Latin American Journal of Pharmacy Regular Article (formerly Acta Farmacéutica Bonaerense) Received: May 6, 2011 Revised version: May 9, 2011 Lat. Am. J. Pharm. 30 (7): 1366-71 (2011) Accepted: May 17, 2011

Accumulation and Distribution of Diterpenic Acids in Leaves of tomentosa

Ramón E. ROBLES-ZEPEDA 1, Edmundo LOZOYA-GLORIA 2, Mercedes G. LÓPEZ 3, & Jorge MOLINA-TORRES *3

1 Departamento de Ciencias Químico Biológicas, Universidad de Sonora, Hermosillo, Sonora, México. 2 Departamento de Ingeniería Genética, 3 Departamento de Biotecnología y Bioquímica, Centro de Investigación y de Estudios Avanzados del IPN, Unidad Irapuato, Km. 9.6 Libramiento Norte, Irapuato, Guanajuato, México.

SUMMARY. has been used for at least last five centuries in traditional medicine in Mexico as a remedy for reproductive impairments. The accumulation of diterpenic acids in M. tomentosa leaves was determined. Using Scanning Electron Microscopy, the type and distribution of glandular tri- chomes (GTs) was observed on the abaxial and adaxial sides of leaves. GTs and non-glandular trichomes (NGTs) were observed on leaf surface, but the latter are confined to the leaves abaxial side. On the adaxial surface, only non-glandular trichomes were observed. Accumulation in GTs and leaf lamina of kaurenoic (KA) and grandiflorenic (GFA) acids was determined by Gas Chromatography coupled to an Electron Im- pact Mass Spectrometric Detector (GC/EI-MSD). GC-MSD analysis indicated that GTs accumulated KA and GFA, with KA accumulation being at a higher level than GFA in these structures. Attention on GFA and KA is due to their importance as growth regulator precursors with potential pharmacological applications.

INTRODUCTION The Montanoa belongs to the tribe of the family. This genus contains 25 species distributed in Mexico, Cen- tral America and northern part of South Ameri- ca. Because the many autapormorphic features this genus has been difficult to locate within the Figure 1. Chemical structures of kaurenoic acid (1) 1 subtribes of Heliantheae . Montanoa tomentosa and grandiflorenic acid (2). Cerv. is usually a small shrub, but is also known as a vine and in Costa Rica one subspecies is sometimes found as a small tree 2. Known as The aerial surface of most contains tri- zoapatle or cihuapatli, is widely distributed in chomes, which can be divided into glandular Mesoamerica. It has been used for at least five (GT) and non-glandular trichomes (NGT) 9. GTs centuries in traditional medicine in Mexico to are secretory structures varying in size, form, lo- treat diverse female health disorders 3. It is par- cation, and function in different plants species. ticularly useful in inducing childbirth 4. Some Glandular trichomes have a spherical morpholo- authors 5,6 attribute the pharmacological proper- gy after reaching the mature stage. Their charac- ties of Montanoa tomentosa to two diterpenic teristic morphology develops as a result of the acids: grandiflorenic (GFA) [kaura-9 (11), 16-di- accumulation of secretory products 10. They en-18-oic acid] and kaurenoic (KA) [ent-kaur-16- store chemical compounds with diverse func- en-18-oic acid] (Fig. 1). These diterpenic acids tions, including defence 11. Accordingly, tri- have been fully chemically characterized previ- chomes density, disposition, and morphology ously 7,8. are known to influence the level of resistance to

KEY WORDS: Asteraceae, Glandular trichomes, Grandiflorenic acid, Kaurenoic acid, Montanoa tomentosa, Zoa- patle. * Author to whom correspondence should be addressed. E-mail: [email protected]

