Seasonal and Intraspecific Variation of Flavonoids and Proanthocyanidins in Cecropia glaziovi Sneth. Leaves from Native and Cultivated Specimens§ Pilar Ester Luengas-Caicedoa, Ferna˜o Castro Bragab, Geraldo Ce´lio Branda˜ob, and Alaı´de Braga de Oliveirab,* a Universidad Nacional de Colombia, Facultad de Ciencias, Departamento de Farmacia, Ciudad Universitaria, Bogota´, Colombia b Faculdade de Farma´cia, Departamento de Produtos Farmaceˆuticos, Universidade Federal de Minas Gerais, Av. Antoˆ nio Carlos, 6627, CEP 31.270-901 Belo Horizonte, MG, Brazil. E-mail: [email protected] * Author for correspondence and reprint requests Z. Naturforsch. 62c, 701Ð709 (2007); received February 2/March 23, 2007 Cecropia glaziovi Sneth. (syn. C. glaziovii, C. glazioui) (Cecropiaceae) is a South American medicinal plant whose antihypertensive activity is attributed to its flavonoid and proanthocy- anidin contents. The seasonal and intraspecific variations of these two classes of compounds in C. glaziovi leaves were assayed by spectrophotometry in samples of young and mature leaves collected from native, cultivated and micropropagated trees in the dry and rainy peri- ods of the year. The total flavonoid and proanthocyanidin contents ranged from (0.64 ð 0.21)% to (3.44 ð 0.45)% and (2.23 ð 0.92)% to (5.36 ð 0.95)%, respectively, among the assayed populations. The flavonoid contents in native plants did not differ statistically be- tween young and mature leaves within the same season, whereas it was higher in both young and mature leaves collected in the dry compared to those collected in the rainy period. For cultivated specimens, the results pointed to higher contents in the dry season, whereas no significant difference was observed for leaves of micropropagated (clone) plants collected in both periods. For the assayed populations, higher proanthocyanidin contents were found in the dry season, excepting the micropropagated (clone) plants, whose contents did not differ significantly between the dry and the rainy periods. Leaves of micropropagated (clone) and cultivated specimens showed less intraspecific variation in the flavonoid and proanthocyani- din contents than those from native trees. These features suggest that, as expected, cultivation of C. glaziovi is of great interest providing raw herbal material of better uniform quality. Key words: Cecropia glaziovi Sneth., Flavonoids, Proanthocyanidins, Seasonal and Intraspe- cific Variation Introduction mum. Except for this last one, the five genera are The genus Cecropia comprises about 100 species morphologically distinct from both Moraceae and occurring in tropical America, from Mexico to Urticaceae (Sytsma et al., 2002).Cecropia glaziovi Southern Brazil (Berg, 1996). This genus has been Sneth. (syn. C. glazioui, C. glaziovii) is popularly placed in the order Urticales, family Cecropiaceae, named “embau´ ba” and “tore´m” in Brazil; “gua- which was morphologically considered as inter- rumo” and “yarumo” in Colombia and Equator; mediate between the Moraceae and Urticaceae “yongol” in Peru; and “yagrumo” in Venezuela (Berg, 1978). Morphological comparisons and re- (Pio Correˆa and Penna, 1969; Pe´rez-Arbela´ez, cent plastid DNA studies indicate that the Cecro- 1996). This species and other Cecropia spp. (C. piaceae in the broad sense are more closely re- adenopus, C. pachystachya, C. peltata and C. holo- lated to the Urticaceae than to the Moraceae and leuca) are traditionally used in several Latin are derived from the Urticaceae. This family is American countries as antihypertensive, cardio- constituted by six genera, Cecropia, Coussapoa, tonic, diuretic and anti-asthmatic. It is a tree of 8Ð Musanga, Myrianthus, Pourouma and Poikilosper- 16 m height, with a slender, hollow, whitish trunk and reddish buds. In Brazil, the species is widely found in the central and southern regions (Pe´rez- § Part of the thesis presented by P. E. Luengas-Caicedo Arbela´ez, 1996; Lorenzi and Matos, 2002). to Curso de Po´ s-Graduac¸a˜oemCieˆncias Farmaceˆuti- The antihypertensive activity of an aqueous ex- cas Ð CPGCF, UFMG, Belo Horizonte, MG, Brazil. tract and butanolic fraction from C. glaziovi leaves 0939Ð5075/2007/0900Ð0701 $ 06.00 ” 2007 Verlag der Zeitschrift für Naturforschung, Tübingen · http://www.znaturforsch.com · D 702 P. E. Luengas-Caicedo et al. · Cecropia Flavonoids and Proanthocyanidins has been pharmacologically demonstrated in iso- tion inhibition, anti-inflammatory, anti-allergic, an- lated preparations, animal models and humans ticarcinogenic, anti-arthrithic, anxiolytic and anti- (Franck, 1998; Franck et al., 1996; Lapa et al., 1999; hypertensive activities (Di Carlo et al., 1997; Rocha et al., 2002). This effect is considered to be Robards and Antolovich, 1997). Recent advances