Chemical markers and chromatographic patterns of three species of Baccharis (Asteraceae) used in traditional medicine of Argentina.

RESER, A. L; COMINI, L.; BARBOZA G. E.; NUÑEZ MONTOYA, S.

Rosario

Congreso; 2º Reunión Internacional de Ciencias Farmacéuticas –RICiFA 2012; 2012

Universidad Nacional de Córdoba y Universidad Nacional de Rosario

Introduction: Our country was one of the pioneers from Mercosur to define and regulate all activities involving plant drugs (PD) and their products (preparations and phytotherapic medicines, FM) (1-4). Thus, the current regulatory, in agreement with the WHO guidelines (5, 6), sets that the guarantee of quality, safety and efficacy of the PD and its products, is based on three fundamental aspects, besides the application of GMP: 1) Botanical identification with scientific name, recognizing their morphological and micrographic characteristics (Botanical quality control). 2) Identification and quantification of the active principles (AP) if they are known or alternatively identifying against chromatographic patterns (Chemical quality control). 3) Hygienic Control, which means that the product is suitable for human consumption. When the laboratories of products based on medicinal plants have to perform the botanical and chemical quality control of native PD, face the problem of the lack or inaccessibility to botanical descriptions (morphological and micrographic) and chemical studies that identify the PA or any chemical marker. This is compounded by the lack of chromatographic patterns, especially for native plants, whose PA are unknown. The objectives of this work are: a) Collect all botanical, chemical and biological information, scientific as "traditional", existing about three species of Baccharis: two species encoded in FA VI as "carqueja": Baccharis crispa Spreng. and Baccharis articulata (Lam.) Pers (7), and a third species: B. trimera (Less) DC. b) Perform chemical profiles of PD by several chromatographic techniques, identifying if it is possible some chemical compound that allows the characterization of each species and in turn its differentiation from the other two (chemical marker).   Materials and Methods: The literature search was conducted by consulting databases and secondary sources with scientific rigor, including also the "traditional" information. Botanical identification was carried out by G. Barboza, PhD (Fac. Cs. Exactas, Físicas y Naturales, UNC). After that, the appropriate drying and conservation practices were performed. From 100 g of each vegetal species (crushed aerial parts), extracts with different solvents (hexane, benzene, CHCl3, EtOH) were prepared by maceration. These extracts were used to obtain the chromatographic patterns by thin layer chromatography (TLC), paper chromatography (PC) and high performance liquid chromatography (HPLC), using different stationary and mobile phases according to the polarity of the extracts analyzed. HPLC was performed on a Varian Star Pro (Model 210, Series 04 171) equipped with a UV-V detector.   Results: We carried out a systematization of the information collected about the three plants, which allowed to establish a chemical compound that is specific for each one, and therefore, characterizes the species and differences it from the other two (chemical marker). Thus, the acacetina flavonoid [1] and the diterpene bacchotricuneatina A [2] are found only in B. articulata, the pilloína flavonoid [3] is distinctive of B. crispa, and the rutine flavonoid [4] is only present in B. trimera. The presence of 1 was identified in EtOH extract of B articulata against standard (Sigma) by TLC on silica gel G254 (Merck), using three different mobile phases (8), this result was confirmed by HPLC (9). For the identification of 2, the benzene and EtOH extracts of B. articulata were analyzed by TLC against standard (Dr. Wagner) (10); this result was also confirmed by HPLC (11). Compound 3 was purified from the hexane extract of B crispa, following the methodology described in the literature (7, 12); it was subsequently characterized by TLC on silica gel G254 using four mobile phases (12), and by means of its spectrum UV-Vis in EtOH (12). We established the presence of 4 in EtOH extract of B. trimera by CP against standard (Dr. Cabrera), which was eluted with 15% HAc in Whatman No. 1, and was revealed with UV/vapor NH3-UV (11). We determined the absence of 1, 2, 3 and 4 in the respective extracts from the other two species with the same chromatographic techniques.   Conclusions: For each species of Baccharis, a compound that characterizes each species and differentiates it from the other two was identified; corroborating by several chromatographic techniques that acacetina and bacchotricuneatina A are chemical markers of B. articulata, pilloína for B. crispa and rutine for B. trimera. In this way, we provide a useful tool for identification of these plant species, which is applicable in the quality control of their preparations and phytotherapeutic medicines.   References: 1) Especialidades medicinales. Resolución n ° 144/98. Ministerio de Salud y Acción Social. (Boletín oficial, de 09/03/98). 2) Normas para la habilitación de establecimientos elaboradores, envasadores/fraccionadores e importadores de Medicamentos Fitoterápicos. Disposición reglamentaria n° 2671/99, Administración Nacional de Medicamentos, Alimentos y Tecnología Médica- A.N.M.A.T. (Boletín Oficial, de 26/05/99). 3) Guía de Buenas Prácticas de Fabricación y Control para Productos Fitoterápicos. Disposición reglamentaria n° 2672/99, Administración Nacional de Medicamentos, Alimentos y Tecnología Médica- A.N.M.A.T. (Boletín Oficial, de 26/05/99). 4) Normas para la implementación del registro de Medicamentos Fitoterápicos. Disposición reglamentaria n° 2673/99. Ministerio de Salud, Presidencia de la Nación. Administración Nacional de Medicamentos, Alimentos y Tecnología Médica, A.N.M.A.T. (Boletín oficial, de 26/05/99). 5) Pautas para la evaluación de los medicamentos herbarios. WHO/TRM/91.4. World Health Organization (19-21/06/1991). 6).Quality Control Methods for Medicinal Plant Materials. WHO-Pharm/92.559. World Health Organization (1992). 7) Ministerio de Salud, Presidencia de la Nación. Administración Nacional de Medicamentos, Alimentos y Tecnología Médica (A.N.M.A.T.). Farmacopea Nacional Argentina, Codex Medicamentarium Argentino. 6° ed. Ciudad Autónoma de Buenos Aires: El Ministerio y A.N.M.A.T; 1978.  p. 213-15. 8) Cañigueral Folcará S. Contribución al estudio de los polifenoles de especies del género Salvia L. [Tesis doctoral]. Barcelona: Universidad de Barcelona, 1986. p. 206-207. 9) Apablaza Escalona C. Diseño de la estandarización química y evaluación de la actividad analgésica tópica de un extracto activo de Buddleja globosa Hope, Buddlejaceae, matico. [Tesis doctoral]. Santiago de Chile: Universidad del Salvador, 2006.p. 44-45. 10) Wagner H, Seitz R, Lotter H, Herz W. New furanoid Ent- Clerodanes from Baccharis Tricuneata. J. Org. Chem. 1978; Vol 43 (17): 3339-3345. 11) Harborne JB. Phytochemical Methods – A Guide to Modern Techniques of Plant Analysis. 2da ed. London: Chapman and Hall; 1984. 12) Bandoni AL, Medina JE, Rondina RVD, Coussio JD. Genus Baccharis L. I: Phytochemical Analysis of a non-polar Fraction from Baccharis crispa Sprengel. Planta Med. 1978; Vol 34: 328-31.