Morphoanatomical Characterization and Antimicrobial Activity of Tillandsia Imperialis (Bromeliaceae)

Núm. 31, pp. 21-29, ISSN 1405-2768; México, 2011

MORPHOANATOMICAL CHARACTERIZATION AND ANTIMICROBIAL ACTIVITY OF TILLANDSIA IMPERIALIS (BROMELIACEAE)

J. Alejandro Vite-Posadas1, Alicia E. Brechú-Franco1, Guillermo Laguna-Hernández1, M. Gabriela Rojas-Bribiesca2 and H. Reyna Osuna-Fernández1. 1Laboratorio de Estructura y Fisiología de Plantas, Facultad de Ciencias, Universidad Nacional Autónoma de México, Ciudad de México; 2Laboratorio de Microbiología, Centro de Investigación Biomédica del Sur (CIBIS), IMSS, Xochitepec, Morelos, México.

ABSTRACT trichomes, tetracyclic stomata and non- sclerotic hypodermis with spherical silica Tillandsia imperialis C.J. Morren ex Roezl is bodies. Microbiological research revealed a bromeliad used in Mexican folk medicine specifi c antimicrobial activity against re- mixed with other plant and animal parts as a spiratory pathogenic microorganisms such good remedy for respiratory diseases, which as Staphylococcus aureus and Streptococ- are one of the main causes of morbility and cus pyogenes as well as six other bacteria. mortality in the rural population of Mexico. These results indicate the existence of anti- The commercialization of medicinal plants microbial compounds in the extracts and a for therapeutic use must comply with high correlation between the traditional uses of standards for quality, safety and effi cacy; this plant and the experimental data. structural, microbiological and pharmaceuti- cal studies make a decisive contribution to Key words: antimicrobial activity, me- this quality control. The aim of this study dicinal plants, structural and histochemical was to analyze the morpho-anatomical char- characteristics, Tillandsia imperialis. acteristics and histochemistry of bracts and leaves of Tillandsia imperialis and evaluate RESUMEN their antimicrobial activity to corroborate the effectiveness of this plant for traditional Tillandsia imperialis (Bromeliaceae) es una uses with experimental data. Crude extracts planta utilizada en la medicina tradicional from the infl orescences and leaves were mexicana para tratar ciertas afecciones evaluated on Staphylococcus aureus (ATCC respiratorias, las cuales son una de las prin- 29213), Streptococcus pyogenes (ATCC cipales causas de morbilidad y mortalidad 08668), Escherichia coli (ATCC 25922), en la población de nuestro país. No obstante, Streptococcus faecalis (ATCC 29212), Sal- no se han realizado investigaciones que monella Typhi (ATCC 06539), Klebsiella corroboren su uso en la medicina tradi- pneumoniae (ATCC 13883) and Candida cional. Se realizó un estudio estructural e albicans (ATCC 10231). Results showed histoquímico de las brácteas y hojas de esta that T. imperialis possesses important mor- especie para aportar información útil para pho-anatomical characteristics useful for su correcta determinación farmacognóstica identifi cation, including mesic and peltate y se evaluó la actividad antimicrobiana