1366 ISSN 0326-2383 Latin American Journal of Pharmacy - 30 (7) - 2011 insects 12. Secondary metabolites in trichomes served in a Leo S420 scanning electron micro- are ecologically bioactive compounds of interest scope (Leo Electron Microscope, Cambridge, as pesticides, pharmaceuticals, flavours and fra- UK) at 10 kV. For the adaxial surface samples grances 13. for scanning study were sputter-coated with Montanoa tomentosa leaves have a pleasant copper and analyzed in a Jeol (JSM 6400) at 10 odour that is enhanced after mechanical dam- kV. age. Stereomicroscopic observation of the sur- face of leaves of M. tomentosa confirmed the Glandular trichomes diterpenic acids presence of abundant glandular trichomes con- content taining terpene volatiles 14. In the present work, A general procedure was used to evaluate we studied the diterpenic acids profile of the the bulk content of GTs and within the leaf lam- glandular trichomes and lamina of basal, middle ina. First, a surface extraction was carried out and apical zones at different development with dichloromethane in order to collect the stages of the leaves of M. tomentosa. Our goal GTs content as previously described 15. Briefly, was to find out the leaves distribution of kau- each leaf was submerged in 20 mL dichloro- renoic acids, putative bioactive components, re- methane for 20 s. Then, the solvent was evapo- sponsible of the ethno medicinal use of this rated under a nitrogen stream and the GT con- species. tent re-suspended in 1 mL of the same solvent and kept at 4°C for further analysis. After the MATERIAL AND METHODS surface extraction with dichloromethane, the re- Plant material maining leaf section was ground in liquid nitro- To determinate the accumulation of diter- gen. Five mL of hexane:ethyl acetate (85:15) penic acids in Montanoa tomentosa Cerv. leaves were added and the mixture, sonicated for 15 were collected from field-grown mature one- min (Branson 1200 sonicator. Branson Ultrason- year-old plants cultivated at CINVESTAV-IPN, ics Corp., Danbury, Conn. USA) and then de- Unidad Irapuato (N 20° 43’ 8.9”, W 101° 19’ canted. The plant debris was re-extracted with 42.6” altitude 1740 m), Guanajuato, México. the same solvent and the combined extract was Samples were collected from September to De- concentrated under a nitrogen stream. The cember after the rainy season. Dr. Jerzy Rze- residue was re-suspended in 1 mL of same sol- dowski from the Instituto de Ecología, Centro vent for gas chromatography analysis of diter- Regional del Bajío, Pátzcuaro, Michoacán, Méxi- penic acids. The latter fraction was named lami- co, performed the species authentication. A na extract. Both, GT and lamina extract content voucher specimen (IEB-57689) was deposited at analysis are the result of the average of three in- that Institute. Leaves were collected in four size dependent determinations each. Data were anal- ranges: 7 to 7.5 cm, 9 to 9.5 cm, 14 to 14.5 cm, ysed using analysis of variance (ANOVA) with and 15 to 15.5 cm. These were classified as Tukey media comparison tests. new, young, mature and old leaves, respective- ly. Within these size ranges, the smaller leaves Grandiflorenic and kaurenoic acids were found in a higher position in the plant, evaluation whereas the older leaves were found at lower The identification and quantification of these level. In order to evaluate the GTs distribution, acids was accomplished as follows: 100 mL of each leaf was divided in three zones of approxi- each surface or laminar extract was dissolved in mately the same length termed as basal, middle hexane:ethyl acetate (85:15), and the diterpenic and apical part. The evaluation of GTs density acids present derivatized adding 100 mL of a in the different leaf zones was performed with mixture of BSTFA [bis-(trimethyl) sililtrifluoroac- the aid of a stereomicroscope (Nikon SMZ-2B, etamide from Sigma Aldrich Química, Toluca, Schott KL500 lamp). The average number of Mex.] and TMCS (trimethylchlorosilane from All- GTs was calculated. Three replicas were evalu- tech, D.F. Mex.) in a 100:5 v/v ratio and heated ated for each leaf size. for 30 min at 60 °C. The derivatized compounds were analyzed using a gas chromatograph Scanning electron microscopy (SEM) (Hewlett-Packard 6890 Series II Gas Chromato- Each leaf section from M. tomentosa plants graph System) equipped with a capillary column was lyophilized (Freezone 6, Labconco, Kansas HP-1 (30 m x 0.25 mm x 0.25 mm) coupled to City, Missouri, USA) for 48 h. Samples for the an electron impact mass selective detector GT SEM were sputter-coated with gold and ob- (Hewlett-Packard 5973 MSD), and employing a

