Flavonoids and Triterpenes from the Nest of the Stingless Bee Trigona spinipes

No nordeste do Brasil, a abelha-sem-ferrão Trigona spinipes perfura o tronco de espécimens cultivados de Eucalyptus citriodora para a produção de exudatos. A investigação química do extrato etanólico do ninho de T. spinipes permitiu o isolamento dos triterpenos cicloartanos ácido magniferólico e ácido 3 -hidroxi-24-metilenocicloartan-26-óico, além dos flavonóides 3’-metilquercetina, sakuranetina, éter 7-metil campferol, tricetina e éter 7-metil aromadendrina como compostos majoritários. O isolamento de sakuranetina, éter 7-metil campferol e éter 7-metil aromadendrina do ninho de T. spinipes e do exudato de Eucalyptus citriodora, sugere esta espécie como origem botânica dos constituintes do ninho destas abelhas-sem-ferrão no nordeste do Brasil. A caracterização estrutural dos compostos isolados foi realizada utilizando-se métodos espectrométricos e comparação com dados da literatura.


Introduction
More than 500 species stingless bees of subfamily Meliponinae are of pantropical distribution, however, the great diversity of species from the tribe Trigonini and all from the trib e Meliponini are found in the Neotropics. 1 Indigenous stingless bee species from South America collect resinous material from plants and mix it with beewax and soil to form "geopropolis" or "divine elixir", terms suggested for the Meliponinae honeys. 2,3In a general way, they build more complex nests than Apis mellifera, although there are a great variety of forms, size and place of construction.The majority of species use closed cavities to build their nests, but some species of the genus Trigona build completely exposed aerial nests with an entrance normally build of wax and mud, and a passage way generally built with geopropolis ending at the storage pots. 4ntil now, chemical reports are concentrated to the honey bee Apis mellifera and only a few information is known about the chemistry of stingless bees, in spite of the estimated number of over 500 Neotropical species. 5eekeeping of stingless bees or "meliponiculture" is well practiced in Guatemala, Mexico and Venezuela and have been subject of an increasing interest for future research activities, especially because geopropolis seems to possess biological potentialities similar to that of honey bee propolis. 2,3The medicinal uses of geopropolis in folk medicine include properties related to digestive, respiratory, female fertility, skin and visual disorders.Pollen and cerumen of the nests are also used in therapy and the larvae of Melipone and Trigona species are used in local diets. 5,6In addition, the knowledge of the botanical origin of propolis and geopropolis is important to beekeepers to assure that bees have specific plants in their flight range, because the determination of the chemical types of their honey products based on the source plant is important for the biological activity associated with its chemistry.
Previous analysis of geopropolis from the indigenous stingless bees Firesomellita varia, Melipona favosa, Melipona compressipes, Scaptotrigona depilis and Paratrigona anduzei in tropical Venezuela, revealed a phenolic profile characterized by the occurrence of polyprenylated benzophenones. 7In the chemical investigation of geopropolis of the Brazilian stingless bees Melipona compressipes, Melipona quadrifasciata anthidioides and Tetragona clavipes, Bankova et al. showed by GC-MS analysis that the chemical composition of three samples was completely different among them. 8Diterpenes acids were found in all samples of geopropolis, and their amounts were significant in M. quadrifasciata anthidioides and T. clavipes.On the other hand, the pentacyclic triterpene -amyrin was identified as the main component in T. clavipes, the flavonoid pinobanksin in M. compressipes and aromatic aldehydes in Melipona quadrifasciata anthidioides, respectively.Surprisingly, the prenylated benzophenones characteristic of geopropolis from Venezuela were absent in geopropolis from Brazil, including the one from Melipona compressipes that was analyzed in both tropical areas.These findings indicated that the chemical composition of geopropolis from stingless bees from Brazil varied with the location of the bee species, and also with the available flora as the most important factor for the composition.
In the Northeast of Brazil, the indigenous stingless bee Trigona spinipes is known as "arapuá", "irapuá" or "bee-of-dog".They possess black coloration measuring around 5 to 6.5 mm in length.In our previous studies of the exudates from a Eucalyptus citriodora specimen, it was observed that T. spinipes usually visit the plant injuring its bark in order to make it exudates.In this work we report the isolation of cycloartane triterpenes and flavonoids as the main constituents of an entire nest of T. spinipes, in order to suggest that Eucalyptus citriodora is one of the botanical sources of the chemical constituents of this kind of stingless bees from Northeast of Brazil.

