. Stem anatomy is described for thefirst time for this species.

Leaf anatomy (Fig. 2, D-I)Cross-section shows the epidermis is uniseri-

ate on both sides; adaxial epidermal cells arepolygonal, with their anticlinal walls straight andcovered with relatively thick cuticle, while thoseof abaxial surface are irregular, with wavy anti-clinal walls, and covered with a thin cuticle. Theleaves are hypostomatic, because the upper epi-dermis has no stomata, whereas the lower onehas a large number of stomata, anomocytic be-cause the surrounding (4-5) cells not differ fromother epidermal cells; these characters areshared by other species of the genus; unlikeAchyrocline species, in G. cheiranthifoliumstomata are located at the same level as otherepidermal cells, or just sunk. Both epidermalsides have glandular and non-glandular tri-chomes of types described above, although

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more abundantly in the lower face. Mesophyllstructure is isobilateral, with 2 layers of palisadechlorenchyma to the epiphyll, and only one ofshort, tabular cells to the hypophyll, while thecenter is occupied by 2-3 layers of spongyparenchyma. Chlorenchymatic cells bear abun-dant lipidic globules. Vascular bundles are col-lateral; the side veins are totally immersed in themesophyll, surrounded by a conspicuousparenchymatic sheath; the midrib is prominenttoward the hypophyll, and is protected by capsof angular collenchyma in contact with bothepidermal sides. Leaf anatomy of this entity isreported for the first time in this study.

Chorology and habitatIt is widespread in South America, from

Southern Brazil and Bolivia, Uruguay to centralArgentina and Chile, between sea level and2,500 m asl, in loose soils, sandy to rocky, inthe biogeographical provinces Yungas, Chaque-ña, Monte, Pampeana, Patagónica and their eco-tones. It has been documented in the Argen-tinean provinces of Buenos Aires, Córdoba, Cor-rientes, Entre Ríos, Jujuy, La Pampa, La Rioja,Mendoza, Neuquén, Río Negro, Salta, San Luis,Santa Cruz, Santa Fe and Tucumán.

PhenologySimilar to that of the preceding species, ex-

cept plants behave as biennials in places ofgreatest height and coldest winters.

Ethnomedical usesLeaves, stems and “flowers” (in fact, capit-

ulescences) are used in folk medicine, especial-ly as a diaphoretic / febrifuge, antitussive andespecially as a depurative 18,26; the infusion asan emmenagogue and abortifacient 18; its use asa digestive and hepatic 18 probably due to theirmorphological similarity with G. gaudichaudi-anum and some species of Achyrocline.

Leaf ArchitectureVenation of both species is pinnate, campto-

drome, with a prominent, straight midrib, and 2-4 basal lateral primary veins emerging from thebase of the blade as ramifications of the midrib,running nearly parallel to it, diverging progres-sively and more forward curve inward and final-ly anastomose with secondary veins, these arisefrom the midrib and in the same way as the lat-eral primary run nearly parallel, diverging thenwith an angle of 50 °. The tertiary veins, which

are reticulate, and the higher-order ones de-crease progressively in importance to the mar-gin, with arches from second to fourth order.The last marginal venation is generally button-hole. It has some intersecondary veins, too. Di-vergence angles of the ramifications of 3rd. orderand above are acute, but little below 80 °. Theareoles are highly developed, from quadrangularto polygonal, and the venules (veinlet termina-tion in each areola) vary from simple to 1-2branched. The differences between the twospecies are reduced to the basal lateral primaryveins arising from the petiole vascular bundle inA. tomentosa, keeping parallel to the midrib by ashort length, diverging at an angle up to 20 º,while in G. cheiranthifolium at least two lateralveins are already present in the decurrence ofthe leaf on the stem, accompanying to the midribby longer distance, and finally separating it witha more acute angle of divergence that in A. to-mentosa. To the present knowledge, this is thefirst description of leaf architecture of these enti-ties, according the nomenclature of Hickey 27.

