Genet Resour Crop Evol (2007) 54:849–854 DOI 10.1007/s10722-006-9161-x ORIGINAL PAPER Seed germination and triterpenoid content of Anemopaegma arvense (Vell.) Stellfeld varieties Ana M. S. Pereira Æ Antonieta N. Saloma˜o Æ Ana H. Januario Æ Bianca W. Bertoni Æ Saulo F. Amui Æ Suzelei C. Franc¸a Æ Antonio L. Cerdeira Æ Rita M. Moraes Received: 30 September 2005 / Accepted: 13 April 2006 / Published online: 7 December 2006 Ó Springer Science+Business Media B.V. 2006 Abstract Anemopaegma arvense (Vell.) Stell- time. Storing catuaba seeds at low temperatures feld, Bignoniaceae, is a native species of the (–20 and –196°C) has not affected emergence and Brazilian savanna (Cerrado) and is commonly survival. These findings suggested that A. arvense known as catuaba among the local farmers. Seeds seeds have an orthodox behavior resisting well to of three varieties were collected in different dehydration and low temperature storage. Three localities and submitted to germination and stor- catuaba varieties were characterized morpholog- age studies in attempting to domesticate this ically and chemically. The presence of triterpenes species as a medicinal crop for small farmers such as oleanolic acid and betulinic acid were located in Brazilian Cerrado. Germination tests identified and quantified in these varieties. Pre- revealed that catuaba seeds presented a dor- vious report has shown that these compounds mancy period of 6 weeks, and 63% of the seed- have promising anticancer activities and herein lings have emerged after 12 weeks of the planting the results point that the aerial parts yielded more triterpenes than the roots. The combination of A. M. S. Pereira Æ A. H. Januario Æ B. W. Bertoni Æ higher capacity and preferential accumulation of S. F. Amui Æ S. C. Franc¸a triterpenes in the aerial parts of catuaba makes Plant Biotechnology Center, University of Ribeirao this plant a potential candidate for agricultural Preto, Av. Costabile Romano, 2201, Ribeirao Preto, production or in situ sustainable harvests as a SP 14.096-380, Brazil promising alternative to the destructive collection A. N. Saloma˜o of the natural population. Brazilian Department of Agriculture, Agricultural Research Service, Embrapa-Cenargen, C.P. 02372, Keywords Anemopaegma arvense Æ Brasilia, DF 70770-900, Brazil Bignoneaceae Æ Catuaba Æ Medicinal plant Æ A. L. Cerdeira Oleanolic acid Æ Seed germination Æ Triterpenes Brazilian Department of Agriculture, Agricultural Research Service, Embrapa-Environment, C.P. 69, Jaguariuna, SP 13820-000, Brazil Introduction R. M. Moraes (&) National Center for Natural Products Research, Catuaba, Anemopaegma arvense (Vell.) Stellfeld, Research Institute of Pharmaceutical Sciences, School Bignoniaceae, is a native species of the Brazilian of Pharmacy, The University of Mississippi, University, MS 38655, USA savanna (Cerrado). Cosmetic products containing e-mail: [email protected] catuaba extracts were patented as being 123 850 Genet Resour Crop Evol (2007) 54:849–854 anti-wrinkle, preventing skin aging, stimulating 47°24¢39;00¢¢W, Alt. 1,017 m). Specimens col- hair growth and blocking sunscreen (Kokou et al. lected in each location were pressed and depos- 2000; Shimizu 2001; Yamashita and Fujita 2002; ited at the herbarium of the Biotechnology Mio et al. 2003). Among local people catuaba has Department, Ribeirao Preto University with the also been used as a stimulant, aphrodisiac, and for following voucher numbers HPM-UNAERP 028, the treatment of hypertension (Hamet 1937). HPM-UNAERP 093 and HPM-UNAERP 0750. Phytochemical study revealed that catuaba Twigs of these three accessions were collected extracts contain catechin and cinchonains for morphological and chemical characterization. (Uchino et al. 2004). Per accession 25 measurements were made for To supply the demand for roots, the commercial the various characters. Based on the morpholog- part of catuaba, the Cerrado’s natural resources ical characters the accessions were identified are being depleted. Greater damage than overex- as different varieties; Anemopaegma arvense ploitation is the relentless destruction of its habitat var. petiolata Bur., Anemopaegma arvense var. since the area has become the new agricultural puberula P.DC. and Anemopaegma arvense var. frontier in Brazil for sowing cash crops such as glabra P.DC. soybean and corn in Brazil (Myers et al. 2000). Recent surveys show that this ecosystem is a rich Germination studies biome with 7,000 plant species but just a few of them have been investigated regarding physiology, One week after seeds were collected, they were growth and development behavior, propagation submitted to germination studies using sand as and sustainability (Melo and Eira 1995; Gemaque substrate. Seeds were sown into planting trays at et al. 2002; Botezelli et al. 2000). depths of 1 cm in three replicates of 100 seeds Although, the cosmetic industry has claimed with PlantimaxÒ substrate composed of the mix- therapeutic properties of