1367 ROBLES-ZEPEDA R.E., LOZOYA-GLORIA E., LÓPEZ M.G. & MOLINA-TORRES J.

HP 7683 Series injector. Helium was used as the in length from 250 to 400 µm, and containing carrier gas, at a constant flow rate of 1 ML/min. from four to seven cells. The NGTs, being The injector temperature was maintained at 200 longer than the GTs, extend over the latter (Fig. °C. The oven temperature profile was the fol- 2A). lowing: 3 min at 150 °C, followed by a tempera- ture increment rate of 4 °C/min up to 300 °C, Glandular trichomes terpenes content then kept at 300 °C for 20 min. The mass selec- The accumulation of KA and GFA in the GTs tive detector consisted of a quadrupole analyzer of the different leaves studied was evaluated with the electron ionization source set at 70 eV. and presented in Figure 3A and B, respectively. Scan parameters were: mass range from 30 to A higher ratio of KA to GFA was observed in all 550 amu with 5.36 scans per second, and the leaf zones. For the apical zone of new leaves, temperature of source and quadrupole were 230 the KA content was 2.17 ng/GT (± 0.61) where- and 150 °C, respectively. The mass spectra iden- as only 1.1 ng GT–1 (± 0.18) of GFA was found tification and calibration curves for these com- in GTs of the same new leaves zone. This rela- pounds were made by comparison with authen- tion was maintained in GTs from the other two ticated standards obtained as a gift from Dr. zones as well as in all leaf development stages Raúl Enriquez (Instituto de Química, UNAM, tested. New leaves presented higher levels of México). these two acids. A larger amount of these acids was observed in the apical zone of all leaves. In RESULTS young leaves, both diterpenic acid levels in the Morphology and distribution of glandular apical zone were about half of those observed trichomes observed by SEM in new leaves. The KA and GFA content in the Using a stereomicroscope, the GTs on the apical part of mature leaves was 0.84 (± 0.03) abaxial surface were observed distributed along and 0.42 ng/GT (± 0.03), respectively. A lower the leaf lamina, on secondary, tertiary, and qua- level of these acids was observed in mature ternary veins with the exception of the main leaves, whereas within old leaves, the middle vein where, above all, NGTs were observed. and basal zones had the lowest levels of the The GTs have a green-yellowish colour and a acids (Fig. 3). spherical shape. The adaxial and abaxial leaf surfaces of Montanoa tomentosa leaves were analyzed by SEM. The abaxial surface contains numerous GTs and NGTs as shown in the mi- crograph (Figure 2A). On the adaxial surface mainly NGTs were observed, but in lower quan- tities than for the abaxial surface (Figure 2B). As observed in SEM micrographs, glandular heads display a smooth surface. The characteris- tic spherical shape may be due to the material accumulated inside the glands. GTs heads had a diameter ranging from 35 to 60 µm. NGTs of both surfaces are multicellular and erect, varying

Figure 2. Morphology and distribution of trichomes Figure 3. Kaurenoic and grandiflorenic acids content on the surface of Montanoa tomentosa leaves. A. of glandular trichomes in the apical, middle and basal Non-glandular and glandular trichomes on the abaxial zones of Montanoa tomentosa leaves at different de- side of a mature leaf. B. Non-glandular trichomes on velopmental stages. A. Kaurenoic acid. B. Grandiflo- the adaxial surface. In A the bar equals 100 µm. In B renic acid. Results are the average of three indepen- the bar equals 10 µm. dent determinations.