Macroscopic features of the commercialdrugs (Figs. 1, J and 2, L)

For both species, the drug of the commerceconsists of the flowering tops, and often onlythe inflorescences (capitula) grouped in capit-ulescences, always dried; depending on the de-gree of milling the drug can be made up mostlyof single flowers isolated, accompanied by frag-ments of peduncles, receptacles and phyllaries.Its general color is pale yellow (when prevailcapitula) to grayish or whitish (when prevailstem and leaf), the smell is highly aromatic andthe taste is bitter. The appearance is that of dis-ordered clusters of foliar and flowering ele-ments, matted by the abundance of the longflagelliform trichomes that form the most con-spicuous pubescence of both species.

Participation of these species in the marketBy means of the sampling of “Marcelas” in

herbal shops, pharmacies and local markets, aswell as in the stock maintained by street ven-dors and connoisseurs, in more important citiesand towns in Argentina and some boundingcountries, the authors have recorded the per-centage share of both species in the herbal mar-ket in the region. Has been determined in thiswork that Acyrocline tomentosa, Gnaphaliumcheiranthifolium and mixtures thereof includeapproximately 10 % of the herbs marketed un-

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der the collective nouns “Marcela”, “Marcela ma-cho”, “Marcelita”, and “Vira-vira”, and phoneticspelling variations recorded in the whole coun-try, that botanically correspond mostly tospecies of the genera Gnaphalium and Achyro-cline. From more than 300 samples obtainedfrom various Argentine markets and neighboringcountries under the above vulgar mentionednames, both in simple formulations as mixturesof herbs, 33 of them correspond to the speciesstudied, or their associations, and the relativepercentage share is shown in Table 1.

Quantitative micrographic parameters Values, expressed in Table 2, are useful

in raw drug identification and processed herbalmedicines obtained from them, especially whenthe material is milled or reduced to powder.

As observed in other species of Achyroclineand Gnaphalium, the leaves of both speciesstudied are hypostomatic, therefore abaxial epi-dermal surface showed between 12 to 13.5

Proven botanical identity% * Claimed Commercial

identity procedure

Simple 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 9.4 “Wira-wira” H,P,M/Fsatureioides

Gnaphalium cheiranthifolium + G. 31.3 “Marcela macho” H,V/Cgaudichaudianum

“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.

ParameterAchyrocline Gnaphaliumtomentosa cheiranthifolium

Upper epidermal surface Stomatal number . mm–2 0 0

Lower epidermal surface Stomatal number . mm–2 12.9 ± 0.7 16.6 ± 0.5Stomatal index (13.2) 13.8 (14.4) (14.7) 15.1 (15.6)

Veinlet termination number . mm–2 4.5 ± 0.5 20.3 ± 0.9Vein-islet number . mm–2 7.5 ± 0.8 14.6 ± 0.5Palisade ratio (4.7) 4.9 (5.2) (6.7) 6.8 (7.2)

Table 2. Quantitative micrographic parameters of samples of Achyrocline tomentosa and Gnaphalium cheiran-thifolium.

stomata per mm2 in A. tomentosa, with a rateclose to 14 stomata, while in G. cheiranthifoli-um these values reach 16 to 17 stomata permm2, with a stomata index near 15. The veinlettermination number ranged between 4 and 5 forA. tomentosa, varying between 19.5 and 21.2 insamples of G. cheiranthifolium, while the vein-islet number per mm2 was between 6.7 and 8.3in A. tomentosa, and between 14 and 15 in G.cheiranthifolium. Finally, the palisade ratioshowed an average of almost 5 parenchymaticcells by each epidermal cell in A. tomentosa,and ca. 7 parenchymatic cells by each epider-mal cell in G. cheiranthifolium. In brief, thestomata numbers and rates did not differ signifi-cantly in samples of the two species, whereasthe veinlet termination number, vein-islet num-ber and to a lesser extent, the palisade ratiowere useful to distinguish (with highly signifi-cant differences) among the samples of the twospecies. These parameters are reported for thefirst time for both species.

