(Aloysia Citriodora Palau) from Argentina Boletín Latinoamericano Y Del Caribe De Plantas Medicinales Y Aromáticas, Vol

Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas ISSN: 0717-7917 [email protected] Universidad de Santiago de Chile Chile

Gattuso, Susana; van Baren, Catalina M.; Gil, Alejandra; Bandoni, Arnaldo; Ferraro, Graciela; Gattuso, Martha Morpho-histological and quantitative parameters in the characterization of lemon verbena (Aloysia citriodora palau) from Argentina Boletín Latinoamericano y del Caribe de Plantas Medicinales y Aromáticas, vol. 7, núm. 4, julio, 2008, pp. 190-198 Universidad de Santiago de Chile Santiago, Chile

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Artículo Original | Original Article

Morpho-histological and quantitative parameters in the characterization of lemon verbena (Aloysia citriodora palau) from Argentina.

[Parámetros morfo-histológicos cuantitativos en la caracterización de lemon verbena (Aloysia citriodora palau) de Argentina] Susana GATTUSO1*, Catalina M. van BAREN2, Alejandra GIL3, Arnaldo BANDONI2; Graciela FERRARO2 y Martha GATTUSO1 1. Cátedra de Botánica, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario Suipacha 531, S2002LRK, Rosario, Argentina. 2. Cátedra de Farmacognosia-IQUIMEFA. Facultad de Farmacia y Bioquímica.UBA-CONICET. Junín 956, 2º piso. C1113AAD Buenos Aires Argentina. 3. Facultad de Agronomía, Universidad de Buenos Aires, Av. San Martín 4453, (C 1417 DSE) Buenos Aires - Argentina. *Contacto: [email protected] .

Received | Recibido 20/02/2008; Corregido | Corrected 24/04/2008; Aceptado | Accepted: 14/05/2008.

Abstract

Lemon verbena, Aloysia citriodora Palau (Verbenaceae) is worldwide used due to the sensorial and medicinal properties of its leaves and essential oil. Micrographic identification of several samples from the most representative growing areas in our country was made in order to define the quality of a typical Argentine lemon verbena. Plant materials were fixed, freehand sectioned and stained with Safranin and Fast Green. Leaves are simples, their blades exhibit anomocytic stomata on the abaxial side, non-glandular and glandular trichomes and dorsiventral mesophyll. The stem, in incipient secondary growth, presents epidermis, angular collenchyma and the vascular bundles which are open collaterally. The morphoanatomical characterization, stomatic index, pallisade ratio and length of stem elements to be used as quantitative parameters of the leaves and stems were determined, in order to contribute to the identification of the plant material.

Key words: Aloysia citriodora, lemon verbena, morpho-anatomical, standardization. Resumen

Lemon verbena, Aloysia citriodora Palau (Verbenaceae) es usada mundialmente por las propiedades medicinales y sensoriales de sus hojas y aceite esencial. Se realizó la identificación micrográfica de numerosos especímenes provenientes de áreas de crecimiento más representativas de nuestro país, con el propósito de definir la calidad de un lemon verbena, típicamente argentino. El material vegetal fue fijado, seccionado a mano alzada y teñido con Safranina-Fast green. Posee hojas simples, sus láminas presentan estomas anomocíticos en su hipofilo, pelos glandulares, eglandulares y un mesófilo dorsiventral. El tallo, de escaso crecimiento secundario, posee epidermis uniestrata, colénquima angular y haces colaterales abiertos. La caracterización morfoanatómica, el índice estomático, la proporción de empalizada y las magnitudes de los elementos foliares y caulinares que se determinaron, contribuyen a la correcta identificación del material vegetal. Palabras clave: Aloysia citriodora Palau, estandarización, lemon verbena, morfo-anatómico.