21 Núm. 31: 21-29 Marzo 2011 de los extractos crudos obtenidos de las in developing countries (Salud Pública infl orescencias y hojas para corroborar su Méx., 2004; WHO, 1998). Phytotherapy as uso en la medicina tradicional. La actividad a part of Therapeutics requires the rational antimicrobiana de los extractos crudos se development of herbal medicinal products evaluó sobre Staphylococcus aureus (ATCC with guaranteed quality, safety and effi cacy, 29213), Streptococcus pyogenes (ATCC along with accurate and assessable informa- 08668), Escherichia coli (ATCC 25922), tion as well as appropriate education in this Streptococcus faecalis (ATCC 29212), fi eld (Cañigueral, 2006). The confi rmation Salmonella Typhi (ATCC 06539), Klebsi- of the authenticity of the botanical samples ella pneumoniae (ATCC 13883) y Candida requires pharmacognostic studies. Few albicans (ATCC 10231). Las características scientifi c investigations have analyzed the morfoanatómicas útiles que se detectaron en leaf, stem or root anatomy, which is useful T. imperialis son tricomas mésicos, pelta- to guarantee the authenticity of medicinal dos, estomas tetracíclicos e hipodermis no botanical materials and in turn contributes to esclerótica con cuerpos esféricos de sílice. their identifi cation and characterization for El estudio antimicrobiano reveló actividad the quality control of the products derived contra microorganismos patógenos de vías from these plants (Arambarri & Mandrile, respiratorias como Staphylococcus aureus 1999). It is necessary to make botanical y Streptococcus pyogenes lo cual corrobora tests for plant materials from markets to y valida su uso en la medicina tradicional avoid the falsifi cation or substitution in the de México. use of any medicinal botanical material. Data acquired in scientifi c experimentation Palabras clave: actividad antimicrobiana, should guarantee and provide approaches estructura e histoquímica, planta medicinal, for quality control, as recommended by the Tillandsia imperialis. World Health Organization and the Mexican Health system. INTRODUCTION In spite of the diversity of medicinal herbs, For many people around the world, tra- the scientifi c knowledge available is very ditional medicine is still used and it is an limited as in the case of bromeliads. This important medicinal resource (McGaw et group exhibits great diversity of habitats al., 2000). Medicinal plants used in the in- and represents the third most important bo- digenous cultures from developing countries tanical family of Mexican monocotyledons are numerous and diverse (Nigenda et al., (Pulido-Esparza et al., 2004). Bromeliads 2001). The traditional medicine in Mexico have been used since ancient times and plays an important role in medical practice are currently being used by Mexicans as where approximately 40 million of people food, forage, ornamental and medicinal employ traditional remedies (García, 2002) plants. Tillandsia imperialis Morren ex which include a rich variety of medicinal Roezl (Bromeliaceae) is a tank epiphytic plants. Some of them are used for the treat- bromeliad found in tropical mountane cloud ment of respiratory infections, one of the forest, distributed throughout southeastern fi rst causes of hospitalization in Mexico Mexico, Salvador and Honduras. In Mexico and responsible for fi fty percent of deaths is known locally as “tencho”, and has been

22 Vite-Posadas, J.A. et al.: Morphoanatomical characterization and antimicrobial activity of Tillandsia imperialis widely exploited in fl orist trade. Infusions Pigmentation type was determined using the made from the leaves of T. imperialis are Benzing and Friedman’s protocol (1981). used in Mexican traditional medicine to treat respiratory ailments along with other Anatomical Characterization plants including Gardenia jasminoides, Physalis pubescens, Juglans regia, Hibis- Leaf paradermal and longitudinal fresh cus rosa-sinensis, and Rosa gallica as well sections of T. imperialis were prepared by as Dasypus novemcinctus (an armadillo), hand with single-edge razor blades. Other (Chino & Jácquez, 1986). samples were fi xed in FAA (Formalin, acetic acid, ethanol 100-50-500 mL in 1L), washed Scientifi c studies supporting the medicinal and dehydrated in increasing concentrations use of this plant and verifi ng its pharmaco- of ethanol solution (30°-50°-70°-96°-100°) logical effectiveness are lacking. The aim and included in Paraplast MR. Sections were of our work was to analyze comparative processed with the Johansen´s method morpho-anatomical characteristics of this (1940), and histochemical studies were bromeliad and evaluate its biological activ- carried out on some samples and were ob- ity, so corroborate the traditional uses of this served in a light microscope (Axiostar plus plant with experimental data. Carl Zeiss).

MATERIAL AND METHODS Preparation of Extracts

Plant Material Infl orescences and leaves were dried at am- bient temperature and powdered. Ten grams Tillandsia imperialis C.J. Morren ex Roezl of the powder were extracted three times 8h plants were bought within local markets of each one sequentially with organic solvents: Mexico City between February and April hexane, dichloromethane and methanol (J.K. 2004. The voucher specimen was depos- Baker) using a Soxhlet apparatus. The crude ited inside the herbarium of Universidad extracts were then evaporated to dryness Autónoma Metropolitana Iztapalapa in under vacuum conditions and the percentage Mexico (UAM-IZ), with the accession yield for each extract was determined. number 55702, (botanical identifi cation) by Ph.D. Adolfo Espejo (UAM-IZ). Microorganisms Evaluation Test Microorganisms Color Distribution and Foliar Pigmentation Six standard microorganisms strains were The color of bracts and leaves were com- used for testing antimicrobial activity: pared in vivo with the help of a standard Staphylococcus aureus (ATCC 29213), color handbook (Kornerup & Wanscher, Staphylococcus aureus OPS (clinical isolate; 1963). Lamina and sheath leaf pigmentation identifi ed and obtained from the Laboratorio was examined on foliar adaxial face as much de Microbiología, Centro de Investigaciones as foliar abaxial face. For detailed analysis, Biomédicas del Sur, IMSS), Staphylococcus each plant part (bract and leaf) was divided aureus AHD (clinical isolate), Streptococ- in three regions: apical, middle and basal. cus pyogenes (ATCC 08668), Escherichia