Results and Discussion
In our chemical investigation of the nest of the stingless bee Trigona spinipes from Northeast of Brazil, we have isolated and characterized by NMR the mixture of the cycloartane triterpenes, magniferolic acid (1) and 3 -hydroxy-24-methylenecycloartan-26-oic acid (2), besides the flavonoids 3'-methyl quercetin (3), sakuranetin (4), kaempferol 7-methyl ether (5), tricetin ( 6) and aromadendrin 7-methyl ether (7) as the main compounds.Although flavonoids are widespread in the propolis constitution of temperate zones, until now only the flavanone pinobanksin has been found in geopropolis of M. favosa from Venezuela and M. compressipes in Brazil. 7,8Pentacyclic triterpenes alcohols have been isolated from geopropolis of Tetragona clavipes, 8 and those with a cycloartane-type skeleton have been found only in Egyptian and Brazilian propolis from honey bees from southeastern region. 9,10However, this is the first report on the occurrence of magniferolic acid (1) and 3 -hydroxy-24-methylenecycloartan-26-oic acid (2) from stingless bees sources.
As well documented in the literature, the main sources of propolis and geopropolis in South America, West Asia and North America are resin exudates from trees.Bud exudates of Populus species are the main source of flavonoids of propolis from temperate zones.In Venezuela, where Populus are not native plants, stingless bees and honey bees visit Clusia specimens in order to collect a resin excreted at the bases of their flowers stamens. 11As a consequence, the chemical composition of both tropical propolis and geopropolis is particularly characterized by the presence of polyprenylated benzophenones, in accordance to the chemical constituents identified from Clusia flowers. 8n the Brazilian southeast, the profiles of flavonoids and other phenolic compounds were quite similar when samples of propolis and the exudate of Baccharis dracuncufolia were compared, revealing this species as the botanical origin for propolis production in this region. 12t was observed that Trigona spinipes bees from Northeast of Brazil visit specimens of Eucalyptus citriodora to collect the "kino", a dark resin exudate excreted after the bark is injured.In a previous chemical investigation of the kino from E. citriodora we have isolated the flavonoids sakuranetin, narigenin, aromadendrin 7-methyl ether, aromadendrin, -D-glucopyranoside. 13,14The isolation of sakuranetin ( 4), kaempferol 7-methyl ether (5), and aromadendrin 7-methyl ether (7) in both the nest and E. citriodora exudates, clearly evidenced their chemical relationship.
The above findings suggest that T. spinipes from the Northeast of Brazil does store biologically active compounds in their nests, and the kino of E. citriodora is one of its botanical sources.However, it is evident that further studies on the chemical constituents of stingless bees from different geographic areas in the Northeast region are needed in order to confirm the chemical relationship between this class of bees and the flora available to them as a source of resin.

General
Melting points were obtained on a Mettler FP82HT apparatus and are uncorrected.IR spectra were recorded using a Perkin Elmer 1000 FT-IR spectrophotometer.The mass spectra were obtained on a Hewlett-Packard 5971 mass spectrometer by electron impact ionization (70 eV). 1 H and 13 C NMR where recorded on a Bruker Avance DRX-500 (500 MHz for 1 H and 125 MHz for 13 C).Silica gel 60 (Merck, kiesegel 60 F254, 0.20 mm) were used for analytical TLC.Silica gel 60 (Merck, 230-240 mesh) was used for column chromatography.All compounds were visualized on TLC by spraying with vanillin/perchloric acid/EtOH followed by heating.

Trigona spinipes nest
The nest of the stingless bees Trigona spinipes Fabricius was collected in Fortaleza, state of Ceará, Northeast of Brazil.The bees were identified by Dr. Daniel Santiago Pereira of Universidade Federal Rural do Semi-Árido (UFERSA).