Characterization of lemon verbena Gattuso et al.

climatic conditions of their habitat (Molina et al., INTRODUCTION 2003) our research group has undertaken this study in The genus Aloysia Ortega ex Jussieu belongs to order to define analytical parameters for the morpho- the Verbenaceae. This genus comprises about 30 histological characterization of lemon verbena species, distributed in the American continent from growing in Argentina. These specifications could be the south of the United States down to Chile and used for the pharmacopoeial standardization of this Argentina. Species belonging to this genus are plant. shrubs, rarely trees, with three leaves per node and small flowers usually assembled in racemose MATERIALS AND METHODS inflorescences (Botta, 1978). Aloysia citriodora Palau (Aloysia triphylla Plant material (L'Hér.) Britt., Verbena triphylla L'Hérit., Zapania Fresh and dried plant materials were used for the citriodora Lam., Lippia citriodora (Ort.) H.B.K., macroscopic, micro-morphologic and quantitative Lippia triphylla (L'Hérit) Kuntze, Aloysia sleumeri studies. Samples were obtained from the Herbarium Mold., Verbenaceae) (Botta, 1978) is native to the collection of the University of Rosario (UNR), geographical region that includes territories Argentina. belonging to Argentina, Uruguay, Paraguay and ARGENTINA: Santa Fe Province: San Jerónimo Chile. It is commonly known as “lemon verbena”, Dept, Loc. Pto Gaboto, 24/I/2006, Gattuso, S 781 “cedrón”, “cidron”, “hierba Luisa” or “verveine (UNR); 19/II/2006, Gattuso, S 785 (UNR). Rosario odorante” according to the country. The importance Dept, Loc. Rosario, 5/X/1996, Gattuso, S 676 (UNR). of lemon verbena can be inferred from the number of Buenos Aires Province, General Madariaga Dept, commercial crops present in different European, Loc. Pinamar, 13/I/1995, Gattuso, M 137 (UNR). African and South American countries. Its Further plant materials used in this study, were significance is due to the sensorial and medicinal collected by our group from stake accessions, properties of its leaves and essential oil. The dried originally collected from Buenos Aires, Mendoza, plant and its extracts are included in different food Salta and San Luis, and grown in the Experimental and medicinal preparations. Its processed leaves are Field of the Facultad de Agronomía (Universidad de used to manufacture tea and as an ingredient of Buenos Aires). alcoholic beverages or other non-alcoholic herbal drinks. It is also often included in phytomedicines as Methods a result of its accepted traditional uses based on Fresh material consisting of leaves and stems were reported pharmacological activities such as digestive fixed in F.A.A. (formaldehyde, ethanol, acetic acid, and diuretic (Arias and Costas, 1948, Duke, 1985, water, 2:10:1:3,5). Dried herbarium material was Torrent Martí, 1985), antispasmodic (Arias and hydrated by boiling it in water with a few drops of Costas, 1948, Torrent Martí, 1976, 1985), detergent. Transverse sections of the stems measuring carminative and sedative, (Soraru and Bandoni, 1978, 10 μm were prepared with a manual microtome and Ratera and Ratera, 1980; Wannmacher et al., 1990a, stained with Safranin and Fast Green (Strittmatter, 1990b, Alonso Paz et al., 1992, Mors et al., 2000), 1979). The leaves were imbedded in paraffin and antimicrobial and local analgesic (Hieronymus, 1882, serial transverse sections, 10 to 12 μm thick, were Dellacasa and Bandoni, 2003). performed and stained with Fast Green – Safranin It is codified by the Argentine Pharmacopoeia (Ruzin, 1999). Epidermis was obtained by scraping (1978), Francaise Pharmacopeia (1996) and fixed material and then stained with diluted Fast Argentine Alimentary Codex. (1969-2007). Green and Cresyl Violet (Strittmatter, 1980). Furthermore, it is included in the FDA's GRAS list, Sections were mounted in synthetic balsam. The i.e. the list of food additives which are Generally stems were macerated and leaves were cleared by Regarded As Safe (Newall et al., 1996). Recently a conventional methods (Boodle, 1916, Strittmatter, monograph under the name: lemon verbena leaf was 1973). The terminology proposed by Hickey (1979), included in the European Pharmacopoeia (2007). was used for the description of leaf architecture. The In view of the commercial value of lemon distribution of calcium oxalate crystals was observed verbena, and considering that plants can display with a polarizing microscope (Johansen, 1940, significant genetic biodiversity and variations in they O´Brian and McCully, 1981). Original drawings were morpho type due to differences in the edaphic or made with a drawing device. Symbols proposed by