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Achyrocline tomentosa Gnaphalium cheiranthifolium

Bioform, functional Chamaephytes (subshrubs), branched, Hemicryptophytes (biennial herbs) totype and general green-grayish to green-yellowish chamaephytes (short-lived perennials),appearance shortly branched at the base,

albo-tomentose to yellowish

Trichomes Short vesiculate glandular trichomes; Long glandular uniseriate andnon-glandular flagelliform (whip-like); biseriate trichomes; non-glandularshort non-glandular flagelliform (whip-like)

Nomophylls Shortly decurrent, blade broadly lanceolate, Long decurrent, blade linear-lanceolate,oblanceolate to narrowly elliptical, 60-80 x 3-8 (15) mm, subconcolor,50-120 x 2-15 mm, discolor (upper surface greyish-tomentose on both sidesyellowish-green and lower surfacealbo-tomentose)

Capitulescences Several-headed dense glomerules, Few-headed dense glomerules, apicalarranged in corymbs or cymes terminaland/or axillary

Capitula Very numerous, narrowly cylindric Few, large (4-6 mm long x 5-6 mm dia.),to subfusiforms, pauci-(4-6) flowered campanulate to hemispheric, pluriflora

Phyllaries 8-9 phyllaries in 3 series, whitish-yellow or Numerous, in 4-5 series, scarious,brownish, the outer woolly and sparsely citrine bordered and trichomesglandular, the inner only glandular 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, Glabrous, smooth to finely tuberculate,striated, pappus 1-seriate with bristles pappus 1-seriate with hairs simple,simple, capillary, strigose slender, barbelate at base

Upper epidermal side Polygonal cells with anticlinal walls Cells with anticlinal walls somewhatstraight; cuticle thick and striated sinuous

Lower epidermal side Anticlinal cell walls windings Cells larger, anticlinal walls very curvy

Mesophyll structure Bifacial Isobilateral

Divergence of basal Angle ca. 20° Angle < 20°lateral primary veins

Stomata Anomocytic, raised Anomocytic, from slightly elevatedto something depressed

Stomatal number.mm–2 12.9 – 13.6 16.1 – 17.1(hypophyll)

Palisade ratio 4.7 – 5.2 6.7 – 7.2

Vein-islet 6.7 – 8.3 14.1 – 15.1number.mm–2

Veinlet termination 4 – 5 19.4 – 21.2number.mm–2

Table 3. Diacritic morphoanatomic and micrographic characters for Achyrocline tomentosa and Gnaphaliumcheiranthifolium.

682


Analysis of dissociated tissues (Figs. 1, K-N,and 2, M-P)

Authentic material of the two species hasshown cellular elements common to both, asscalariform vessels, fibrotracheids, fibers, sclerei-ds, and the remains of whip-shaped trichomesand parenchyma, while the diacritical elementswere adaxial epidermal cells with straight anti-clinal walls, and wavy in the abaxial epidermalcells, conspicuously raised stomata, short vesic-ulate glandular trichomes and short non-glandu-lar biseriate trichomes for Achyrocline tomen-tosa, while that G. cheiranthifolium abaxial epi-dermal cells show anticlinal walls somewhat sin-uous, and very curvy in the adaxial epidermalcells; stomata from slightly elevated to sunkenin the epidermis; and long biseriate glandulartrichomes.

CONCLUSIONSAttributes of the two species studied that al-

low their characterization and differentiation atmorphological, anatomical and quantitative mi-crographic levels, are summarized in Table 3.

Acknowledgements. Authors thank to anonymousreviewers whose comments have benefited themanuscript, as well as to the economic support of theProjects 2-4-8702 SECyT-UNSL and 22-Q-616 SPU-ME.

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