23 Núm. 31: 21-29 Marzo 2011 coli (ATCC 25922), Streptococcus faecalis RESULTS (ATCC 29212), Salmonella Typhi (ATCC 06539), Klebsiella pneumoniae (ATCC Color and Foliar Pigmentation 13883) y Candida albicans (ATCC 10231). Detailed results of bract and leaf pigmen- All bacteria were maintained on Tryptic tation are shown in Fig 1. Soy agar (TSA Merck) at 37°C. Deﬁ bri- nated sheep blood (5%) was added to the The leaf color is not uniform; it varied medium for Streptococcus pyogenes. Yeast considerably depending upon the surface was maintained on Sabouraud 4% dextrose and position along the axis. Reddish color agar (SDA Merck). (11E8, 11F8) abundantly dominated in both the adaxial and the abaxial foliar apical faces Antimicrobial Assays (Fig. 1A, 1D), green pigments (30D7, 30D8) were majorly found in the leaf’s middle Antibacterial and antifungal activities were region (Fig. 1B. 1E). At the basal region tested by two-fold dilution method (Rojas (Fig. 1C, 1F) a pale yellow color was de- et al., 2001). Extracts were dissolved in tected near to stem (1A2, 2A2). Cyanic color 5% dimethylsulfoxide (DMSO; Merck) (10D8, 12E8, 14F5, 15F5) was localized and added to melted agar culture medium between the middle and basal foliar region. in Petri dishes at the following ﬁ nal con- Pigmentation pattern was linear and it was centrations: 0.5, 1.0, 2.0, 4.0, 8.0 mg/mL. darker in the adaxial face (Fig. 1F). Api- The antimicrobial assay was carried out cal bracts pigments included reddish color on Muller-Hilton agar (MHA Merck); in (9A8, 9C8) and pale pink (8A3), they were the case of Streptococcus pyogenes sheep more brilliant at abaxial face (Fig. 1G) than blood (5%) was added to Mueller-Hilton the adaxial face (Fig. 1I). Yellowish white medium. Microbial suspensions with 0.5 color (1A2) was found at the basal region McFarland standard turbidity equivalents (Fig. 1H, 1J) similar to the leaf basal region. were prepared by suspensions of the growth from Brain Heart Infusion (BHI BBL). Sus- Anatomical characterization and histo- pensions were further diluted 1:20 to obtain chemical test a concentration of 104 colony-forming units (CFU)/mL for the bacteria and yeast. The The leaf presented a single epidermis with diluted inoculum was applied with a loop peltate trichomes located on both abaxial calibrated to deliver 0.002 mL, resulting in and adaxial foliar surfaces with a symmetric a spot covering a circle of 5-8 mm diam- linear arrangement. The trichomes presented eter. The plates were incubated for 24 h at cell rings 4 + 8 + 32 mesic anatomy (Fig. 37°C. Gentamicin and Nystatin (5 and 10 2A). The epidermal cells of abaxial surface μg/mL respectively) and 5% DMSO were showed straight thick inner periclinal walls used as reference standards. Observations whereas outer ones were thinner (Fig. 2B). were performed in duplicate and results Only abaxial epidermal cells presented a expressed as the lowest concentration of single siliceous spherical body in each cell plant extract that produced a complete (Fig. 2C). Tetracyclic stomata were distrib- suppression of colony growth, the minimal uted only in the apical abaxial leaf epidermis inhibitory concentration (MIC). (Fig. 2D).

24 Vite-Posadas, J.A. et al.: Morphoanatomical characterization and antimicrobial activity of Tillandsia imperialis

Fig.1. Color distribution and pigmentation pattern of leaf (A, B, C, D, E, F) and bracts (G, H, I, J) of T. imperialis.

Fig 2. A) Leaf scale trichomes; B) Transversal section of the leaf of basal region, ab: abaxial face; ad: adaxial face leaf; C) Epidermal cells with silica bodies; D) Abaxial face with stomata; E) Adaxial hypodermis and a base of trichome; F) Tannins in abaxial hypodermis. Scale bar: 50 μm (A, D, E, F); 100 μm (B); 10 μm (C).