related to a blockade of the voltage-gated calcium in the knowledge on the neuropharmacological channels in vascular smooth muscles (Lapa et al., and cardiac effects of flavonoids point out to their 1999; Rocha et al., 2002). It is suggested that fla- potential for the management of various psychiat- vonoids and proanthocyanidins are involved in the ric conditions and cardiac insufficiencies including antihypertensive acitivity of C. glaziovi leaves by the treatment of hypertension, arrhythmia and ta- acting as inhibitors of angiotensin-converting en- chycardia (Johnson and Beart, 2004; WHO, 2002). zymes (ACEs) (Lacaille-Dubois et al., 2001). Considering the importance of C. glaziovi as a Anxiolytic-like effects were also reported for Brazilian medicinal plant and the previous phar- extracts of this species (Rocha et al., 2002). macological investigations that have confirmed its It is well known that the qualitative and quanti- antihypertensive and anxiolytic effects, possibly tative contents of secondary metabolites in a plant related to flavonoids and proanthocyanidin, along show marked variation, that is regulated by intrin- with the lack of information on chemical parame- sic factors (ontogeny and phenology) and also by ters for drug quality control, the main goal of the abiotic (e.g. light, moisture, nutrient availability) present study was to evaluate the seasonal and in- and biotic factors such as different physiological traspecific variation of the total flavonoid and pro- and growth stages (Harborne, 1993; Brooks and anthocyanidin contents in the leaves of this spe- Feeney, 2000; Sosa et al., 2005; Calixto, 2000). In cies. the case of plants used for medicinal purposes, all these factors must be considered, besides the post- Materials and Methods harvesting managements (Sharapin, 2000). Flavonoids are ubiquitous secondary products Plant material of plants and about 4,000 representatives are Five different populations of C. glaziovi were known (Harborne, 1977; Witzell et al., 2003). The used in the quantitative assays. Three populations production of flavonoids shows a wide qualitative of adult native specimens were harvested in the and quantitative variation between plant organs state of Minas Gerais, Brazil: 1) Parque Estadual and is dependent on plant growth, environmental do Rio Doce (PERD); 2) campus Pampulha, Uni- factors and stress stimuli such as UV irradiation, versidade Federal de Minas Gerais (UMFG), Belo drought and high temperatures (Sosa et al., 2005; Horizonte; and 3) Serra da Piedade, Caete´,intwo Witzell et al., 2003). Besides the physiological role periods within a year, at the end of the rainy pe- of plant flavonoids, they have been recognized as riod (RP; MarchÐApril) and of the dry period part of the defense strategies being responsible for (DP; SeptemberÐOctober). Two other groups of important ecological functions such as antimicro- trees of similar ages were selected from popula- bial, antifungal, antioxidant, allelopatic and de- tions of cultivated C. glaziovi. The first one was toxificant of heavy metals. Consequently, they are harvested in the vicinity of Ubatuba, state of Sa˜o involved in plant-animal and plant-plant biochem- Paulo, Brazil, and the second one was originated ical relationships, as well as in nutrient cycles from micropropagation of a plant obtained by tis- (Sosa et al., 2005; Simmonds, 2003). Flavonoids are sue culture methods. Initial plant material for in frequently found in fruits and vegetables and vitro assays was obtained from one plantlet de- therefore are part of the human diet. They are also rived from seed germination as previously de- responsible for the pharmacological effects of sev- scribed (Alves, 1993). All plants derived from this eral medicinal plants. As a consequence of their process resulted in a clone. These cultivated plants chemical diversity and biological functions, there were established at the experimental field of Cen- is an increasing interest in this group of phyto- tro de Pesquisas Quı´micas, Biolo´ gicas e Agronoˆ m- chemicals as chemotaxonomic markers, as well as icas (CPQBA), Campinas, state of Sa˜o Paulo, Bra- in their ecological role and beneficial health ef- zil. Young (from buds; YL) and mature leaves fects in chronic and degenerative diseases. They (from lower half part of the trees; ML) were col- disclose a wide pharmacological profile including lected from native, cultivated and micropropa- antioxidant, free radical scavenger, lipid peroxida- gated trees. Sampling was of 1 to 10 individuals P. E. Luengas-Caicedo et al. · Cecropia Flavonoids and Proanthocyanidins 703 Table I. Collection data for Cecropia glaziovi samples. Collection sitea Collection Development Sample codec Herbarium periodb stage of leaves voucher