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Metcalfe and Chalk (1972) were used in the diagrams. Surface view of epidermis The epidermal cells of the adaxial face show Statistics straight, anticlinal walls of 4 to 5 sides (Fig. 1, D). ANOVA was performed to determine differences Epidermic abaxial cells present anticlinal walls of stomata index and palisade ratio of Aloysia slightly undulating with anomocytic stomata and citriodora from different regional origin. Post hoc striate cuticule around of the stomata (Fig. 1, E; Fig. Scheffe’s test was applied for multiple comparisons 2 E). Two types of trichomes are present on both whenever differences were significant. Statistic sides of the leaf surfaces. Non glandular trichomes: a- significance admitted p <0.05. Mean ± Standard the upper epidermis shows numerous conical, Deviation (SD) were from 5 replications of each unicellular, thick-walled, verrucose, cystolithic species. Data were processed by commercial software trichomes, each arising from a rosette of cells at the STATGRAPHICS Plus-5.0 (2000). base, with cystoliths, (Fig.1, D; Fig. 2 F); b- the lower epidermis shows simple, short, unicellular, RESULTS thickened, cystolithic trichomes, (Fig. 1, E, F). Glandular trichomes: a- with foot simple or Macroscopical characteristics compound; stalk, 1 to 2 celled and head unicellular, Aloysia citriodora Palau is an erect, deciduous cell mostly broader than long, outer walls convex, perennial shrub which grows to a height of 1 to 3 thin, smooth, content scanty and translucent, in both meters and exudes a powerful lemony scent. The epidermis, more abundant in the lower one, (Fig. 1, leaves are simple with short petioles. They are E, Fig. 2 E). b- with foot simple, head small, narrow, lanceolate and 3 form at each node of the relatively narrow, unicellular, contens dense, stems. The lamina is pale green, 5 to 10 cm long, commonly persistent. Outer walls slightly convex, entire with a rough margin and slight teeth, having thin, smooth. Cuticular vesicle restricted to the apex, parallel veins at 90° to the mid-rib and flat bristles persistent or collapsing early, they occur widely and along the edges. The inflorescence is a slim terminal are aggregated in depressions on the lower surface panicle, the flowers consisting of a small, tubular (Fig. 1 E; Fig. 2 E). calyx with 4 long narrow teeth and a pale, greenish- mauve corolla divided into 4 acuminate lobes (Fig. 1 Cross-sections A) It is dorsiventraly compressed. Unstratified adaxial epidermis with cuticule thin and smooth, Plant material of interest without stomata can be observed. Mesophyll is Dried leaves. They are thin and fragile, deep- dorsiventral, with a 2-3 layer of pallisade green in color. Shortly petiolate, ovate-lanceolate, parenchyma (Fig. 1, I, J; Fig. 2 A). The spongy acuminated, measuring 5-10 cm long, with glandular parenchyma cells are located next to the abaxial hairs, shortly pilose on the lower surface. epidermis, loosely arranged, with many intercellular Microscopical characteristics spaces between them. The midrib consists of a collateral vascular bundle. The lower epidermis is unstratified and presents stomata in columns (Fig.1, Leaf anatomy K; Fig. 2 D).

Foliar architecture Stem anatomy The analysis of the foliar architecture shows Transverse sections show a circular margin with 6 pinnate, camptodromous, brochidodromous venations ribs (Fig. 1 L). The epidermis is unstratified, with (Fig.1, A, B) The primary vein is stout and shows a stomata which are composed of rectangular, rolled straight course. Secondary veins are thick and show a cells, trichomes are scarce. At the level of the ribs straight course. The marginal ultimate venation is and in a subepidermal position, 3-4 layers of angular looped. Limited, simple and curved venules. Well- collenchyma can be observed (Fig. 1 L). Cork usually developed areola randomly arranged, quadrangular, appears near the phloem (Fig. 1 M). Vascular bundles polygonal (Fig 1, C). are open collaterally and are arranged forming a eustele, reinforced by conspicuous sclerenchymatic fibers. . Bol. Latinoam. Caribe Plant. Med. Aromaticas Vol. 7 (4) 2008 192 Characterization of lemon verbena Gattuso et al.