1368 Latin American Journal of Pharmacy - 30 (7) - 2011

DISCUSSION Montanoa tomentosa (zoapatle) is a medici- nal plant widely utilized from ancient times in the Mesoamerica cultures. It has been used for at least last five centuries in traditional medicine in Mexico to treat diverse female health disor- ders 3 and there have been reported several studies related to its medicinal properties 16,17. However, there are not previous reports about the accumulation of the diterpenic acids in the Montanoa tomentosa leaves. The presence of glandular and non-glandular trichomes is common in the Asteraceae species, however, the distribution of GT and NGTs in M. tomentosa is different from that observed in oth- er species containing this type of structures. The majority of the Helianthus species, in the same Heliantheae tribe as Montanoa, possess GT on leaves, predominantly on the abaxial leaf sur- face, but several lack them on vegetative plant parts 18. Mentha x piperita has GT on both sides of the leaves, but in developed leaves the num- ber of GTs is major on the side abaxial that Figure 4. Kaurenoic and grandiflorenic acids content 19 in the foliar lamina deprived of glandular trichomes adaxial . To date, the presence of GTs on the in the basal, middle and apical zones of Montanoa abaxial leaf surface, as M. tomentosa, has not tomentosa leaves. A. Kaurenoic acid. B. Grandiflo- been reported. renic acid.. Results are the average of three indepen- Several studies have demonstrated that GTs dent determinations. can accumulate organic compounds such as ter- penes, which are either synthesized or stored in Lamina diterpenic acids content these organelles. Sesquiterpene lactones are In order to determine if the presence of characteristic metabolites of the Asteraceae and these diterpenic acids was specific to GTs, the have being used in systematic studies using me- content of these compounds in the foliar lamina chanical GTs needle microsampling 18. On GC (leaves left without trichomes after the surface there are not successful determination reports of extraction with dichloromethane) was evaluated. sesquiterpene lactones as they are evaluated by The absence of GTs was confirmed by observ- HPLC. Up to day, no reports on mono or diter- ing the surface of the dichloromethane-extracted penes in the GTs in Asteraceae family have leaves under the stereomicroscope as the sol- been published. vent removed the cuticular GTs and its contents. GTs are diverse in terms of their biosynthetic However, after this surface treatment, the pres- activities when compared among plant families. ence of KA and GFA was still detected in the In Mentha x piperita, isolated trichome cells lamina deprived of GTs (Fig. 4A and B, respec- were able to synthesize monoterpenes from ex- tively). The accumulation ratio of these acids in ogenously administered sugar 20. Enzymatic ac- the foliar lamina was the inverse to that ob- tivities of the monoterpene biosynthetic path- served in the surface GTs, with higher GFA to way have been detected in GTs employing cell- KA ratios in all leaf stages of development and free enzyme assays 21. Moreover, some terpenes zones analyzed. Diterpenic acid levels in foliar have been detected only in GTs. The sesquiter- lamina were from two to three times higher pene artemisinin has been isolated from the GTs than in GTs at any developmental stage and of Artemisia annua 13,22, and Nepeta racemosa were highest in the apical zone. In general, the accumulates nepetalactone, a sesquiterpene lac- content for GFA in the apical zone was approxi- tone, exclusively in GT 23. On the other hand, it mately 4 mg/g of dry weight, with the exception has been demonstrated that cannabinoids from of young leaves where the content was about 5 Cannabis sativa are biosynthesized and accu- mg/g of dry weight. In both cases, the presence mulated in the storage cavity of the glandular of these acids increased acropetally irrespective- trichomes 24. Kaurenoic acid, together with oth- ly of whether the leaves had trichomes or not. er kaurene derivatives have been reported in