25 Núm. 31: 21-29 Marzo 2011

Under the epidermis was found a mono- romethanic extracts from inflorescence layer of hypodermal cells (Fig 2E) with (MIC 0.5mg/mL) which showed a signifi - colorless thin-walled and spherical cells in cant antibacterial activity by inhibiting the the abaxial face; the adaxial hypodermal growth of bacteria at a relatively low MIC, cells were rectangular or elongated. The so compounds of low polarity maybe are hypodermal cells of the basal region of the the responsible for the inhibitory response. leaf presented condensed tannin (red-brown The most active inhibitory value against S. colour), but only at the abaxial face (Fig. aureus AHD (clinical isolate) was obtained 2F). This microscopic features are useful with the hexanic extract from the infl ores- for the characterization and botanical de- cence (MIC 8 mg/mL). scription of T. imperialis and will help in the right identifi cation of the commercial Streptococcus faecalis was inhibited by hex- herbal drug samples. anic infl orescence extract (MIC 8mg/mL). Klebsiella pneumoniae and Salmonella typhi Antimicrobial Assays were inhibited by methanolic extracts from leaves and infl orescences (MIC 8mg/mL). The results of the antimicrobial activity of In general, inflorescence extracts were T. imperialis extracts are shown in Table more active than leaf extracts. These results 1. The highest yield was obtained with the indicate the existence of antimicrobial com- methanolic extract in both leaves and infl o- pounds in the extracts and show a correlation rescence. Both hexane and dichloromethane between the traditional uses of this plant and extracts from infl orescence presented a high the experimental data. inhibitory activity. According to previous antimicrobial studies carried out at the CI- DISCUSSION BIS laboratory (IMSS), crude plants extracts with MIC values between 2.5 and 8mg/mL According to the results, the color pattern (Navarro et al., 1996; Rojas et al., 2001; of the leaves of T. imperialis is considered Salinas et al., 2009) have led to the isolation an ephemeral cyanic pigmentation (Benz- of strong antimicrobial compounds. Consid- ing and Friedman, 1981). The cell rings ering that in this study only crude extracts (4 + 8 + 32) of the peltate trichomes with were employed, extracts with MIC values of a symmetric linear arrangement, match up 8mg/mL or below were considered active. with the cell arrange in other genus like Catopsis, Glomeropitcairnia, Guzmania All extracts assayed (save the dichlorometh- and Vriesea (Tomlinson, 1969). T. impe- ane extract from the leaves) exhibited anti- rialis presents important morphoanatomical microbial properties against at least one of characteristics, some of which are common the tested microorganisms at concentrations to the taxonomic family such as the mesic of 8 mg/mL or below. None of the extracts trichomes, tetracyclic stomata, non-scle- showed antimicrobial activity against Can- rotic hypodermis and spherical silica bodies dida albicans and Escherichia coli. Three which are mainly distributed in the abaxial extracts inhibited Staphylococcus aureus: epidermis which contrast with the silica the hexanic extract from the leaves (MIC bodies deposited in the hipodermis in T. 4mg/mL) and the hexanic and dichlo- andicola (Prychid et al., 2004). The number

26 Vite-Posadas, J.A. et al.: Morphoanatomical characterization and antimicrobial activity of Tillandsia imperialis

Table 1. Results of the antimicrobial activity of T. imperialis extracts.

Extracta Extract Antimicrobial activity MIC (mg/mL) yield Gram (+) Gram (-) Fungi (%)b S.a. S.a.1 S.a.2 S.p. S.f. K.p. S.t. E.c. C.a.c Leaf H 14.78 4 >8 >8 >8 >8 >8 >8 >8 >8 D 29.57 >8 >8 >8 >8 >8 >8 >8 >8 >8 M 34.54 >8 >8 >8 >8 >8 8 8 >8 >8 Inflorescence H 6.50 0.5 8 >8 8 8 >8 >8 >8 >8 D 6.85 0.5 >8 >8 >8 >8 >8 >8 >8 >8 M 35.08 >8 >8 >8 >8 >8 8 8 >8 >8 Gentamicind 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 NTe Nystatin NT NT NT NT NT NT NT NT 0.010

a H: Hexane extract; D: Dichloromethane extract; M: Methanol extract. b Dry residue of the extract in terms of dry starting material. c Tested strains: S. a., Staphylococcus aureus; S. a. 1., Staphylococcus aureus AHD (clinical isolate); S a. 2., Staphylococcus aureus OPS (clinical isolate); S. p., Streptococ- cus pyogenes; S. f., Streptococcus faecalis; K. p., Klebsiella pneumaniae; S.t., Salmo- nella Typhi, E. c., Escherichia coli; C. a., Candida albicans. d Positive controls e NT: not tested