Figure 1. Aloysia citriodora Palau. A-K leaf. A, vegetative branch. B-F, superficial view of the lamina. B, foliar architecture. C, last marginal venation and areolae. D, adaxial epidermis with conical, unicellular, thick-walled, verrucose, cystolithic trichomes, each arising from a resette of cells at the base, with cystoliths. E, abaxial epidermis. F, simple trichomes, slightly verrucose, with cystoliths of calcium carbonate. G-M, cross section. G, glandular trichomes with unicellular head, in depressions on the leaf surface. H, glandular trichomes with unicellular foot and head 1-2 cells. I, schematic representation of the limb. J, detail of the limb indicated in I. K, stomata on columns. L-M, stem in cross section. L, schematic representation of the stem. M, detail of the stem indicated in L. N-R, dissociated cellular elements. N, fibre. O, vessel. P, tracheids. Q, parenchyma cells. R, xilematic parenchyma. S, collenchimatic cells. Scale bars 1 to B. 2 to I, L. 3 to D-H, J- K, M-S. 4 to C.

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Figure 2. A-E: Aloysia citriodora Palau. Photomicrographs by light microscope. Transverse sections of leaf. A, main nerve, B, C, D mesophyll, in B, glandular trichomes in depressions on the leaf surface; C, glandular trichomes with unicellular foot and head 1-2 cells, D, stomata on columns. E-F, superficial view of the lamina. E, abaxial epidermis with glandular trichomes. F, adaxial epidermis whith conical, unicellular, thick-walled, verrucose, cystolithic trichomes, each arising from a resette of cells at the base, with cystoliths. a, glandular trichomes with unicellular head, in depressions on the leaf surface. b, glandular trichomes with unicellular foot and head 1-2 cells. c, cystoliths. e, epidermis. pp, pallisade parenchyma. s, stomata.

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Tabla 1: Stomatic index and pallisade ratio data of reference plant material, experimental samples and the different materials studied.

Origin of the material Stomatic index Pallisade ratio Buenos Aires 5,45 (7,05) 10,20 8,00 (9,31) 10,50 Santa Fe 5,16 (9,23) 13,43 5,50 (6,70) 8,25 San Luis 8,33 (10,38) 12,69 9,25 (10,60) 12,25 Mendoza 8,64 (10,35) 12,09 11,50 (11,81) 12,50 Salta 8,93 (10,73) 13,73 7,50 (8,20) 8,75 Jujuy 7,27 (9,67) 11,11 10,75 (14,30) 17,25 Interval for Argentine Material 5,16 - 13,73 5,50 - 17,24 Rancagua (Chile) 7,14 (8,97) 11,67 8,25 (9,65) 11,75 Talca (Chile) 5,77 (7,57) 9,26 7,00 (8,40) 10,25

Figure 3: Bar graph of stomatic index and pallisade ratio of Aloysia citriodora samples of different origin. Mean ± SD. p < 0,05 (∗).

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Figure 4: Pallisade ratio Aloysia citriodora samples of addition, according to this last author, the leaves have different origin. Test multiple comparisons. Scheffe (95%). a striking aromatic characteristic. The leaf venation, BA, Buenos Aires; JU, Jujuy; ME, Mendoza; RA, Rancagua (Chile); SA, Salta; SF, Santa Fe; SL, San Luis; TA, Talca is brochidodromous type and quantitative parameters (Chile). of vein islet index is 24, 50 - 29,10 - 33,75 (Albrecht et al. 2005). Metcalfe and Chalk (1972) mentioned for the genus Aloysia the occurrence of anomocytic stomata, we could ascertain this feature in this species. Trichomes are epidermal outgrowths of considerable value for taxonomic purposes. In A. citriodora there are unicellular, thick-walled cystolithic trichomes, trichomes arising from rosette of cell at the base, and they are by far the most prevalent and numerous subsessile glandular trichomes with a globular head according to Metcalfe and Chalk (1972) and Barbosa et al., (2001). According to Metcalfe and Chalk (1972) members of the family usually have dorsiventral mesophyll and include several (2-3) layers of pallisade parenchyma,