A. arvense, cultivation ture 2:1:1:1 v/v (manure, vegetable coal, soil, and still needs to be further encouraged. Thus it was sand) and kept in a glasshouse at 75% relative included into a conservation research program humidity, temperature at 30 ± 3°C and daily mist funded by the Biodiversity Research Program of irrigation for 30 min. Germination was evaluated Sao Paulo, Brazil (BIOTA). The major effort of weekly during 3 months. When cotyledons have this research program is to preserve the gene pool emerged, seeds were counted as germinated. of therapeutically relevant plant species. Pereira Root length was measured at the end of the et al. (2003) reported an in vitro propagation and experiment. Seeds of each variety were consid- conservation protocols to preserve catuaba elite ered as a treatment and 100 seeds were used per biotypes. In order to understand the gene pool replication of a treatment. Treatments were rep- variability of the species, the present study was licated three times (Table 2). Data were submit- conducted to characterize morphologically and ted to Tukey test (P £ 0.05). chemically the three varieties found in their For studying seed storage behavior the seeds native habitats and to study seed germination were dehydrated to 5.5% moisture content in an behavior of this species. air forced dryer and then exposed to tempera- tures of –20 and –196°C (liquid nitrogen) for 6 months. This procedure was repeated four Materials and methods times using 20 seeds per replication. After the storage of low temperature seeds were placed on Catuaba seeds were collected during the year of wet paper towel and kept in the dark at 25°C for 2002 in three regions in Brazil, in Uberlandia germination. Data were analyzed by Tukey test County, Minas Gerais state (Lat. 26°04¢21;00¢¢N, (P £ 0.05). Glasshouse germination studies were Long. 48°08¢58;00¢¢W, Alt. 747 m), in Rio Verde conducted at 30°C and germination after storage County, Goias state (Lat. 18°08¢43;30¢¢N, Long. at 25°C because preliminary studies have shown 49°57¢26;90¢¢W, Alt. 523 m) and in Pedregulho that this was the best range for germination and County, Sao Paulo state (Lat. 20°28¢20¢¢N, Long. the extreme temperatures were chosen. 123 Genet Resour Crop Evol (2007) 54:849–854 851 Growth measurements and yield of triterpenes leaves (Ferrreira 1973). Presence of hair in the stem and leaves are well noticed in puberula Seeds were sowed in plastic containers (10 l), 50 variety, while glabra has no hair. Plants of both seeds with three replications, containing soil/sand varieties puberula and glabra are the same height substrate at 1:1 ratio. These containers were kept but shorter than petiolata. in the greenhouse at average temperature of 30°C Seeds of the Bignoniaceae are distinguished and daily irrigation for 30 min. Growth mea- from related families due to the absence of surements from soil surface up to the apical endosperm; fruits often show conspicuously meristem were taken from aerial and under- wings, which are commonly perceived as winged ground parts at 5, 8 and 30 months after the seeds seeds (Laurence 1963). In evaluating the growth emerged. and development of the catuaba seedlings, data Plants were harvested and dried at 50°C, roots revealed that the presence of wings is not critical and aerial parts were ground separately into a fine for emergence. In fact, seedlings of winged seeds powder using coffee grinder. Ground plant produced shorter roots than non-winged ones material (100 mg) was extracted with methanol (data not shown). Thus, wing removal of the seeds by maceration for 30 min under sonication. The might be a helpful practice to obtain well-rooted solvent was evaporated to dryness and the residue plants. In addition, the results in Table 2 revealed dissolved in methanol. Each sample was extracted that after sowing, seeds of catuaba were dormant in triplicates. Extracts were analyzed by HPLC for 42 days. After this period, seeds started to analysis performed by Shimadzu LC-10AD VP, emerge and most sprouted in the period up to day Column LC-18 Shim-pack 25 cm · 4.6 mm 84 after planting. The speed of germination varied (5 lm); mobile phase MeOH 85% and 15% among the three varieties. Cotyledons of puberula (H2O + 0.1% CH3COOH) for 15 min, MeOH and petiolata were noticed 28 days after planting 100% (5 min) at 0.9 ml/min. Absorbance was while glabra seedlings required 12 days. At measured at 210 nm according to the procedure 49 weeks after planting, (84 days), germination described by Bolta et al. (2000). The pentacyclic percentage among these catuaba varieties triterpenoid standards oleanolic acid, betuline (Table 2) was alike, but roots were more devel- and betulinic acid and ursolic acid were pur- oped in seedlings of variety petiolata. chased from Sigma. Seed technologists have classified seeds into orthodox, intermediate and recalcitrant species HPLC-mass spectrometry analysis depending on their storage behavior after dehy- dration (Kermode 1990).