1369 ROBLES-ZEPEDA R.E., LOZOYA-GLORIA E., LÓPEZ M.G. & MOLINA-TORRES J. the GTs of sonchifolia, in the same technical support in SEM. This work was supported tribe Heliantheae (Asteraceae), interestingly the by CONACYT scholarship 86454 to R.E.R.Z., and the morphology of GTs and NGTs on the adaxial project No 28029N financial support. surface is similar to our species in study 25. It is possible that in M. tomentosa, these REFERENCES diterpenic acids are biosynthesized by GT cells 1. Bremer, K. (1994) Asteraceae, Cladistics & and accumulated in the glandular space. The Classification. Timber Press Portland, Oregon, lipophilic nature of KA and GFA supports this USA. possibility since their transport from the intercel- 2. Funk, V.A. (1982) The systematics of Montanoa lular or vascular tissue to GT on the leaf surface (Asteraceae, Heliantheae), Memoirs of the would be unlikely. It is not known whether KA New York Botanical Garden. Vol. 36. Bronx, and GFA have a specific function in GTs. Differ- N.Y. ent authors have speculated that KA could have 3. Lozoya, X. (1999) Xiuhapatli, Herba officinalis. a protective function in these glands 25, although Ed. Secretaría de Salud-UNAM, México, D.F. no experimental evidence was produced to sup- pp. 33-37. port this proposal. On the other hand, in the fo- 4. Gallegos, A. (1983) Contraception 27: 211-5. liar lamina, the role of KA in the gibberellins 5. Bejar, E., R. Enriquez & X. Lozoya (1984) J. Ethnopharmacol. 11: 87-97. biosynthetic pathway is well known 26. Isolated 6. Enriquez, R.G., E.G. Mirya, B. Ortíz, I. León, KA has also been shown to have antibiotic ac- G. Magos, A. Peña, et al. (1996) Planta Med. tivity against Gram-positive bacteria 27, Try- 62: 569-71. 28 panosoma cruzi , as well as cyto- and embry- 7. Reynolds, W.F., R.G. Enriquez, L.I. Escobar & 29 otoxicity . Its analgesic properties are even X. Lozoya (1984) Can. J. Bot. 62: 2421-5. higher than some commercial drugs 30, having 8. Lu, T., D. Vargas, S.G. Franzblau & N.H. Fisch- additionally antispasmodic activity 31. In con- er (1995) Phytochemistry 38: 451-6. trast, GFA has been suggested to have an in- 9. Werker, E. (2000) Adv. Bot. Res. 31: 1-35. ducting effect on childbirth 4,5, discrete in vitro 10. Turner, G.W., J. Gershenzon & R.B. Croteau antibacterial activity 32 and herbicide effect 33, (2000) Plant Physiol. 124: 655-63. but no other activity has been reported for this 11. Gannon, A.J. & C.E. Bach (1996) Environ. En- acid. tomol. 25: 1077-82. Based on the previous reports, it is evident 12. Lambert, L., R.M. Beach, T.C. Kilen & J.W. the diversity of biological activities that display Todd (1992) Crop Sci. 32: 463-6. both diterpenic acids and the relevancy that 13. Duke, M.V., R.N. Paul, H.N. Elsohly, G. Sturtz they could have in a future. In this respect, and & S.O. Duke (1994) Int. J. Plant. Sci. 155: 365- still when the KA is ubiquitous to all the plants, 72. due to the fact that is a precursor of the gib- 14. Robles-Zepeda, R.E., E. Lozoya-Gloria, M.G. López, M.L. Villarreal, E. Ramírez-Chávez & J. berellins biosynthesis 26,34, the relative concen- Molina-Torres (2009) Fitoterapia 80: 12-7 trations for KA and GFA in the Montanoa 15. Bisio, A., A. Corallo, P. Gastaldo, G. Romussi, species observed in this work are high. This jus- Ciarallo G., N. Fontana, et al. (1999) Ann. Bot- tifies partially the fact that some species of London. 83: 441-52. genus Montanoa are used to induce menses, 16. Montoya-Cabrera, M.A., A. Simental-Toba, S. 35,36 abortion and/or labor . Sánchez-Rodríguez, P. Escalante-Galindo & A. In conclusion, the medicinal plant M. tomen- Aguilar-Contreras (1998) Gac. Med. Mex. 134: tosa was shown to have two types of trichomes, 611-5. GT and NGT; the GTs were homogenously dis- 17. Carro-Juárez, M., I. Lobatón, O. Benítez & A. tributed on abaxial side of the leaves and accu- Espíritu (2006) J. Ethnopharmacol. 106: 111-6. mulate partiality the diterpenic acids GFA and 18. Spring, O. (1989) Biochem. Syst. Ecol. 17: 509- KA, responsible of the medicinal properties. A 17. different pool of these acids is present within 19. Maffei, M., F. Chialva & T. Sacco (1989) New the leaf tissue, being this latter pool bigger but Phytol. 111: 707-16. with a different proportion of these compounds. 20. McCaskill, D., J. Gershenzon & R. Croteau (1992) Planta 187: 445-54. Acknowledgements. We thank Mr. Francisco Solorio 21. Gershenzon, J., M. Maffei & R. Croteau (1989) from Instituto de Investigaciones Metalúrgicas, Uni- Plant Physiol. 89: 1351-7. versidad Michoacana de San Nicolás de Hidalgo and 22. Lommen, W.J., E. Schenk, H.J. Bouwmeester & M. C. Dolores Elena Alvarez Gasca from Facultad de F.W. Verstappen (2006) Planta Med. 72: 336- Arquitectura, Universidad de Guanajuato for their 45.

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