according with the two strata found in this study. The Dissociated stems stem organization of A. citriodora observed in this study corresponds to a typical herbaceous pattern The following cellular elements can be observed (Barbosa et al., 2001, Bonzani et al., 2003). in this preparation: 632 µm long fibers (Fig.1 N); These results together with the quantitative long vessels with appendage and simple terminal parameters accomplish the morpho-histological plates measuring 360 x 56 µm (Fig. 1 O); tracheids characters stated in European Pharmacopeia. measuring 552 µm (Fig.1 P); thin walled parenchyma cells (Fig.1 Q); xylem axial parenchyma (Fig.1 R); CONCLUSION collenchyma cells (Fig. 1 S). The following diacritical macroscopic and micro DISCUSSION morphological characteristics and quantitative parameters were established after the analysis of the The correct taxonomical nomenclature of the studied plant material. The presence of the below species was the first feature to harmonize in the described elements is considered to be useful for the search for data that could contribute to the botanical identification of the A. citriodora. normalization of lemon verbena. Palau (1784) used the binomial Aloysia citrodora in the original Morphological characteristics identification of the species. However, in most bibliographical references it is recurrently and almost Three leaves per node, simple, entire, lanceolate, universally named as Aloysia citriodora. We petiolate. Stem terete, with ribs. Flower white, small, consulted several taxonomists about this situation, arranged in a cymose inflorescence. among them, Dr. Charlie Jarvis and Dr. Norman Robson (Kew Royal Botanic Gardens), as authorities Anatomical characteristic in taxonomical nomenclature. They both agreed on Leaves: Venation pinnate, camptodromous, the use of Aloysia citriodora because it appears to be brochidodromous. Adaxial epidermis with anticlinal the result of the correction of the former name to thin walls and strains. Abaxial epidermis with conform the current nomenclatural botanical rules, anticlinal thin and sinuous walls. Anomocytic i.e., if a name is the result of the conjunction of two stomata, slightly prominent or in column. Non or more words, in this case, “citro” and “odora”, it is glandular trichomes: a- the upper epidermis there are necessary to add a link vowel “i” or “o” between the numerous conical, unicellular, thick-walled, Latin or Greek words, respectively. For this reason verrucose, cystolithic trichomes, each arising from a we assume that Aloysia citriodora Palau is the correct rosette of cells at the base, with cystoliths, b- the taxonomical nomenclature for this species. The leaf lower epidermis shows simple, short, unicellular, morphology and phyllotaxy of A. citriodora are thickened, cystolithic trichomes, Glandular similar to the description made by Botta (1979). In trichomes: a- with foot simple or compound; stalk, 1 Bol. Latinoam. Caribe Plant. Med. Aromaticas Vol. 7 (4) 2008 196 Characterization of lemon verbena Gattuso et al.