and the arrangement of the trichomes cells bromeliads such as Nidularium and Tilland- are specifi c characteristics for T. imperia- sia, fl avonoids and triterpenoids (Delaporte lis which are useful for identifi cation and et al., 2004) have been registered as major differentiation from other species. These chemical components (Delaporte et al., microscopic characters are important for 2004). Cabrera and Seldes, 1997 and Can- establishing an appropriate foliar anatomical tillo-Ciau et al., 2001, identifi ed cycloartane identifi cation of T. imperialis. triterpenoids in extracts of T. usneoides and T. fasciculata. Adrián-Romero and The pharmacological research established Blunden (2001) detected high contents of the specifi c biological activity of the extracts glycinebetaine in other Tillandsia species, of T. imperialis against respiratory microor- which have been reported to play a role in ganisms such as Staphylococcus aureus and pathogen-resistance mechanisms/strategies Streptococcus pyogenes as well as suggested in plants. Possibly this kind of compounds the presence of antimicrobial compounds are responsible for the observed micro- in the extracts. These results correlate with bial inhibition in T. imperiallis, however, the traditional uses of this plant. Neverthe- experimental research to identify active less, phytochemical analyses of this species compounds in this bromeliad, responsible haven’t been reported. In other species of for the microbial inhibition observed, must

27 Núm. 31: 21-29 Marzo 2011 be carried out, especially in the extracts that Arambarri, A.M., E.L. Mandrile, 1999. “Til- exhibited good antibacterial activity such as landsia L. (Bromeliaceae): Anatomy the ones effective against Staphylococcus and ethnopharmacology”. Acta Hort., aureus AHD (clinical isolate). 503: 133-140.

CONCLUSION Benzing, D.H., W.E. Friedman, 1981. “Pat- terns of foliar pigmentation in Bro- The microscopic characters which help meliaceae and their adaptive signifi - out on an accurate foliar identifi cation cance”. Selbyana, 5(3-4): 224-240. and differentiation for other species of T. imperialis are mesic trichomes, tetracyclic Cabrera, M.G., A.M. Seldes, 1997. “Short stomata, a non-sclerotic hypodermis, and side-chain cycloartanes from Tilland- spherical silica bodies. The pharmacologi- sia usneoides. Phytochemistry, 45(5): cal research reached out to determine the 1019-1021. specifi c biological activity of the extracts of T. imperialis against respiratory micro- Cantillo-Ciau, Z., W. Brito-Loeza, L. organisms, demonstrated the existence of Quijano. 2001. “Triterpenoids from antimicrobial compounds in the extracts Tillandsia fasciculata”. J. Nat.Prod., and show a positive correlation between 64: 953-955. the traditional uses of this plant and the scientifi c experimental data. Cañigueral, S., 2006. “Las monografías de calidad seguridad y efi cacia en el uso racional de los preparados a base ACKNOWLEDGMENTS de plantas medicinales”. Fitoterapia 6(S1): 25-29. All the authors are grateful to M.F.P. Ana Isabel Bieler Antolin from Laboratorio de Chino, V., R, Jácquez, 1986. “Contribución Microcine, Fac. de Ciencias, UNAM. We al conocimiento de la fl ora medicinal also thank to Q.A. Veronica Muñoz Ocotero de Quimixtlán, Puebla”. Tesis de from Laboratorio de Fitoquímica, Facultad licenciatura. ENEP-Iztacala, UNAM, de Ciencias, UNAM, M.C. Aurora Zlotnik Ciudad de México, 344 pp. for her assistant in the anatomical technique. The authors thank Dr. Adolfo Espejo for the Delaporte, R., M. Sarragiotto, O. Takemura, taxonomic identifi cation of the plant and to G. Sánchez, B. Filho, C. Nakamur, Dr. Robert A. Bye Boetler for the English 2004. “Evaluation on the antioede- write revision. matogenic, free radical scavenging and antimicrobial activities of aerial LITERATURE CITED parts of Tillandsia streptocarpa Baker (Bromeliaceae)”. J. Ethnopharmacol., Adrian-Romero, Maricela., G. Blunden, 95: 229-233. 2001. “Betaine distribution in the Bromeliaceae”. Biochem. syst. ecol., García, E.I, 2002. Catálogo de plantas me- 29: 305-311. dicinales de un mercado de la ciudad

28 Vite-Posadas, J.A. et al.: Morphoanatomical characterization and antimicrobial activity of Tillandsia imperialis

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Recibido: 16 noviembre 2009. Aceptado: 16 diciembre 2010.