to 2 celled and head unicellular, cell mostly broader Córdoba, Serie Especial I, Ed. Graphyon, Córdoba, than long. b- with foot simple, head small, relatively Argentina, pp. 8-11. narrow, unicellular, content dense, commonly Bonzani N, Filippa E, Barbosa G. 2003. Estudio anatómico persistent, occur in depressions on the lower surface. comparativo de tallo en algunas especie de Mesophyll dorsiventral, hypostomatic. Verbenaceae. Anales del Instituto de Biología Universidad Autónoma de México, Serie Botánica 74(1): 31-45. Quantitative assessments Boodle LA. 1916. A method of macerating fibres. Roy. The following values were established for the Bot. Gard. Kew Bull. Misc.Inform. 5: 108-110. cellular elements of macerated stems: vessel bordered Botta S. 1979. Las especies argentinas del género Aloysia pits, with a 360 x 56 µm appendix, 552 µm tracheids (Verbenaceae). Darwiniana 22(1-3): 67-108. and 632 µm fibres. Código Alimentario Argentino. (1969-2007) : Ed. De la The data resulting from the analysis of the Canal y Asoc, Buenos Aires, Argentina.. stomatic index and pallisade ratio of reference plant Dellacasa E, Bandoni AL. 2003. Hierbaluisa. Aloysia citriodora Palau. Revista de Fitoterapia. 3(1): 19-25. material, experimental samples and the different Duke JA. 1985. Handbook of Medicinal Herbs. Ed. CRC chemotypes studied are summarized in Table 1 Press, Florida, Estados Unidos, p. 33. grouped according to their origin. European Pharmacopoeia 2007. Supplement 5.8, Monograph: Lemon verbena leaf Strasbourg: Council Statistical analysis. of Europe. Stomata index sample data yielded non significant Farmacopea Nacional Argentina, VI edición, 1978, Buenos differences among them (Fig.3). Statistical process of Aires, Argentina. palisade ratio in the same samples (Fig.3, Fig. 4), Francais Pharmacopeé X Ed. (1996), Maisonneuve, París, Francia. showed that the sample values from Jujuy (JU) was Hieronymus J. 1882. Plantas Diafóricas de la Flora higher, a fact that could be explained by the arid Argentina. Bo.l Acad. Nac. de Ciencias Córdoba, environment, demonstrating that regarding the Tomo IV, pp. 404-405. structure, leaves are the most important organ Hickey J. 1973. Classification of the architecture of reflecting the environmental influence. Samples from Dicotyledons leaves. Am. J. Bot. 60: 17-33. Mendoza (ME) and San Luis (SL) showed a similar Johansen DA. 1940. Plant microtechnique. Mc Graw-Hill, behaviour. The sample from Santa Fe (SF) presented New York, pp. 184-202. significant differences with the former ones, Metcalfe CR, Chalk L. 1972. Anatomy of Dicotyledons. expressing itself as a mesophyte, since the palisade Vol. I Clarendon Press, Oxford, Inglaterra, pp. i-iii. ratio value correspond to environments with middle Metcalfe CR, Chalk L. 1972. Anatomy of Dicotyledons. Vol. II. Clarendon Press, Oxford, Inglaterra, pp. 1030- temperature and humidity. The remaining samples 1041. are the reflection of the edaphically characteristics of Molina AC, Viturro CI and Heit C. 2003. I. Estudio the communities in which they develop, therefore, químico de aceites esenciales de Aloysia triphylla. determining a great variety of ecological niches Resúmenes del XIV Congreso de la Sociedad evidenced by intermediate values for each analysed Argentina de Investigación en Química Orgánica index. (SAIQO), Santa Fe, Argentina. 07 al 10 de noviembre de 2003 REFERENCES Mors WB, Rizzini CT, Alvarez P. 2000. Medicinal Plants of Brazil. Reference Publications INC, Algonac, Albrecht RA, Bassols GB, Gurni AA. 2005. Aplicación de Michigan, Estados Unidos, p. 30. parámetros cuantitativos en el estudio de Aloysia Newall CA, Anderson LA, Phillipson JD. 1996. Herbal citrodora Palau (Verbenaceae). Acta Farm. Medicines. A Guide for Health-care Professionals. Ed. Bonaerense 24(2): 239-241. The Pharmaceutical Press, London Alonso Paz E, Bassagoda MJ, Ferreira F. 1992. Uso O´Brian TP, McCully ME. 1981. The study of plant Racional de las Plantas Medicinales. Ed. Fin de Siglo, structure, principles and selected methods. Montevideo, Uruguay, pp. 42-43. Termarcarphi Pty Ltd, Melbourne, Australia, pp. 5, 1- Arias H, Costas F. 1948. Plantas Medicinales. Ed. 5, 14. Biblioteca Nueva, Buenos Aires, Argentina, pp. 34- Palau A. 1784. Parte Práctica de Botánica del Caballero 35. Carlos Linneo I. Imprenta Real, Madrid, España. Barbosa G, Bonzani N, Filippa E, Luján M, Morero R, Ratera EL, Ratera MO. 1980. Plantas de la Flora Bugatti M, Decolatti N, Arizar Espinar L. 2001. Atlas Argentina Empleadas en Medicina Popular. Ed. Histo-morfológico de Plantas de Interés Medicinal de Hemisferio Sur, Buenos Aires, Argentina, pp. 62-64. Uso Corriente en Argentina. Museo Botánico Bol. Latinoam. Caribe Plant. Med. Aromaticas Vol. 7 (4) 2008 197 Characterization of lemon verbena Gattuso et al.

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